Human ACE2 gene knock-in mouse model, its production method and use

Abstract

Provided are mice with the human ACE2 gene knocked into their genomes, methods for their production, and their use in evaluating the in vivo efficacy of antiviral agents and vaccines against SARS-CoV-2. Human ACE2 was precisely inserted into the mouse genome at the site of the mACE2 gene, establishing a novel knock-in mouse model for the human ACE2 gene at the site of the mACE2 gene.

Description

Technical field:

[0001] The present invention relates to mice with the human angiotensin-converting enzyme II (hACE2) gene knocked into their genomes, as well as methods for producing and using the same. Specifically, the present invention accurately inserts the hACE2 gene into the mouse mACE2 gene, establishing a novel knock-in mouse model with the hACE2 gene at the mACE2 gene site. The hACE2 knock-in mice are genetically stable and express only hACE2, thereby eliminating potential interference caused by mACE2 expression. This model can be used to evaluate the in vivo efficacy of antiviral agents and vaccines against SARS-CoV-2, as well as to study the infection mechanism of the new coronavirus. Background technology:

[0002] Coronaviruses belong to the genus Coronavirus (CoV) of the family Coronaviridae. They are positive-stranded, single-stranded RNA viruses with an envelope, approximately 80-120 nm in diameter, with an average diameter of 100 nm. They are spherical or elliptical, and exhibit pleomorphism. Spikes are present on the viral envelope, resembling the entire virus's corona, though the spikes differ significantly between different coronaviruses. Tubular inclusion bodies can sometimes be seen within coronavirus-infected cells. Coronaviruses have the largest genetic material of all RNA viruses. Existing data indicate that there are hundreds of species of coronaviruses, most of which are confined to animals. However, these viruses can occasionally cross into humans and cause disease.

[0003] Symptoms of COVID-19 are similar to those of patients infected with SARS-CoV or MERS-CoV, including fever, chills, cough, malaise, myalgia, headache, diarrhea, vomiting, and nausea. However, some patients may not experience typical clinical symptoms in the early stages of infection. Typical pathological features of severe cases include a prolonged inflammatory response, including destruction and desquamation of alveolar pneumocytes, formation of hyaline membranes, interstitial inflammatory infiltrates, and interalveolar hemorrhage. Multinucleated giant cells may also be present in the patient's tissues. CT images of most COVID-19 patients show typical features of ground-glass opacities and bilateral patchy shadows in the lungs. The infection can lead to immune dysfunction and multiple organ failure.

[0004] Vaccines are one of the most effective means of controlling disease epidemics. After the outbreak of the novel coronavirus, China has attached great importance to the research and development of novel coronavirus vaccines and has laid out the development of various types of vaccines, which are currently in the animal experiment stage. Animal models that can reproduce clinical characteristics are of great significance for vaccine development, antibody evaluation, research on viral infection mechanisms, and the formulation of epidemic prevention and control strategies. A variety of animals have been tried to construct susceptible or disease animal models of SARS-CoV-2, including non-human primates, ferrets, Syrian hamsters, New Zealand white rabbits, naturally immunodeficient mice, and wild-type mice. However, with the exception of two non-human primates, rhesus monkeys and marmosets, other animal species, including wild mice, are not susceptible to it.

[0005] Given that small animal models, such as rat and mouse models, have good economic performance and easy accessibility, they have obvious advantages over expensive and difficult-to-obtain non-human primate large animal models in routine scientific research, early vaccine evaluation and drug screening.

[0006] There is an urgent need in this field for preclinical animal models, especially mouse models, that simulate human pathogenesis caused by SARS-CoV-2 virus infection to develop vaccines and therapies against SARS-CoV-2 virus. SUMMARY OF THE INVENTION

[0008] The present invention provides an hACE2 gene knock-in mouse model in which mACE2 function is destroyed, which can well simulate the human disease progression caused by SARS-CoV-2 virus infection and is a useful small animal model for evaluating anti-SARS-CoV-2 viral agents and vaccines.

[0009] The first aspect of the present invention relates to a method for constructing an hACE2 knock-in mouse model that disrupts mACE2 function, the method comprising: (a) inserting the hACE2 gene at a specific site in the mACE2 gene in the mouse genome; (b) obtaining F1 offspring mice that are homozygous or heterozygous for the hACE2 gene at the mACE2 gene in the mouse genome; and (c) optionally, obtaining offspring of the mouse in (b). The mouse strain is not particularly limited, for example, including but not limited to mouse strains known in the art such as C57BL / 6, BALB / c, ICR, and KM mice. In one embodiment, the mACE2 gene in the mouse genome is selected from any exon of the mouse mACE2 gene, for example, exon 2 of the mACE2 gene, preferably 1-100 nt after the ATG of exon 2. In another embodiment, the method obtains a mouse that is homozygous for the hACE2 gene at exon 2 of the mouse mACE2 gene, for example, the hACE2-KI / NIFDC mouse of the present invention.

[0010] In one embodiment, the site-directed insertion is produced by using a nuclease agent that generates a double-strand break at the mACE2 gene in the mouse genome (e.g., at exon 2 of the mouse mACE2 gene), preferably, the nuclease agent is a transcription activator-like effector nuclease (TALEN), a zinc finger nuclease (ZFN) or a CRISPR / Cas system (e.g., a CRISPR / Cas9 system).

[0011] In another embodiment, step (a) comprises:

[0012] (i) introducing a CRISPR / Cas system (e.g., a CRISPR / Cas9 system) and donor DNA into a mouse zygote, wherein the CRISPR / Cas system (e.g., a CRISPR / Cas9 system) is capable of introducing a double-strand break at a target site at exon 2 of a mouse mACE2 gene in the mouse genome, and the donor DNA comprises the hACE2 gene;

[0013] (ii) introducing each zygote obtained in (i) into a surrogate mouse for pregnancy, and allowing the surrogate mouse to give birth to offspring;

[0014] The zygote and surrogate mouse can be from any mouse strain. In one embodiment, the zygote is from C57BL / 6 mice, BALB / C mice, ABI mice, etc., and the surrogate mouse is from KM mice, ICR mice, etc.;

[0015] (iii) using mice identified as hACE2-positive among the offspring of surrogate mice as founder mice, and mating the founder mice with the background mouse strain from which the zygote originated (e.g., mating the female founder mice with wild-type male mice from the background of which the zygote originated; or mating the male founder mice with wild-type female mice from the background of which the zygote originated) to obtain hACE2-positive F1 offspring, which are mice homozygous or heterozygous for the hACE2 gene, also referred to as F1 offspring-positive mice;

[0016] (iv) Optionally, interbreeding the F1 progeny positive mice obtained from (iii) to obtain mice homozygous for the hACE2 gene at exon 2 of the mouse mACE2 gene.

[0017] In one embodiment, the CRISPR / Cas system (e.g., CRISPR / Cas9 system) introduced into the mouse zygote is Cas mRNA (e.g., Cas9 mRNA) and guide RNA; Cas protein (e.g., Cas9 protein) and guide RNA; or a DNA construct comprising an expression cassette of a Cas protein (e.g., Cas9 protein) encoding gene and / or an expression cassette of a guide RNA, preferably, the Cas protein (e.g., Cas9 protein) encoding gene is operably linked to one or more polynucleotides encoding a nuclear localization signal.

[0018] In one embodiment, the donor DNA introduced into the mouse zygote is an expression cassette comprising the hACE2 gene; preferably, the donor DNA introduced into the mouse zygote is a targeting vector comprising the hACE2 gene; more preferably, the targeting vector comprises a 5' homology arm, an hACE2 gene, an element that enhances mRNA stability and translation efficiency (e.g., a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE)), a poly A sequence, and a 3' homology arm; most preferably, the targeting vector comprises a 5' homology arm, an hACE2 gene, an internal ribosome entry site (IRES) sequence and / or a 2A sequence, a reporter gene (e.g., a tdTomato reporter gene), a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), a poly A sequence, and a 3' homology arm; wherein the homology arm is a sequence homologous to the genomic sequence at exon 2 of the mouse mACE2 gene.

[0019] In one embodiment, the method for constructing a novel mouse model of hACE2 knock-in further comprises, in step (b), identifying the genotype and phenotype of mice heterozygous or homozygous for the hACE2 gene at the mACE2 gene in the mouse genome (e.g., exon 2 of the mouse mACE2 gene). In one embodiment, a mouse homozygous for the hACE2 gene is obtained, also referred to as a hACE2-KI / NIFDC mouse. In one embodiment, the genotype of the knock-in mouse is identified by PCR, Southern blotting, etc., the expression of hACE2 mRNA in the knock-in mouse is identified by reverse transcription polymerase chain reaction (RT-PCR), and / or the expression of hACE2 protein and its distribution in the mouse are identified by Western blotting and bioluminescence imaging (BLI).

[0020] The second aspect of the present invention relates to the use of the hACE2 gene knock-in mouse model constructed by the above method for evaluating the in vivo efficacy of antiviral drugs and vaccines against SARS-CoV-2, such as evaluating the in vivo protective efficacy of the vaccine, or evaluating the in vivo efficacy of the drug. For example, evaluating the in vivo preventive and / or therapeutic efficacy of antiviral drugs and vaccines against SARS-CoV-2, including determining the in vivo efficacy of the antiviral agents and vaccines by evaluating the body weight of hACE2 gene knock-in mice, the viral load in various tissues of the mice, the viral titer, the degree of histopathological lesions including histopathological scores, immunofluorescence staining and / or immunohistochemical analysis of viral load.

[0021] In one aspect, the present invention relates to a method for evaluating the in vivo protective efficacy of a vaccine against SARS-CoV-2 virus using the mouse model described herein, comprising the following steps:

[0022] 1) Obtain the hACE2 mouse model described herein;

[0023] 2) administering the SARS-CoV-2 virus vaccine to be evaluated to one group of hACE2 mouse models obtained in 1), and administering an adjuvant to another group of mouse models obtained in 1) as a control;

[0024] 3) Infecting the mouse model vaccinated with the vaccine and adjuvant obtained in 2) with a SARS-CoV-2 virus strain. In some embodiments, the infection is performed after vaccination, and the titer of antibodies produced against the vaccine and the increase in titer are monitored, and the antibody level is monitored until a certain level is reached.

[0025] 4) Detecting the degree of SARS-CoV-2 viral infection in the two groups of mouse models in 3). In some embodiments, the weight change and / or birth rate of each group of mice are detected from the date of infection. In some embodiments, the detection of the degree of viral infection includes detecting indicators such as viral titer, pathological changes in animal tissues, and / or changes in cytokines. In some embodiments, the detection requires dissecting each group of animals and performing the detection in the collected target organs. In some embodiments, the detection is performed 3-10 days after infection, depending on the characteristics of the vaccine. In a specific embodiment, the weight change and birth rate of each group of mice are monitored until the date of infection; 3-10 days after infection (the detection time varies depending on the different characteristics of the vaccine), each group of animals is dissected, the target organs are collected, and the viral titer, pathological changes, cytokine changes (such as cytokines related to inflammatory responses) and other indicators are detected.

[0026] 5) Comparing the degree of viral infection between the vaccine-immunized group and the adjuvant-immunized group, if the degree of SARS-CoV-2 viral infection in the mouse model administered with the vaccine to be evaluated is lower than that in the control mouse model administered with the control adjuvant, it indicates that the vaccine to be evaluated has in vivo protective efficacy. In some embodiments, the viral titer of the vaccine-immunized group and the adjuvant-immunized group are compared, the degree of lesions in the two groups of models are compared, and / or the cytokine production is compared. If, compared with the adjuvant-immunized group, the viral titer of the vaccine-immunized group is lower, the degree of lesions is milder, and the level of cytokines (e.g., cytokines related to inflammatory responses) is lower or closer to that of normal mice, it indicates that the vaccine to be evaluated has in vivo protective efficacy.

[0027] In a specific embodiment, the method of the present invention for evaluating the in vivo protective efficacy of a vaccine against SARS-CoV-2 virus comprises the following steps:

[0028] 1) Obtain the hACE2 mouse model described herein;

[0029] 2) administering the SARS-CoV-2 virus vaccine to be evaluated to one group of hACE2 mouse models obtained in 1), and administering a control adjuvant to another group of mouse models obtained in 1) as a control;

[0030] 3) After vaccination, monitoring the titer of antibodies produced against the vaccine to be evaluated and the process of titer increase. When the antibody level reaches a certain level, infecting the vaccine and adjuvanted mouse model obtained in 2) with the SARS-CoV-2 virus strain;

[0031] 4) Monitor the weight changes and birth rate of mice in each group from the day of infection. 3-10 days after infection (depending on the characteristics of the vaccine, the detection time varies), dissect the animals in each group, collect target organs, and detect viral titers, pathological changes, cytokine changes, and other indicators.

[0032] 5) The following demonstrates that the vaccine to be evaluated has protective efficacy in vivo: the viral titers of the vaccine-immunized group and the adjuvant-treated group are compared, and the SARS-CoV-2 virus titer in the mouse model administered with the SARS-CoV-2 virus vaccine is lower than the degree of SARS-CoV-2 virus infection in the control mouse model administered with the control adjuvant; for example, the degree of lesions and cytokine production in the two groups of models are compared: the degree of lesions in the vaccine-immunized group should be relatively mild, with lower cytokine levels or closer to those in the normal mouse group, while the adjuvant-treated group has more severe pathological changes and higher levels of cytokines related to inflammatory responses.

[0033] In some embodiments, the method further comprises testing the immunogenicity of the vaccine on ordinary mice or the mouse model of the present invention before administering the vaccine in step 2).

[0034] In some embodiments, the "adjuvant" used is generally a corresponding solvent as a control for the vaccine, such as PBS or physiological saline.

[0035] The present invention also relates to a method for evaluating the in vivo efficacy of antiviral drugs against SARS-CoV-2 virus using the mouse model described herein, comprising the following steps:

[0036] 1) Obtain the hACE2 mouse model described herein;

[0037] 2) Infecting the hACE2 mouse model obtained in 1) with the SARS-CoV-2 virus strain;

[0038] 3) Optionally, testing the extent of SARS-CoV-2 infection in the mouse model described in 2). In some embodiments, testing the extent of viral infection includes measuring indicators such as weight change, viral titer, pathological changes in animal tissues, and / or changes in cytokines, or testing viral infection by immunofluorescence staining. In some embodiments, testing is performed by sacrificing a portion of a group of mice. In some embodiments, the extent of viral infection in mice is tested in vivo.

[0039] 4) The mouse models obtained in 2) are divided into groups for treatment, wherein one group of mouse models is administered with a test drug for the SARS-CoV-2 virus (e.g., administered in three dose groups: high, medium, and low), and another group of mouse models obtained in 2) is administered with a vehicle (e.g., PBS or saline) as a control;

[0040] 5) Detecting the extent of SARS-CoV-2 infection in the two mouse models described in 4). In some embodiments, detecting the extent of viral infection includes measuring body weight changes, viral titers, pathological changes in animal tissues, and / or changes in cytokines, or detecting viral infection by immunofluorescence staining. In some embodiments, the detection requires dissecting each mouse model group and performing detection in the collected target organs.

[0041] 6) The effect of the antiviral drug is measured by the following indicators: the degree of SARS-CoV-2 virus infection in the mouse model administered with the test drug is lower than the degree of SARS-CoV-2 virus infection in the control mouse model administered with the vehicle, indicating that the test drug is effective; the degree of SARS-CoV-2 virus infection in the mouse model administered with the test drug is reduced (the degree of virus infection detected in step 3) vs. the degree of virus infection detected in step 5) is greater than the degree of SARS-CoV-2 virus infection in the control mouse model administered with the vehicle (the degree of virus infection detected in step 3) vs. the degree of virus infection detected in step 5), indicating that the test drug is effective. In some embodiments, if the viral titer of the mouse model in the group administered with the drug is reduced, the degree of tissue pathological changes is reduced, the level of cytokines (such as cytokines related to inflammatory response) is lower or closer to that of normal mice, and the weight loss is slowed compared to the control group administered with the vehicle, it indicates that the test drug is effective. In other embodiments, if the virus titer of the mouse model in the group administered with the drug is reduced more, the degree of tissue pathological changes is alleviated more, the level of cytokines (such as cytokines related to inflammatory responses) is reduced more or closer to that of normal mice, and the weight loss is slowed down to a greater extent compared to the changes in virus titer, degree of tissue pathological changes, cytokine changes and body weight changes in the control mouse model administered with the vehicle, it indicates that the test drug is effective.

[0042] The present invention also relates to a method for evaluating drugs against the SARS-CoV-2 virus using the mouse model described herein, comprising the following steps:

[0043] 1) Obtain the hACE2 mouse model described herein;

[0044] 2) Infecting the mouse model obtained in 1) with the SARS-CoV-2 virus strain;

[0045] 3) Detecting the degree of SARS-CoV-2 infection in the mouse model described in 2). In some embodiments, detecting the degree of viral infection includes measuring indicators such as weight change, viral titer, pathological changes in animal tissues, and / or changes in cytokines, or detecting viral infection by immunofluorescence staining. In some embodiments, the detection is performed by sacrificing a portion of a group of mice. In some embodiments, the degree of viral infection in mice is detected in vivo.

[0046] 4) administering a test drug against the SARS-CoV-2 virus to a group of mouse models obtained in 2) (e.g., administering three dose groups of high, medium, and low doses);

[0047] 5) Detecting the extent of SARS-CoV-2 infection in the mouse model described in 4). In some embodiments, detecting the extent of viral infection includes measuring indicators such as weight change, viral titer, pathological changes in animal tissues, and / or changes in cytokines, or detecting viral infection by immunofluorescence staining. In some embodiments, such detection requires dissecting the mouse model and performing detection in the collected target organs.

[0048] 6) The efficacy of the antiviral drug is measured using the following indicators: a lower degree of infection in the mouse model tested in 5) compared to the degree of infection in the mouse model tested in 2) indicates that the drug is effective. In some embodiments, if the viral titer in the mouse model tested in 5) is reduced, the degree of tissue pathological changes is alleviated, or the level of cytokines (e.g., cytokines related to inflammatory responses) is lower or closer to that of normal mice, compared to the indicators tested in 2), the drug is effective.

[0049] The present invention also relates to a method for screening drugs against the SARS-CoV-2 virus using the mouse model described herein, comprising the following steps:

[0050] 1) Select possible antiviral drugs against SARS-CoV-2 virus;

[0051] 2) evaluating the in vivo efficacy of the drug against SARS-CoV-2 in a mouse model, for example, using the evaluation method of the present invention;

[0052] 3) Select the drug evaluated as effective in 2) as a drug against the SARS-CoV-2 virus.

[0053] In one embodiment, drugs against SARS-CoV-2 virus (anti-SARS-CoV-2 viral drugs) include antibodies against SARS-CoV-2 virus, such as SARS-CoV-2 viral antiserum.

[0054] In some embodiments, the antibody, vaccine, or drug to be evaluated or candidate can be "administered" in any manner. The preferred mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal (ip), intramuscular) administration. In a preferred embodiment, administration is by intravenous infusion or injection. In another preferred embodiment, administration is by intramuscular, intraperitoneal, or subcutaneous injection.

[0055] In some embodiments, the "level of viral infection" in a mouse model is determined by, for example, measuring viral load in various tissues of the mouse (e.g., by RT-qPCR), histopathological scoring, immunofluorescence staining, and / or immunohistochemical analysis of viral load. In some embodiments, the detection of the level of viral infection includes detecting indicators such as weight loss, viral titer, pathological changes in animal tissues, and / or changes in cytokines.

[0056] In one embodiment, the infection in step 2) comprises intragastric infection or intranasal infection, for example, by nasal drops or gavage. In one embodiment, the infection dose in step 2) depends on the dose of the virus. In this experiment, the infection dose of nasal drops is 10 4 -10 5 PFU, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, or 9) × 10 4 PFU or 10 5 PFU, the infection dose for oral administration is 10 6 -10 7 PFU, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, or 9 × 10 6 PFU or 10 7 PFU.

[0057] The "vehicle" used is generally the corresponding solvent used as a control for the drug, such as PBS or physiological saline.

[0058] In some embodiments, the present invention also relates to the use of the mouse model described herein for evaluating the efficacy of a vaccine against the SARS-CoV-2 virus, or for screening a vaccine against the SARS-CoV-2 virus.

[0059] In some embodiments, the present invention also relates to the use of the mouse model described herein for evaluating the efficacy of drugs, such as antibodies, against the SARS-CoV-2 virus, or for screening drugs, such as antibodies, against the SARS-CoV-2 virus. Description of the accompanying drawings:

[0061] Figure 1A : Shows the constructed targeting vector.

[0062] Figure 1B : Shows the enzyme digestion identification map of the constructed targeting vector.

[0063] Figure 1C : Shows the knock-in strategy of the targeting construct at the mACE2 gene in the mouse genome as exemplified.

[0064] Figure 2A : Shows the pre-cut diagram of the pCS-3G vector to which the sgRNA is to be linked.

[0065] Figure 2B : Shows the activity of multiple designed sgRNAs, and Con represents the blank control.

[0066] Figure 3 : Shows the Southern blot results of F1 offspring mice.

[0067] Figure 4 : Shows the expression of hACE2 in the lung, kidney, spleen, and liver of F1 offspring mice and compares it with the expression of mACE2 protein in mice.

[0068] Figure 5 : Shows the overall expression pattern of hACE2 in F1 offspring young (4.5 weeks old) and older (30 weeks old) mice.

[0069] Figure 6A : Shows the expression of mACE2 mRNA in various organs of F1 offspring mice compared with wild-type controls.

[0070] Figure 6B : Shows the expression of hACE2 mRNA in various organs of F1 offspring mice compared with wild-type controls.

[0071] Figure 6C : shows the expression of hACE2 mRNA in F1 offspring young (4.5 weeks old) and older (30 weeks old) mice.

[0072] Figure 7 Figure 2 shows the situation of hACE2 humanized mice infected intranasally with SARS-CoV-2: Figure A: Wild-type C57BL / 6 mice, young (4 weeks old) and older (28 weeks old) hACE2-KI / NIFDC mice (n=3) were anesthetized with 50 mg / kg sodium pentobarbital via the intraperitoneal route and then infected intranasally with 40,000 pfu of SARS-CoV-2. Mice were sacrificed on day 6 after infection, and lung, brain, laryngotrachea, spleen, kidney, liver, small intestine and serum tissues were collected. Figure B: Tissue distribution of SARS-CoV-2 viral RNA. Viral RNA copies were analyzed by RT-qPCR on a portion of each tissue and serum. Figure 7C: Mouse lung paraffin sections were immunofluorescently stained for SARS-CoV-2 S protein (green), ACE2 (red), and DAPI (blue). Figure D: Immunofluorescence staining. Tissue paraffin sections were immunofluorescently stained for PDPN cell markers (indigo), ACE2 (white), CC10 (green), SPC (gold), and b-tubulin (purple). The white box is magnified on the right. Solid yellow arrows, red arrows, and white arrows indicate SARS-CoV-27ACE27CC10+ cells, SARS-CoV-27CC10+ cells, and ACE27CC10+ cells, respectively.

[0073] Figure 8 Figures show pathological findings and inflammatory responses in hACE2-KI / NIFDC humanized mice infected intranasally with SARS-CoV-2. Panels A and B: Histopathological analysis demonstrates mild inflammation in hACE2-KI / NIFDC humanized mice. Panel A: Inflammatory cell infiltration (yellow arrows), thickened alveolar septa, and distinctive vascular damage (blue arrows) were observed in both young and older hACE2-KI / NIFDC humanized mice. More alveolar epithelial cell lesions and focal hemorrhages (green arrows) were observed in older mice. (Right panel) Semi-quantitative histological analysis of H&E-stained lung sections on day 6 (right panel). Panels B and C: IHC staining analysis. Increased neutrophils (Neu+) and macrophages (CD68+) were observed in hACE2-KI / NIFDC humanized mice. Semi-quantitative neutrophil and macrophage counts were performed in the right panel. Panel D: Cytokine expression in serum from SARS-CoV-2-infected mice. Statistical significance was analyzed by unpaired Student's t-test. *, P < 0.05.

[0074] Figure 9 Shown are hACE2-KI / NIFDC humanized mice infected intragastrically with SARS-CoV-2. Figure 9 A: Wild-type C57BL / 6 mice and hACE2-KI / NIFDC mice (n=3) were infected with 4×10 6 pfu of SARS-CoV-2. Mice were killed on day 5 after infection, and the main tissues including lung, brain, larynx and trachea, spleen, kidney, liver, small intestine and serum were obtained; Figure 9 B: Tissue distribution of SARS-CoV-2 viral RNA. Viral RNA copies were analyzed in a portion of each tissue and serum by RT-qPCR. Figure 9 C: Immunofluorescence staining of mouse lung paraffin sections against SARS-Cov-2 S protein (green) and DAPI (blue). Figure 9 D: In hACE2-KI / NIFDC humanized mice, interstitial inflammation was observed, accompanied by thickening of the alveolar septa (yellow arrows). No significant vascular damage was observed in either wild-type or hACE2-KI / NIFDC humanized mice (blue arrows). Histopathological analysis of mouse lungs. Semiquantitative analysis was performed in the right panel. Statistical significance was analyzed by unpaired Student's t-test. *, P < 0.05.

[0075] Figure 10 Shown are body weight changes in hACE2-KI / NIFDC humanized mice infected intranasally with SARS-CoV-2.

[0076] Wild-type C57BL / 6 mice (young (4 weeks old) and old (28 weeks old)) and hACE2-KI / NIFDC humanized mice (n=3) were intranasally infected with 40,000 pfu SARS-CoV-2, and body weight changes were monitored daily.

[0077] Figure 11 Figure 2 shows IFA detection of SARS-CoV-2 viral infection in the brains of hACE2 humanized mice infected intranasally with SARS-CoV-2. Wild-type C57BL / 6 mice and older hACE2-KI / NIFDC humanized mice (n=3) were intranasally infected with 40,000 pfu SARS-CoV-2. They were sacrificed on day 6 after intranasal infection and brain tissue was collected. Paraffin sections of collected mouse brain tissue were immunofluorescently stained for SARS-CoV-2 S protein (green) and DAPI (blue).

[0078] Figure 12 This figure shows IFA detection of SARS-CoV-2 infection in the brains of hACE2-KI / NIFDC humanized mice. Wild-type C57BL / 6 mice and IFNαβγ-positive C57BL / 6 mice (n=3) were intranasally infected with 40,000 pfu of SARS-CoV-2. Mouse weight changes were monitored daily. Mice were sacrificed on day 6 of intranasal infection, and major tissues including lung, brain, larynx and trachea, spleen, kidney, liver, small intestine, and serum were collected. Figure 12 A: Tissue distribution of SARS-CoV-2 in mice after intranasal administration. Viral RNA copies were analyzed in a portion of each tissue and serum by RT-qPCR. B: Body weight changes of mice after SARS-CoV-2 infection.

[0079] Figure 13 The weight data of mice before challenge (0 dpi), 1 day and 3 days after challenge and before autopsy (4 dpi) are shown. It can be seen that there is no obvious decrease in the weight of heterozygous and homozygous mice.

[0080] Figure 14 The figure shows the lung weight ratio calculated by weighing the mouse body weight and the total weight of the lung during autopsy. It can be seen from the figure that the lung weight ratio of the heterozygous mouse group is slightly higher than that of the homozygous mouse group, but the data of the heterozygous mouse group are relatively discrete.

[0081] Figure 15 shows the pathological findings and inflammatory responses in heterozygous and homozygous mice infected intranasally with SARS-CoV-2 (Panel A: homozygous group, Panel B: heterozygous group). As can be seen, there were no significant differences between the groups in the lung and spleen, but the homozygous mice had higher pathological scores in the lung and spleen. In the liver, the homozygous group had more severe pathological damage than the heterozygous group. Overall, the pathological changes in the homozygous mice were more severe than those in the heterozygous mice.

[0082] Figure 16 The figure shows the pathological scores of each group of mice, performed by two blinded pathologists, and the statistical results were plotted. The figure shows that the homozygous mice group had higher scores. Detailed description of the invention:

[0084] I. Definition

[0085] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.

[0086] As used herein, the terms "comprise" or "comprising" are meant to include stated elements, integers or steps, but not to exclude any other elements, integers or steps.

[0087] The term "coronaviruses (CoV)" herein refers to viruses belonging to the genus Betacoronavirus of the family Coronaviridae, with virus particles that are spherical or ellipsoidal with a diameter of approximately 60 to 220 nm. "2019-nCoV" and "SARS-CoV-2" are used interchangeably herein.

[0088] The term "SARS-CoV-2" refers to a membrane-bound, single-stranded, positive-sense RNA virus measuring 80-120 nm in size. Its genome is approximately 29.9 kb long and shares 80% homology with the genome of SARS-CoV, also belonging to the genus Betacoronavirus in the family Coronaviridae. The spike (S) protein encoded by the viral genome primarily mediates viral entry by binding to host cell receptors and determines the virus's host specificity. Recent articles (Wrapp D et al., Cryo-EM Structure of the 2019-nCoV Spike in the Prefusion Conformation, Science, February 19, 2020, published online, pii:eabb2507.doi:10.1126 / science.abb2507) and (Xiaolong Tian et al., Potent binding of 2019 novel coronavirus spike protein by a SARS coronavirus-specific human monoclonal antibody, Emerging Microbes & Infections, 2020, 9:1, p382-385, DOI:10.1080 / 22221751.2020.1729069) have shown that the affinity (K) of the SARS-CoV-2 S protein binding to human ACE2 was detected by Fortebio. D ) is about 15nM, which is comparable to the affinity of SARS-CoV S protein binding to human ACE2. This shows that hACE2 is also the receptor protein for 2019-nCoV to infect the human body and enter cells.

[0089] The terms "SARS-CoV-2 spike protein", "SARS-CoV-2 Spike protein", "SARS-CoV-2S protein" or "SARS-CoV-2" are used interchangeably in this article. SARS-CoV-2S protein is one of the important structural proteins on the surface of the SARS-CoV-2 virus. The SARS-CoV-2S protein contains a receptor binding domain (RBD). SARS-CoV-2 binds to the host cell surface receptor angiotensin-converting enzyme II (ACE2) with the help of this domain, mediating membrane fusion, thereby releasing the viral genome into the cell and rapidly translating the viral replicase and structural proteins in the infected cell using the (+)RNA viral genome sequence as a template. After being packaged into mature virus particles in the endoplasmic reticulum and Golgi apparatus, they are transported to the cell membrane surface through vesicles and released. ACE2 is the main target antigen for SARS-CoV-2 vaccine research.

[0090] The term "human angiotensin-converting enzyme II (hACE2)" belongs to the angiotensin-converting enzyme family of dipeptidylcarboxydipeptidases. The secreted protein catalyzes the cleavage of angiotensin I into angiotensins 1-9 and angiotensin II into the vasodilating angiotensins 1-7. The organ- and cell-specific expression of the hACE2 gene suggests that it may play a role in regulating cardiovascular and renal function, as well as fertility. Furthermore, the encoded protein is a functional receptor for the spike glycoprotein of the human coronavirus HCoV-NL63 and the human severe acute respiratory syndrome coronaviruses SARS-CoV and SARS-CoV-2. The hACE2 gene is located on sex chromosome X p22.2, Gene ID: 59272 (https: / / www.ncbi.nlm.nih.gov / gene / 59272), and the encoded polypeptide is 805 amino acids long (GenBank: BAB40370.1, https: / / www.ncbi.nlm.nih.gov / protein / BAB40370.1).

[0091] The term "guide RNA (gRNA)" refers to an RNA specific for a target DNA that can form a complex with a Cas protein and bring the Cas protein to the target DNA, thereby introducing a double-strand break at the site of the target DNA. In one embodiment, the target DNA is the mACE2 gene in the mouse genome of a mouse.

[0092] In the present invention, the guide RNA can be composed of two RNAs, namely CRISPR RNA (crRNA) and transactivating crRNA (tracrRNA), or the guide RNA can be a single guide RNA (sgRNA) generated by fusing the essential parts of crRNA and tracrRNA.

[0093] The term "recombinant" when applied to, for example, a cell, nucleic acid, protein or vector, indicates that the cell, nucleic acid, protein or vector has been modified by the introduction of a heterologous nucleic acid or protein, or by the alteration of a native nucleic acid or protein.

[0094] The terms "functional fragment," "functionally equivalent fragment," "functionally equivalent fragment," and the like are used interchangeably and refer to a portion or subsequence of a parent polypeptide that retains the functional activity of the parent polypeptide. For example, a functional fragment of a Cas protein retains the ability to produce a double-strand break in the target DNA in conjunction with a guide RNA.

[0095] The term "donor DNA" or "donor nucleic acid sequence" refers to a polynucleotide comprising a polynucleotide sequence of interest to be expressed, which is inserted into a target site in the mouse genome. In certain embodiments, the donor DNA further comprises a sequence homologous to the genomic sequence (also referred to as a "homologous arm"). "Homologous" means a similar DNA sequence. The homologous arm is sufficient for homologous recombination with the homologous genomic sequence to occur. For example, the homologous arm can comprise at least 50-3500 or more bases in length. In one embodiment, the polynucleotide sequence of interest to be expressed is a full-length hACE2 gene or a functional fragment thereof that binds to the SARS-CoV-2S protein.

[0096] The term "zygote" is a eukaryotic cell formed by the fertilization event between two partners. The genome of the zygote is the genome after the DNA in each partner is combined, and it contains all the genetic information required to form a new individual. In multicellular organisms, the zygote is the earliest developmental stage. In one embodiment, the zygote is a mouse fertilized egg.

[0097] "Percent (%) identity" of an amino acid sequence refers to the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the amino acid sequence described in this specification, after aligning the candidate sequence with the specific amino acid sequence shown in this specification and introducing gaps, if necessary, to achieve the maximum percentage identity, and not considering any conservative substitutions as part of the sequence identity.

[0098] The term "operably linked" means that the specified components are in a relationship permitting them to function in their intended manner.

[0099] II. General Methods

[0100] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology (including recombinant techniques), microbiology, biochemistry, zoology, virology, and immunology known and available to those skilled in the art. Such techniques are described in, for example, Molecular Cloning: A laboratory Manual, 3rd Edition (Sambrook et al., 2001) Cold Spring Harbor Press; Oligonucleotide Synthesis (P. Herdewijn, ed., 2004); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Current Protocols in Molecular Biology (FM Ausubel et al., ed., 1987); PCR: The Polymerase Chain Reaction (Mullis et al., ed., 1994); Current Protocols in Immunology (JE Coligan et al., ed., 1991); and Short Protols in Molecular Biology (Wiley and Sons, 1999). Unless otherwise specified, all terms and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0101] III. Construction of an hACE2 knock-in mouse model with disrupted mACE2 function

[0102] The present invention constructs an hACE2 gene knock-in mouse model in which the mACE2 function is destroyed by site-specific knock-in of the hACE2 gene into the mACE2 gene in the mouse genome and expressing the hACE2 protein.

[0103] III.1. Determine the location of hACE2 gene integration:

[0104] The hACE2 gene was site-specifically integrated into the mACE2 gene in the mouse genome. It is known in the art that the mouse mACE2 gene is located on the positive strand of sex chromosome X. It is approximately 49.1 kb in length (Gene ID: 70008), contains 19 exons, and the transcript NM_001130513 is 3566 nt. The coding region (CDS) is located between nt 331 and nt 2748 of the transcript, encompassing exons 3 to 18. The expressed full-length mACE2 protein contains 805 amino acid residues.

[0105] Therefore, in one embodiment, the hACE2 gene is site-specifically knocked into any exon of the mACE2 gene in the mouse genome and / or replaces one or more mACE2 exons, and the hACE2 gene is stably expressed. Since hACE2 is more sensitive to the SARS-CoV-2 virus than mACE2 after infection in mice, and pathological symptoms are more pronounced, disruption of mACE2 is desirable for constructing the mouse model of the present invention.

[0106] In order to achieve site-specific knock-in of the hACE2 gene at the mACE2 gene in the mouse genome, a nuclease reagent specifically targeting the mACE2 gene in the mouse genome can be used to generate a DNA double-strand break, and then the expression cassette containing the hACE2 gene is integrated into the mACE2 gene in the mouse genome through homologous recombination. In some embodiments, the nuclease reagent is a transcription activator-like effector nuclease (TALEN), a zinc finger nuclease (ZFN), or a CRISPR / Cas system. In one embodiment, the nuclease reagent is a CRISPR / Cas9 system.

[0107] III.2. Design and Efficiency Testing of CRISPR / Cas Systems

[0108] In one embodiment, the CRISPR / Cas system comprises a guide RNA or a DNA encoding a guide RNA specific for a target DNA, and a composition of a Cas protein-encoding nucleic acid or a Cas protein, wherein the target DNA is the mACE2 gene in the above-mentioned mouse genome of the mouse genome.

[0109] In one embodiment, the guide RNA is a dual RNA containing crRNA and tracrRNA. In another embodiment, the guide RNA is a single guide RNA (sgRNA) containing portions of crRNA and tracrRNA.

[0110] In one embodiment, the guide RNA further comprises one or more additional nucleotides at the 5' end of the crRNA of the single guide RNA or dual RNA, for example, comprises two additional guanine nucleotides.

[0111] The guide RNA can be transferred into the cell (e.g., the zygote of the present invention) in the form of RNA or DNA encoding the guide RNA. The guide RNA can be in the form of isolated RNA, RNA incorporated into a viral vector, or encoded in a vector. Preferably, the vector can be a viral vector, a plasmid vector, or an Agrobacterium vector, but is not limited thereto.

[0112] The DNA encoding the guide RNA can be a vector comprising a sequence encoding the guide RNA. For example, the guide RNA can be transfected into a cell (e.g., a zygote of the present invention) by transfecting the cell (e.g., a zygote of the present invention) with an isolated guide RNA or a plasmid DNA comprising a sequence encoding the guide RNA and a promoter.

[0113] Alternatively, viral-mediated gene delivery can be used to transfer the guide RNA into cells (e.g., a zygote of the present invention).

[0114] When the guide RNA is transfected into cells (e.g., zygotes of the present invention) in the form of isolated RNA, the guide RNA can be prepared by in vitro transcription using any in vitro transcription system known in the art. The guide RNA is preferably transferred into the cell in the form of isolated RNA, rather than in the form of a plasmid containing the coding sequence of the guide RNA. However, the use of plasmid DNA or virus-mediated gene delivery to transfect the guide RNA is not excluded.

[0115] In one embodiment, Cas9 protein is bound to a specific site of the mouse genome by sgRNA guidance, and Cas9 protein cuts the specific site, and the double-strand break subsequently formed undergoes homologous recombination under the condition of the presence of donor DNA with homology arms, thereby achieving the site-directed insertion of the target gene. After determining the site of hACE2 gene fixed integration, sgRNA is designed according to the integration site. In order to improve the subsequent site integration efficiency, multiple sgRNAs are usually designed, and by transfecting mouse cells, the editing efficiency is calculated (Hwang WY et al., Efficient genome editing in zebrafish using a CRISPR-Cas system, Nat. Biotechnol., 2013, 31 (3): 227-229). The sgRNA with the highest editing efficiency is selected according to the test results for subsequent experiments.

[0116] In a specific embodiment, the specific site of the target mouse genome to which the sgRNA guides the Cas9 protein to bind is the mACE2 gene in the mouse genome (for example, exon 2 of the mACE2 gene), and the Cas9 protein cuts the specific site. The designed and verified sgRNA that efficiently targets exon 2 of the mouse mACE2 gene in the mouse genome comprises the nucleotide sequence shown in GAAAGATGTCCAGCTCCTCCTGG and / or the nucleotide sequence shown in GAAGCAATCAATAACCCCCTGGG.

[0117] The CRISPR / Cas system may comprise a Cas component in the form of a protein or in the form of a nucleic acid encoding a Cas protein.

[0118] In the present invention, the Cas protein may be any Cas protein as long as it has endonuclease or nickase activity when complexed with a guide RNA.

[0119] Preferably, the Cas protein is a Cas9 protein or a variant or a functional fragment thereof.

[0120] The Cas protein may be a protein isolated from an organism such as Streptococcus sp., preferably Streptococcus pyogenes, or a recombinant protein, but is not limited thereto.

[0121] In one embodiment, the Cas protein comprises the amino acid sequence of SEQ ID NO: 6 derived from Streptococcus pyogenes Cas9:

[0122] MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEA

[0123] TRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIV

[0124] DEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKL

[0125] FIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLG

[0126] LTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRV

[0127] NTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQ

[0128] EEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPF

[0129] LKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIER

[0130] MTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFK

[0131] TNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDIL

[0132] EDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGK

[0133] TILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQ

[0134] TVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIGELGSQILKEHP

[0135] VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRS

[0136] DKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAEGGGLSELDKAGFIKR

[0137] QLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINN

[0138] YHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS

[0139] NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQT

[0140] GGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKE

[0141] LLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGN

[0142] ELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADAN

[0143] LDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD(SEQ ID NO:6)

[0144] In another embodiment, the Cas protein comprises an amino acid sequence having at least 50% homology to the amino acid sequence shown in SEQ ID NO: 6, preferably at least 60, 70, 80, 90, 95, 97, 98, or 99% homology to the amino acid sequence shown in SEQ ID NO: 6, but is not limited thereto.

[0145] In the context of the present invention, the Cas protein encoding nucleic acid may be in the form of a vector, such as a plasmid comprising a Cas encoding sequence under a promoter such as CMV or CAG. When the Cas protein is Cas9, the Cas9 encoding sequence may be derived from Streptococcus, preferably from Streptococcus pyogenes. For example, the Cas9 encoding nucleic acid may comprise a nucleotide sequence encoding SEQ ID NO: 6. In addition, the Cas9 encoding nucleic acid may comprise a nucleotide sequence having at least 50% homology to the nucleotide sequence encoding SEQ ID NO: 6, preferably at least 60, 70, 80, 90, 95, 97, 98, or 99% homology to the nucleotide sequence encoding SEQ ID NO: 6, but is not limited thereto.

[0146] III.3. Construction of donor DNA

[0147] After the guide RNA is determined, the donor DNA is constructed according to the action position of the guide RNA. In the donor DNA, the 5' homology arm and the 3' homology arm are sequences homologous to the genomic sequence of 150-3500 nucleotides on the left and right sides of the guide RNA action position, respectively, for effectively mediating homologous recombination. In some embodiments, the 5' homology arm and the 3' homology arm are sequences homologous to the genomic sequence of about 250, 500, 750, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, and 3250 nucleotides in length, respectively. The term "homologous" as used herein, in addition to the case where the sequences are completely (i.e., 100%) identical to each other, as long as they can effectively mediate homologous recombination, therefore, also includes cases where some sequences are different. Usually at least 95% or more, preferably at least 97%, and more preferably at least 99% sequence identity. Through the interaction between the genomic region on the mouse chromosome and the 5' homology arm and 3' homology arm on the donor DNA, the nucleic acid sequence located between the 5' homology arm and the 3' homology arm on the donor DNA is knocked into a specific location of the genome on the mouse chromosome.

[0148] In one embodiment, the donor DNA sequentially comprises a 5' homology arm, an hACE2 gene or a functional fragment thereof, and a 3' homology arm.

[0149] In one embodiment, the donor DNA is a targeting vector. The basic targeting vector serving as the backbone is not particularly limited, as long as it has a prokaryotic replication origin and a selectable marker for vector propagation in bacteria.

[0150] In a preferred embodiment, in order to increase the expression of the hACE2 gene or a fragment thereof, an intron is ligated into the targeting vector to drive the cDNA of the hACE2 gene, which enables ubiquitous expression of hACE2 via the Rosa26 promoter.

[0151] In one embodiment, the targeting vector contains a reporter gene for detecting the expression of the hACE2 gene or its functional fragment in mice after gene knock-in. Preferably, the reporter gene is a fluorescent reporter gene. More preferably, the reporter gene is a tdTomato reporter gene, which fluoresces red upon expression.

[0152] In one embodiment, the targeting vector comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) that enhances the stability of mRNA transcription.

[0153] In a specific embodiment, the targeting vector comprises an operably linked 5' homology arm, an hACE2 gene or a functional fragment thereof, an internal ribosome entry site (IRES) sequence and / or a 2A sequence, a reporter gene (e.g., a tdTomato reporter gene), a WPRE, a poly A sequence, and a 3' homology arm.

[0154] III.4. Obtaining hACE2 gene knock-in mice

[0155] The above-mentioned guide RNA specific for the target DNA or DNA encoding the guide RNA, Cas protein-encoding nucleic acid or Cas protein, and donor DNA are introduced into a mouse zygote; and the zygote is transferred to a surrogate mouse to produce a germline-transmissible hACE2 gene knock-in mouse.

[0156] Zygotes from any mouse can be used in the present invention. Mouse zygotes can be produced by injecting PMSG (pregnant mare serum gonadotropin) and hCG (human chorionic gonadotropin) into 4-7 week old female mice, mating the superovulated female mice with male mice, and collecting the fertilized zygotes from the oviduct. In some embodiments, the zygote is a zygote or fertilized egg from a C57BL / 6 mouse, a BALB / C mouse, or an ABI mouse.

[0157] The CRISPR / Cas system of the present invention and the donor DNA are introduced into the zygote, whereby the target DNA complementary to the guide RNA is cut by the action of the Cas protein, causing the hACE2 gene to be knocked into the target DNA (e.g., any exon of the mouse mACE2 gene in the mouse genome of the present invention, preferably, exon 2 of the mACE2 gene, more preferably, 1-100 nt after the ATG of exon 2).

[0158] In a specific embodiment, the hACE2 knock-in mouse of the present invention is germline transmissible.

[0159] The method for introducing the CRISPR / Cas system of the present invention and donor DNA into the zygote can be any method known in the art, such as microinjection, stem cell insertion, retroviral insertion, etc. Preferably, the CRISPR / Cas system of the present invention and donor DNA can be introduced into the pronucleus of the zygote or fertilized egg using pronucleus microinjection technology. In one embodiment, the CRISPR / Cas system of the present invention and donor DNA are microinjected into the cytoplasm or pronucleus of the zygote or fertilized egg, and the embryos cultured in vitro are used to analyze the insertion of the hACE2 gene at exon 2 of the mouse mACE2 gene in the mouse genome.

[0160] Any surrogate mouse can be used in the present invention. In a non-limiting embodiment, the surrogate mouse is from a KM mouse or an ICR mouse.

[0161] Mice identified as hACE2-positive among the offspring born by surrogate mice were used as founder mice, and the founder mice were mated with the background mouse strain from which the zygotes originated to obtain F1 offspring; then, the F1 offspring-positive mice were intercrossed to obtain mice that were homozygous for the hACE2 gene at exon 2 of the mouse mACE2 gene.

[0162] The genotype and phenotype of homozygous mice with the hACE2 gene knocked into the mACE2 gene in the mouse genome (e.g., exon 2 of the mouse mACE2 gene) can be identified using a variety of methods known in the prior art, including but not limited to identifying the genotype of the knock-in mouse using PCR, Southern blotting, etc., identifying the expression of hACE2 mRNA in various organs or tissues of the knock-in mouse by reverse transcription polymerase chain reaction (RT-PCR), and / or identifying the expression of hACE2 protein by Western blotting, and detecting the fluorescence after expression of a reporter gene such as a tdTomato reporter gene, and detecting the distribution of hACE2 protein by bioluminescence imaging (BLI), etc.

[0163] In one embodiment, a mouse homozygous for the hACE2 gene at exon 2 of the mouse mACE2 gene is obtained. In the mouse, the hACE2 gene is highly expressed in the lungs and kidneys, which is an ideal mouse model.

[0164] IV. Detection of Viral Infection in Mouse Models

[0165] The extent of infection in the mouse models of the present invention after viral infection can be assessed by a variety of methods. In some embodiments, the extent of viral infection is assessed in tissue or serum of the mouse model. In some embodiments, the tissue is selected from the lung, brain, laryngotrachea, spleen, kidney, liver, or small intestine.

[0166] In one embodiment, the extent of viral infection can be represented by the amount of viral RNA in mouse tissues after virus infection, for example, by measuring the viral RNA in various tissues of the mouse model by RT-qPCR.

[0167] In another embodiment, the extent of viral infection is represented by histopathological analysis of mice infected with the virus. For example, by staining tissue sections and examining them with an optical microscope. In one embodiment, pathological analysis is an evaluation of the extent of lung tissue damage, such as by evaluating the extent of lung damage by degeneration of alveolar epithelial cells, expansion of the parenchymal wall, edema, interstitial inflammation, thickening of the alveolar septum, damage to the vascular system, hemorrhage, and / or infiltration of inflammatory cells (e.g., macrophages and / or neutrophils). For example, the severity of lung damage can be divided into mild (grade 1), moderate (grade 2), or grade 3 (significant).

[0168] In another embodiment, immunofluorescence staining is performed on tissues of virally infected mouse models to assess the extent of viral infection and the types of cells infected by the virus. In some embodiments, detection of the SARS-CoV-2 virus is generally performed by detecting the viral spike (S) protein. The receptor molecule ACE2, cell nuclei (DAPI), and alveolar cells are also labeled with different molecular markers to track the cell types infected by the virus.

[0169] In another embodiment, the extent of viral infection is assessed by immunohistochemical assay of mouse tissues after viral infection. In some embodiments, the infiltration of inflammatory cells, such as macrophages and / or neutrophils, is detected by immunohistochemical assay.

[0170] In another embodiment, the degree of viral infection in a mouse model after viral infection is reflected by the expression of cytokines (e.g., cytokines related to inflammation), for example, cytokines such as Eotaxin, GM-CSF, G-CSF, IL-α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p40, IL-12p70, IL-13, IL-17A, IFN-γ, KC, MCP-1, MIP-1α, MIP-1β, RANTES, and TNF-α are detected.

[0171] The following examples are described to assist understanding of the present invention. The examples are not intended to, and should not be interpreted in any way as, limiting the scope of protection of the present invention. Example:

[0172] Ethical Statement

[0173] All animal experiments have been approved by the Laboratory Animal Ethics and Welfare Committee of the Institute of Laboratory Animal Resources, National Institutes for Food and Drug Control (Approval No.: 2018-B-002) and the Laboratory Animal Center of the Academy of Military Medical Sciences (Approval No.: IACUC-DWZX-2020-001).

[0174] Materials and Methods:

[0175] Isolation and Titration of SARS-CoV-2

[0176] The virus was amplified and titrated by standard plaque formation assay on Vero cells. All experiments involving infectious SARS-CoV-2 were conducted in a biosafety level 3 (BSL3) closed laboratory at AMMS.

[0177] Instruments

[0178] PCR instrument (ABI), real-time fluorescence quantitative PCR instrument (the Light Cycler 480, Roche), micromanipulation system (1X71, OLYMPUS), stereomicroscope (SMZ645, NIKON), CO2 incubator (8000DH, THERMO), holding pipette (5175240006, EPPENDORF), and injection needle (930001040, EPPENDORF).

[0179] Quantitative and Statistical Analysis

[0180] Statistical analysis was performed using Prism software (GraphPad). Data are presented as mean ± sem. The statistical tests (t-test or Fisher's exact test) used are described in the relevant legends.

[0181] The experimental animals used in the embodiments of the present invention are as follows:

[0182] SPF-grade C57BL / 6 mice, 4 weeks old, were used to generate embryos. SPF-grade KM mice, 6 - 8 weeks old, were used as recipients for transplanted embryos; hACE2-KI / NIFDC mice, a newly established susceptible mouse model for novel coronavirus. All experimental animals used in this experiment were provided and raised by the Institute of Laboratory Animal Resources, National Institutes for Food and Drug Control [SCXK (Beijing) 2016-004].

[0183] Example 1. Construction of a Targeting Vector Containing the hACE2 Gene

[0184] Human angiotensin-converting enzyme II (hACE2) is a receptor for SARS-CoV-2, which has the ability to mediate SARS-CoV-2 infection of human hosts (Ashour HM et al., Insights into the Recent 2019 Novel Coronavirus (SARS-CoV-2) in Light of Past Human Coronavirus Outbreaks, Pathogens, March 4, 2020;9(3).pii:E186.doi:10.3390 / pathogens9030186; Roujian Lu et al., Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding, www.thelancet.com, published online January 29, 2020, https: / / doi.org / 10.1016 / S0140-6736(20)30251-8).

[0185] In this example, a targeting vector containing the hACE2 gene was constructed to knock the hACE2 gene into the mouse genome and express the hACE2 protein, aiming to obtain gene-knockin mice that can be infected with SARS-CoV-2.

[0186] The pDonor Knockin vector purchased from VectorBuilder was used to connect the 5' homology arm, hACE2 gene (hACE2Genebank ID: 59272), internal ribosome entry site (IRES) sequence, tdTomato reporter gene, woodchuck hepatitis virus posttranscriptional regulatory element (WPRE)), poly A sequence, and 3' homology arm; wherein the 5' homology arm and 3' homology arm are both approximately 1.4 kb in length and are sequences homologous to the 5' genomic sequence and 3' genomic sequence at the mouse to be inserted site, respectively, to obtain a targeting vector.

[0187] IRES sequence 5'-3'

[0188] GGTTGTGGCCATATTATCATCGTGTTTTTCAAAGGAAAACCACGTCCCCGTGGTTCGGGGGGCCTAG

[0189] ACGTTTTTTTAACCTCGACTAAACACATGTAAAGCATGTGTACCGAGGCCCCAGATCAGATCCCATA

[0190] CAATGGGGTACCTTCTGGGCATCCTTCAGCCCCTTGTTGAATACGCTTGAGGAGAGCCATTTGACTCT

[0191] TTCCACAACTATCCAACTCACAACGTGGCACTGGGGTTGTGCCGCCTTTGCAGGTGTATCTTATACAC

[0192] GTGGCTTTTGGCCGCAGAGGCACCTGTCGCCAGGTGGGGGGTTCCGCTGCCTGCAAAGGGTCGCTAC

[0193] AGACGTTGTTTGTCTTCAAGAAGCTTCCAGAGGAACTGCTTCCTTCACGACATTCAACAGACCTTGC

[0194] ATTCCTTTGGCGAGAGGGGAAAGACCCCTAGGAATGCTCGTCAAGAAGACAGGGCCAGGTTTCCGG

[0195] GCCCTCACATTGCCAAAAGACGGCAATATGGTGGAAAATAACATATAGACAAACGCACACCGGCCTTATTCCAAGCGGCTTCGGCCAGTAACGTTAGGGGGGGGGGAGGGAGAGGGG(SEQ ID NO:1) tdTomato sequence 5'-3'

[0196] ctacttgtacagctcgtccatgccgtacaggaacaggtggtggcggccctcggagcgctcgtactgttccacgatggtgtagtcctcgttgtgggaggtgatgtccag

[0197] cttggtgtccacgtagtagtagccgggcagttgcacgggcttcttggccatgtagatggtcttgaactccaccaggtagtggccgccgtccttcagcttcagggcctgg

[0198] tggatctcgcccttcagcacgccgtcgcgggggtacaggcgctcggtggaggcctcccagcccatggtcttcttctgcattacggggccgtcgggggggaagttgg

[0199] tgccgcgcatcttcaccttgtagatcagcgtgccgtcctgcagggaggagtcctgggtcacggtcaccagaccgccgtcctcgaagttcatcacgcgctcccacttga

[0200] agccctcggggaaggacagcttcttgtaatcggggatgtcggcggggtgcttcacgtacgccttggagccgtacatgaactggggggacaggatgtcccaggcga

[0201] agggcagggggccgcccttggtcaccttcagcttggcggtctgggtgccctcgtaggggcggccctcgccctcgccctcgatctcgaactcgtggccgttcatgga

[0202] gccctccatgcgcaccttgaagcgcatgaactctttgatgacggccatgttgttgtcctcggaggaggcggtgccggagctgccgctgccggtgctgccggtgccat

[0203] gccccaggaacaggtggtggcggccctcggagcgctcgtactgttccacgatggtgtagtcctcgttgtgggaggtgatgtccagcttggtgtccacgtagtagtagc

[0204] cgggcagttgcacgggcttcttggccatgtagatggtcttgaactccaccaggtagtggccgccgtccttcagcttcagggcctggtggatctcgcccttcagcacgc

[0205] cgtcgcgggggtacaggcgctcggtggaggcctcccagcccatggtcttcttctgcattacggggccgtcgggggggaagttggtgccgcgcatcttcaccttgtag

[0206] atcagcgtgccgtcctgcagggaggagtcctgggtcacggtcaccagaccgccgtcctcgaagttcatcacgcgctcccacttgaagccctcggggaaggacagct

[0207] tcttgtaatcggggatgtcggcggggtgcttcacgtacgccttggagccgtacatgaactggggggacaggatgtcccaggcgaagggcagggggccgcccttgg

[0208] tcaccttcagcttggcggtctgggtgccctcgtaggggcggccctcgccctcgccctcgatctcgaactcgtggccgttcatggagccctccatgcgcaccttgaagcgcatgaactctttgatgacctcctcgcccttgctcaccat(SEQ ID NO:2)

[0209] The sequence of WPRE 5'-3'

[0210] Gcggggaggcggcccaaagggagatccgactcgtctgagggcgaaggcgaagacgcggaagaggccgcagagccggcagcaggccgcgggaaggaaggtccgctggattgagggccgaagggacgtagcagaaggacgtcccgcgcagaatccaggtggcaacacaggcgagcagccaaggaaaggacgatgatttccccgacaacaccacggaattgtcagtgcccaacagccgagcccctgtccagcagcgggcaaggcaggcggcgatgagttccgccgtggcaatagggagggggaaagcgaaagtcccggaaaggagctgacaggtggtggcaatgccccaaccagtgggggttgcgtcagcaaacacagtgcacaccacgccacgttgcctgacaacgggccacaactcctcataaagagacagcaaccaggatttatacaaggaggagaaaatgaaagccatacgggaagcaatagcatgatacaaaggcattaaagcagcgtatccacatagcgtaaaaggagcaacatagttaagaataccagtcaatctttcacaaattttgtaatccagaggttgatt(SEQ ID NO:3)

[0211] The sequence of polyA 5'-3'

[0212] tccccagcatgcctgctattgtcttcccaatcctcccccttgctgtcctgccccaccccaccccccagaatagaatgacacctactcagacaatgcgatgcaatttcctcattttattaggaaaggacagtgggagtggcaccttccagggtcaaggaaggcacgggggaggggcaaacaacagatggctggcaactagaaggcacag(SEQID NO:4)

[0213] The constructed targeting vector is as Figure 1A shown. Through restriction enzyme digestion identification and sequencing, it was confirmed that the construction of the targeting vector was completed( Figure 1B ).

[0214] In the targeting vector, the tdTomato gene sequence was inserted downstream of the hACE2 gene and the internal ribosome entry site (IRES) sequence, which allowed the co-expression of hACE2 and tdTomato. The poly A sequence and WPRE element were added to enhance mRNA stability and translation efficiency ( Figure 1C ).

[0215] Example 2. Generation of mice with targeted knock-in of the hACE2 gene at the target site

[0216] The targeting vector containing the hACE2 gene constructed in Example 1 is used to target exon 2 of the mouse mACE2 gene at the GRCm38.p6 site on the mouse X chromosome (i.e., the first coding exon of the mACE2 gene) via homologous recombination, aiming to generate hACE2-KI / NIFDC humanized mice. Specifically, hACE2-IRES-tdTomato-WPRE-pA is inserted after the ATG of the first coding exon of the mACE2 gene, while simultaneously deleting 61 amino acids downstream of the first coding exon. This targeting strategy allows the hACE2 protein to be intrinsically expressed under the control of the mACE2 promoter, while disrupting the organizational structure of the mACE2 gene and preventing the expression of the mACE2 protein.

[0217] In order to achieve site-specific knock-in of the hACE2 gene construct into the first coding exon of the mACE2 gene in the mouse genome, a CRISPR / Cas system specifically targeting the first coding exon of the mACE2 gene was used to generate DNA double-strand breaks, and then the hACE2 gene was integrated into the first coding exon of the mACE2 gene through homologous recombination.

[0218] 2.1 Sequencing confirmation of target sequences

[0219] To ensure the efficiency of the designed CRISPR / sgRNA, the target site sequence was amplified by PCR and sequenced in the tail of C57BL / 6 mice to ensure that the designed sgRNA recognition sequence was completely consistent with the DNA sequence of the constructed mouse strain.

[0220] DNA was extracted from C57BL / 6 mouse tails and amplified using the following PCR primers for sequencing:

[0221]

[0222] The results showed that the target sequence determined by C57BL / 6 mouse tail was consistent with the sequences given in Genebank and Ensembl.

[0223] 2.2 CRISPR / sgRNA design and vector construction

[0224] A total of 8 sgRNAs were designed for the target site region of the mACE2 gene, as shown in the following table:

[0225] sgRNA number Sequence (5'-3') sgRNA1-1 GAAAGATGTCCAGCTCCTCCTGG(SEQ ID NO:7) sgRNA2-1 TACTGCTCAGTCCCTCACCGAGG(SEQ ID NO:8) sgRNA3-1 CAAAAGATGGTAAGTTCTTGAGG(SEQ ID NO:9) sgRNA4-1 CAAGTGAACTTTGATAAGACAGG(SEQ ID NO:10) sgRNA5-1 ATCAAAGTTCACTTGCTTCTTGG(SEQ ID NO:11) sgRNA6-1 GGATGGGATCTTGGCGCACGGGG(SEQ ID NO:12) sgRNA7-1 GTCAATTGAATTTAGCCATCTGG(SEQ ID NO:13) sgRNA8-1 GAAGCAATCAATAACCCCCTGGG(SEQ ID NO:14)

[0226] The sgRNAs were ligated into the pCS-3G vector (obtained from ZYbscience) by Gibson method. The vector map is shown in Figure 2A As shown, E. coli DH5α was transformed for amplification.

[0227] 2.3sgRNA Activity Detection

[0228] By UCA TM The activity of sgRNA was detected by the detection method. Figure 2B As shown:

[0229] from Figure 2B The results show that sgRNA1-1) and sgRNA8-1 have relatively high Cas9 / sgRNA activity. The sequence of sgRNA1-1 was selected for the next experiment.

[0230] 2.4 Preparation of sgRNA

[0231] The sequence of sgRNA1-1 was ligated into the plasmid vector pT7 with a T7 promoter, and sgRNA for microinjection into fertilized eggs was prepared by in vitro transcription.

[0232] 2.5 Microinjection of zygotes

[0233] The targeting vector containing the hACE2 gene constructed in Example 1, the sgRNA prepared in Example 2.4 above that efficiently targets exon 2 of the mouse mACE2 gene, and a Cas9 mRNA sequence known in the prior art (e.g., a Cas9 mRNA sequence encoding the amino acid sequence shown in SEQ ID NO: 6) (sgRNA: approximately 50 ng, Cas9: approximately 100 ng) were microinjected into 370 C57BL / 6 mouse fertilized eggs. The fertilized eggs were then implanted into pseudopregnant surrogate KM mice or ICR mice for pregnancy, and the surrogate mice gave birth to offspring.

[0234] PCR amplification of the hACE2 gene was performed on 46 offspring born to surrogate mice using primers specific for the hACE2 gene. The two pairs of PCR primers used are as follows.

[0235]

[0236] The results showed that the DNA amplification fragments of 3 male and 6 female mice were positive. These 9 offspring are also called founder mice (F0 generation).

[0237] The three founder mice were mated with wild-type C57BL / 6 mice to obtain a total of 66 F1 offspring.

[0238] DNA was extracted from the tail tips of F1 offspring mice and digested with two endonucleases, NcoI and StuI. Southern blotting was performed to confirm the presence of off-target (using the WPRE 3' probe) and correct insertion of the hACE2 gene (using the 5' probe) in positive F1 offspring. Since the expected size of the wild-type band is 5.6 kb and the expected size of the hACE2 gene targeted band is 8.0 kb, heterozygous (female mice in this experiment) mice have two bands, while homozygous (male mice in this experiment) mice have only the 8.0 kb band.

[0239] Southern blot results showed that mouse numbers 1EJ68-002, 1EJ68-003, 1EJ68-009, 1EJ68-010, 1EJ68-013, 1EJ68-014, 1EJ68-028, 1EJ68-032, 1EJ68-070, 1EJ68-071, 1EJ68-075, and 1EJ68-077 were all correctly recombined and sequenced correctly, and only one 12.4 kb band was detected using the WPRE probe, indicating that there was no random insertion. A total of 12 F1 positive mice ( Figure 3 ).exist Figure 3 In the naming principle of mouse numbers, for example, 1 in 1EJ68-077 represents the F1 generation (no number represents the F0 generation); EJ68 is the project number within the institution; -077 is the tail-docking mouse number.

[0240] Example 3. Detection of hACE2 gene expression in F1 positive mice

[0241] This example studies the expression of the hACE2 gene in F1 positive mice.

[0242] 3.1 Western blotting

[0243] Mouse tissues were homogenized in 20 mM Tris-HCl pH 7.5, containing 150 mM NaCl, 100 mM EDTA, and 1% Triton X-100, supplemented with phenylmethanesulfonyl fluoride (PMSF) (Beyotime, Shanghai) for Western and IP lysis. Denatured protein lysates were separated on 10% SDS-PAGE gels. After transfer, anti-angiotensin-converting enzyme 2 rabbit monoclonal antibody (Cat#: ET1611-58, diluted 1:1000, Huabio, Cambridge, MA, USA) and anti-GAPDH antibody (1:1000, Abcam, Cambridge, UK) were added, followed by horseradish peroxidase (HRP)-conjugated anti-mouse and anti-rabbit IgG (1:20,000, Abcam, Cambridge, UK). Development was performed using PierceECL Western Blotting Substrate (THERMO FISHER, USA).

[0244] Western blotting results also showed strong hACE2 expression in the lungs and kidneys, and weak expression in the spleen and liver, showing an expression pattern similar to that of the mACE2 protein in mice ( Figure 4 ).

[0245] 3.2 Bioluminescence imaging (BLI) detection

[0246] Mice were anesthetized by intraperitoneal (IP) injection of sodium pentobarbital (240 mg per kg body weight). tdTomato bioluminescence imaging was performed using an IVIS-Lumina II imaging system (Xenogen, Baltimore, MD). The relative intensity of the emitted light was expressed as a color ranging from red (strong) to blue (weak) and was expressed as photon flux in photons / second / cm 2 / sr.

[0247] Benefiting from the co-expression design of the tdTomato gene inserted after the hACE2 gene, the overall expression pattern of hACE2 in young (4.5 weeks old) and older (30 weeks old) mice was visualized by bioluminescent imaging (BLI). Figure 5 ), while wild-type mice lack this fluorescence.

[0248] RNA extraction and real-time quantitative PCR

[0249] To confirm the expression of hACE2 and mACE2 in F1-positive humanized mice, 4.5-week-old and 30-week-old mice were dissected and tissues were obtained and immediately immersed in (Invitrogen, USA) stabilizer and stored at -80 ° C. Total RNA was extracted from each tissue using TRIzol and quantified using a spectrophotometer. Real-time quantitative PCR (RT-qPCR) was performed on a Light Cycler 480II real-time PCR system (Roche, Indianapolis, IN, USA) using a reverse transcription (RT)-PCR kit containing SYBR green dye (Takara, Shiga, Japan). Each reaction was repeated three times. The primer pairs ehACE2 F1 (5'-cgaagccgaagacctgttcta-3') and ehACE2 R1

[0250] Relative hACE2 and mACE2 expression levels were determined using the following mACE2 amplicon: emACE2-F1 (5'-tccagactccgatcatcaagc-3') and emACE2-R1 (5'-tgctcatggtgttcagaattgt-3'). Amplicons were normalized to GAPDH (glyceraldehyde 3-phosphate dehydrogenase) expression.

[0251] As expected, RT-PCR analysis of 4.5-week-old mice revealed that the mACE2 gene was not detected in homozygous hACE2 mice, and the mACE2 gene expression was almost reduced by half in heterozygous mice ( Figure 6A 、 Figure 6B ), significant hACE2 expression was found in the lungs, small intestine, spleen, and kidneys of heterozygous and homozygous hACE2 mice, but not in wild-type mice ( Figure 6A 、 Figure 6B ), strong expression of hACE2 was observed even in mice older than 30 weeks ( Figure 6C ), indicating that this humanized mouse model is genetically and phenotypically stable.

[0252] The positive mice were designated as C57BL / 6-Ace2 em2(hACE2-tdTomato) / NIFDC, abbreviated as hACE2-KI / NIFDC, was further used in subsequent experiments. In the examples herein, "hACE2-KI / NIFDC humanized mice," "hACE2 humanized mice," and "hACE2 mice" are used interchangeably.

[0253] Example 4: Intranasal infection of SARS-CoV-2 in hACE2 humanized mice of the present invention

[0254] according to Figure 7 As shown in A, wild-type C57BL / 6 mice, young (4 weeks old), and old (28 weeks old) hACE2-KI / NIFDC mice (n=3 / group) were anesthetized with 50 mg / kg sodium pentobarbital intraperitoneally and then intranasally infected with 40,000 pfu of SARS-CoV-2. Mice were sacrificed on day 6 after infection, and lung, brain, laryngotracheal, spleen, kidney, liver, small intestine, and serum tissues were collected. The following analyses were performed:

[0255] 4.1 Tissue distribution of SARS-CoV-2 viral RNA.

[0256] Viral RNA copies were analyzed on a portion of each tissue and serum by RT-qPCR. Specifically, viral RNA in each sample was quantified by RT-qPCR targeting the S gene of SARS-CoV-2. RT-qPCR was performed using the One Step PrimeScript RT-PCR kit (Takara, Japan) using the following primers and probes: CoV-F3 (5′-TCCTGGTGATTCTTCTTCAGGT-3′); CoV-R3 (5'-TCTGAGAGAGGGTCAAGTGC-3'); and CoV-P3 (5'-AGCTGCAGCACCAGCTGTCCA-3').

[0257] The results are as follows Figure 7 As shown in b, the details are as follows: high titer of virus can be detected in the lungs, brain, larynx and trachea of hACE2 mice, reaching 10 8 RNA copies / gram tissue, while in other organs, either undetectable or very low levels were detected in control wild-type mice. This viral distribution is similar to that seen in clinical viral infections.

[0258] 4.2 Immunofluorescence staining analysis

[0259] The mouse tissue obtained was fixed with 10% neutral buffered formalin, dehydrated and embedded in paraffin. 3 μM paraffin sections were dewaxed in xylene and rehydrated in a series of graded alcohols (70%, 80%, 90% and 100% ethanol). Antigen retrieval was performed in a citrate buffer (pH 6) at 95°C using a microwave oven (Sharp, R-331ZX) and then cooled at room temperature for 20 minutes. Multiple fluorescent labeling was performed using TSA-dendritic-fluorophore (NEON Allround Discovery Kit for FFPE, Histova Biotechnology, NEFP750).

[0260] Briefly, endogenous peroxidase was quenched in 3% H2O2 for 20 minutes, followed by 30 minutes in blocking agent (specific reagents) at room temperature. The primary antibody (see below for details) was incubated in a humidified chamber at 37°C for 2 hours, and then detected using a secondary antibody TSA-dendritic-fluorophore conjugated to HRP. Afterwards, the cells were washed with water at 95°C in extraction / elution buffer ( Primary and secondary antibodies were completely eliminated by heating the slides for 10 seconds in a 4% PBS (Histova Biotechnology, ABCFR5L). Each antigen was labeled with a different fluorophore in a sequential manner. The multiplex antibody panel (primary antibodies) used in this study were antibodies against the following proteins: ACE2 (Abcam, 1:200), SARS-CoV Spike (Sinobiologic, 1:2000); b-tubulin IV (Abcam, 1:1000), CC10 (Millipore, 1:500), Podoplanin (Sinobiologic, 1:1000), SP-C (Abcam, 1:500), CD68 (Abcam, 1:1000), and Cleaved Caspase-3 (CST, 1:200). After sequential detection of all antibodies, sections were imaged using a Zeiss LSM880 confocal laser scanning microscope. Mouse lung paraffin sections were stained for SARS-Cov-2 S protein (green), ACE2 (red) and DAPI (blue). Figure 7 As shown in C. This shows that the virus does co-localize with the ACE2 receptor, indicating that the virus binds to the ACE2 receptor.

[0261] Paraffin sections of tissue were immunofluorescently stained for PDPN cell markers (indigo), ACE2 (white), CC10 (green), SPC (gold), and b-tubulin (purple). Figure 7 D, where the white box is enlarged on the right. Solid yellow arrows, red arrows, and white arrows represent SARS-CoV-27ACE27CC10, respectively. + cells, SARS-CoV-27CC10 + Cells and ACE27 CC10 + The specific results are as follows: The SARS-CoV-2 S protein preferentially colocalizes with CC10 cells and hACE2 cells, indicating that in this model, CC10-positive Clara cells located in the airways are the primary target cells for SARS-CoV-2 infection. Wild-type mice treated with the same virus infection treatment did not become infected.

[0262] 4.3 Body weight changes of infected mice

[0263] The weight changes of the mice infected with the above were detected. The results are shown in Figure 10 It can be seen that after intranasal infection with the virus, the weight of older mice showed a downward trend, and on the third day after the infection, it dropped by nearly 10%, while the weight loss of young mice was atypical, which may be due to their young age and the fact that their weight was still in the growth stage.

[0264] 4.4 Immunofluorescence staining of infected mice

[0265] The paraffin sections of the collected mouse brain tissue were immunostained for SARS-CoV-2 S protein (green) and DAPI (blue) using the same method as in 4.2. The results are shown in Figure 4. Figure 11 As shown, in hACE2 mice infected with SARS-CoV-2, immunofluorescence staining experiments showed that S protein could be detected in neurons, astrocytes, and microglia, indicating that these cells were infected with the new coronavirus. In summary, the hACE2 mouse model is susceptible to the new coronavirus, and the target organs of infection are Clara cells in the lungs, bronchi, and brain.

[0266] Example 5 Pathological findings and inflammatory response in SARS-CoV-2 infected mice

[0267] In this example, the pathological findings and inflammatory responses in mice infected with SARS-CoV-2 in Example 4 were primarily verified. Histopathological analysis showed that mild inflammation occurred in hACE2 humanized mice.

[0268] 5.1 Immunohistochemistry

[0269] The specific steps are as follows:

[0270] The obtained mouse tissues were fixed with 10% neutral buffered formalin, dehydrated and embedded in paraffin. According to standard procedures, sections with a thickness of 4 μm were stained with hematoxylin and eosin (H&E) for light microscopy. The degree of lung injury was evaluated under a light microscope by degeneration of alveolar epithelial cells, expansion of the parenchymal wall, edema, hemorrhage and inflammatory cell infiltration. The severity of lung injury was semi-quantitatively assessed according to the above items (grade 1, mild; grade 2, moderate; grade 3, significant), and the cumulative score of severity provided a total score for each animal, and the average of three animals was taken as the total score for the group.

[0271] Paraffin-embedded tissues were cut into 4 μm thickness for immunohistochemical staining. The sections were dewaxed and rehydrated, and endogenous peroxidase was inactivated with methanol containing 0.3% hydrogen peroxide for 30 minutes. Antigen retrieval was performed with citrate buffer (pH 6) at 95°C for 30 minutes. After incubation in blocking solution (5% normal goat serum) at room temperature for 10 minutes, the slides were incubated with mouse neutrophil marker antibodies (Santa Cruz Biotechnology, 1:200) and rabbit anti-CD68 polyclonal antibodies (Abcam, 1:800) at 4°C overnight. After washing three times, the sections were incubated with biotinylated anti-IgG at 37°C for 1 hour and then incubated with streptavidin-peroxidase conjugate (Zhongshan Biotechnology Co., Ltd., Beijing, China). Immunoreactivity was detected using 3,3′ diaminobenzidine, and the sections were stained with hematoxylin for observation by microscopy. To semiquantitatively measure macrophage and neutrophil infiltration, 10 randomly selected 40× (neutrophils) or 100× (macrophages) objective fields of the lung parenchyma in each lung section were examined by light microscopy to check for the presence of neutrophils or macrophages. This assessment was performed in a blinded manner. The cumulative score for each animal was expressed as the number of positive fields per 100 fields (%).

[0272] The results are as follows Figure 8 AC. Specifically, the lesions in hACE2 transgenic mice were more typical than those in wild-type mice (grade 2), while those in old transgenic mice were more typical than those in young transgenic mice (grade 3). Semi-quantitative histological analysis of H&E-stained lung sections on day 6 (right) showed that inflammatory cell infiltration (yellow arrows), alveolar septal thickening, and unique vascular system damage (blue arrows) were observed in both young and old hACE2-KI / NIFDC humanized mice. More alveolar epithelial cell lesions and focal hemorrhages (green arrows) were observed in older mice. ( Figure 8 A)

[0273] Panels B and C show IHC staining analysis. The right panel shows a semi-quantitative analysis of neutrophils and macrophages. More neutrophils (Neu+) and macrophages (CD68+) were observed in hACE2-KI / NIFDC humanized mice. Both cell types were more abundant in older mice (30 weeks of age) than in younger mice (4.5 weeks of age). These results demonstrate that hACE2 mice are not only susceptible to the novel coronavirus but also develop pneumonitis-like changes in the lungs, similar to clinical observations.

[0274] 5.2 Cytokine Assay

[0275] A total of 25 μl of serum from each mouse was used for cytokine analysis using a Bio-Plex Pro Mouse Cytokine Grp IPanel 23-Plex (BIO-RAD) according to the manufacturer's instructions. Data were collected on a Luminex 200 and analyzed by Luminex PONENT (Thermo Fisher). Figure 8 Figure D shows cytokine expression in the serum of mice infected with SARS-CoV-2. Statistical significance was analyzed using an unpaired Student's t-test. *, P < 0.05. The results demonstrate that eotaxin, G-CSF, IL-9, and IFN-γ cytokines increased significantly in older mice, while responses were relatively weak in younger mice. These cytokine changes provide further evidence of pathological changes in hACE2 mice following viral infection.

[0276] Example 6: Intragastric infection of SARS-CoV-2 in hACE2-KI / NIFDC humanized mice of the present invention

[0277] according to Figure 9 As shown in A, wild-type C57BL / 6 mice and hACE2-KI / NIFDC humanized mice (n=3) were infected with 4×10 6 pfu of SARS-CoV-2. The mice were sacrificed on day 5 after infection, and the main tissues, lung, brain, larynx and trachea, spleen, kidney, liver, small intestine and serum were obtained (the method is as shown in Example 4).

[0278] The following analysis is performed:

[0279] 6.1 Tissue distribution of SARS-CoV-2 viral RNA.

[0280] A portion of each tissue and serum was analyzed for viral RNA copies by RT-qPCR. Detailed steps are described in Example 4.1.

[0281] The results are as follows Figure 9 As shown in Figure B, the virus was detected in the lungs and bronchi of some mice, but not in other organs. While the background value for this virus was approximately 4.5 log, the virus titers detected in the larynx, trachea, and lungs of some mice reached 6-7 logs, an increase of approximately 2 orders of magnitude, indicating that it can support viral replication.

[0282] 6.2 Immunofluorescence staining of mouse lung paraffin sections for SARS-CoV-2 S protein (green) and DAPI (blue)

[0283] The paraffin sections of the obtained mouse lung tissue were subjected to immunofluorescence staining for SARS-Cov-2 S protein (green) and DAPI (blue). For specific steps, see Example 4.2.

[0284] The results are as follows Figure 9 As shown in Figure C, the details are as follows: SARS-CoV-2 infection can be found in the lung tissue of hACE2-KI / NIFDC humanized mice, indicating that an infection model can also be established through gavage.

[0285] 6.3 Histopathological Analysis of Mouse Lungs

[0286] The mouse lungs were subjected to histopathological analysis as described in Example 5.1. Figure 9 D, where semiquantitative analysis was performed in the right panel. Statistical significance was analyzed by unpaired Student's t-test. *, P < 0.05.

[0287] exist Figure 9 As can be seen in D, interstitial inflammation, accompanied by thickening of the alveolar septa (yellow arrows), was observed in hACE2-KI / NIFDC humanized mice. No obvious vascular damage (blue arrows) was observed in either wild-type or hACE2-KI / NIFDC humanized mice. The pathological changes observed in this model are similar to clinical observations.

[0288] Example 7. SARS-CoV-2 does not effectively infect IFNαβγ-C57BL / 6

[0289] Wild-type C57BL / 6 mice and IFNαβγ-positive C57BL / 6 mice (n=3) (purchased from the China Food and Drug Administration) were intranasally infected with 40,000 pfu of SARS-CoV-2 as described in Example 4. The mice were monitored daily for weight changes. Mice were sacrificed on day 6 of intranasal infection, and major tissues (lung, brain, larynx, trachea, spleen, kidney, liver, small intestine), and serum were collected as described in Example 2 (methods described in Example 2).

[0290] As shown in Example 4.1, a portion of each tissue and serum was analyzed for viral RNA copies by RT-qPCR to obtain the tissue distribution of SARS-CoV-2 in mice after intranasal administration. Figure 12 As can be seen in A, the brain, lungs, trachea and other organs are not infected, and no virus is detected, which is close to the wild type.

[0291] Figure 12 B shows the changes in body weight of mice after infection. It can be seen that the body weight of wild type and IFNαβγ- decreased on the fifth day of infection.

[0292] Example 8 Comparative Analysis of Heterozygous and Homozygous Mice

[0293] hACE2 transgenic mouse F1 offspring were constructed according to the method of Example 2, and 5 heterozygous female mice and 5 homozygous female mice were selected. The following experiments were performed according to the method described in Example 4.

[0294] The 10 mice were infected intranasally with SARS-CoV-2, and their body weights were measured on the first day after infection (1 dpi), the third day after infection (3 dpi), and the fourth day after infection (4 dpi). Figure 13 As can be seen from the figure, there was no significant decrease in body weight in the heterozygous and homozygous mouse groups.

[0295] The mice were killed on day 4 after infection (after weight measurement), and the body weight and total lung weight of the mice were weighed during autopsy. The lung weight ratio was calculated and plotted to obtain Figure 14 From the figure, we can see that the lung weight ratio of the heterozygous mice group is slightly higher than that of the homozygous mice group, but the data of the homozygous mice group is more concentrated and the consistency between homozygous mice is better.

[0296] The lung, liver and spleen tissues of the mice were collected and the tissues were subjected to pathological results and inflammatory reaction analysis. Immunohistochemical assay was performed according to the method of Example 5. The immunohistochemical staining of the tissues is shown in FIG. Figure 15A and Figure 15B The severity of tissue damage was assessed by a double-blind semi-quantitative evaluation by pathologists (grade 1, mild; grade 2, moderate; grade 3, significant). Figure 16 It can be seen that there is no significant difference in the lung and spleen tissues among the groups; in the liver, the pathological damage in the homozygous mice group is more serious than that in the heterozygous mice.

[0297] In summary, it can be seen that both heterozygous and homozygous mice can be used to evaluate the in vivo efficacy of antiviral agents and vaccines against SARS-CoV-2, as well as to study the infection mechanism of the new coronavirus. However, the pathology of homozygous mice is more severe than that of heterozygous mice, making them more suitable as models for subsequent research.

[0298] While certain representative embodiments and details have been shown for purposes of illustrating the present invention, it will be apparent to those skilled in the art that various changes and modifications may be made thereto without departing from the scope of the subject invention. In this regard, the scope of the present invention is limited solely by the following claims.

Claims

1. A method for constructing an hACE2 knock-in mouse model in which mACE2 function is disrupted, the method comprising: (a) The hACE2 gene was inserted into the mACE2 gene in the mouse genome. (b) obtaining F1 offspring mice that are homozygous or heterozygous for the hACE2 gene at the mACE2 gene in the mouse genome; and (c) obtaining offspring of the mouse in (b), wherein the mACE2 gene in the mouse genome is exon 2 of the mACE2 gene, Wherein said step (a) comprises: (i) introducing a CRISPR / Cas9 system and donor DNA into a mouse zygote, wherein the CRISPR / Cas9 system is capable of introducing a double-strand break at a target site at exon 2 of the mouse mACE2 gene in the mouse genome, and the donor DNA comprises the hACE2 gene; (ii) introducing each zygote obtained in (i) into a surrogate mouse for pregnancy, and allowing the surrogate mouse to give birth to offspring; (iii) using mice identified as hACE2-positive among the offspring of the surrogate mice as founder mice, and mating the founder mice with the background mouse strain from which the zygotes originated to obtain hACE2-positive F1 offspring, which are mice homozygous or heterozygous for the hACE2 gene, also referred to as F1 offspring-positive mice; The donor DNA introduced into the mouse zygote is a targeting vector comprising the hACE2 gene, wherein the targeting vector comprises a 5' homology arm, the hACE2 gene, an internal ribosome entry site (IRES) sequence, a reporter gene, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), a poly A sequence, and a 3' homology arm; wherein the homology arm is a sequence homologous to the genomic sequence at exon 2 of the mouse mACE2 gene, The CRISPR / Cas9 system comprises a composition for cleaving DNA at exon 2 of the mouse mACE2 gene as the target DNA, comprising a single guide RNA specific for the target DNA or DNA encoding a single guide RNA, and a Cas protein-encoding nucleic acid or a Cas protein composition, wherein the single guide RNA consists of the nucleotide sequence shown in GAAAGATGTCCAGCTCCTCCTGG, and the Cas protein consists of the amino acid sequence shown in SEQ ID NO:

6.

2. The method of claim 1, wherein the mouse strain is selected from C57BL / 6, BALB / c, ICR or KM mice.

3. The method of claim 1, wherein the mACE2 gene location in the mouse genome is 1-100 nt after the ATG of exon 2.

4. The method of claim 1, wherein the zygote is from a C57BL / 6 mouse, a BALB / c mouse, an ABI mouse and / or the surrogate mouse is from a KM mouse, an ICR mouse.

5. The method of claim 1, wherein the step (iii) comprises mating the female founder mouse with a wild-type male mouse of the background from which the zygote originated; or mating the male founder mouse with a wild-type female mouse of the background from which the zygote originated.

6. The method of claim 1, wherein the target site at exon 2 is 1-100 nt after the ATG of exon 2, and step (iv) obtains a mouse homozygous for the hACE2 gene 1-100 nt after the ATG of exon 2.

7. The method of claim 1, wherein the method further comprises, in step (b), identifying the genotype and phenotype of mice that are heterozygous or homozygous for the hACE2 gene at exon 2 of the mouse mACE2 gene.

8. The method of claim 7, wherein the genotype of the knock-in mouse is identified by PCR and Southern blotting, the expression of hACE2 mRNA in the knock-in mouse is identified by reverse transcription polymerase chain reaction, and / or the expression of hACE2 protein and its distribution in the mouse are identified by Western blotting and bioluminescence imaging.

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