Generation of a humanized mAce2 and a conditional hACE2 mouse models permissive to SARS-COV-2 infection

Abstract

The Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) remains a public health concern and a subject of active research effort. Development of pre-clinical animal models is critical to study viral-host interaction, tissue tropism, disease mechanisms, therapeutic approaches, and long-term sequelae of infection. Here, we report two mouse models for studying SARS-CoV-2: A knock-in mAce2^F83Y,H353K mouse that expresses a mouse-human hybrid form of the angiotensin-converting enzyme 2 (ACE2) receptor under the endogenous mouse Ace2 promoter, and a Rosa26 conditional knock-in mouse carrying the human ACE2 allele (Rosa26^hACE2). Although the mAce2^F83Y,H353K mice were susceptible to intranasal inoculation with SARS-CoV-2, they did not show gross phenotypic abnormalities. Next, we generated a Rosa26^hACE2;CMV-Cre mouse line that ubiquitously expresses the human ACE2 receptor. By day 3 post infection with SARS-CoV-2, Rosa26^hACE2;CMV-Cre mice showed significant weight loss, a variable degree of alveolar wall thickening and reduced survival rates. Viral load measurements confirmed inoculation in lung and brain tissues of infected Rosa26^hACE2;CMV-Cre mice. The phenotypic spectrum displayed by our different mouse models translates to the broad range of clinical symptoms seen in the human patients and can serve as a resource for the community to model and explore both treatment strategies and long-term consequences of SARS-CoV-2 infection.

Figure 1.. Generation of the mouse-human hybrid mAce2^F83Y,H353K mouse model.

(A) Schematic presentation of the approach to generate a mouse-human hybrid allele via CRISPR/Cas9 gene editing. In brief, two guide RNAs directed Cas9-mediated cleavage at codon 83 (exon 3) and codon 353 (exon 18) of Ace2. Edits were introduced by two single-stranded donor DNA oligos. (B) Sanger sequencing confirmed correct editing of the mAce2 allele in a male founder (red boxes). Of note, knocked-in mutations are in obligate cis configuration (14 kb apart) since mAce2 is located on the X chromosome. Silent mutations introducing restriction enzyme sites for genotyping are underlined in red. (C) Expression of Ace2 in multiple tissues, as measured by real-time qPCR in mAce2^F83Y,H353K mice and wild type littermates (WT) and normalized to beta actin, showing that gene editing did not significantly alter the expression level. The ratio of mAce2^F83Y,H353K to endogenous (WT) mAce2 level is plotted in various tissues. Data shown as mean +/− SD, n=3–4 per tissue.

Figure 1.. Generation of the mouse-human hybrid mAce2^F83Y,H353K mouse model.

(A) Schematic presentation of the approach to generate a mouse-human hybrid allele via CRISPR/Cas9 gene editing. In brief, two guide RNAs directed Cas9-mediated cleavage at codon 83 (exon 3) and codon 353 (exon 18) of Ace2. Edits were introduced by two single-stranded donor DNA oligos. (B) Sanger sequencing confirmed correct editing of the mAce2 allele in a male founder (red boxes). Of note, knocked-in mutations are in obligate cis configuration (14 kb apart) since mAce2 is located on the X chromosome. Silent mutations introducing restriction enzyme sites for genotyping are underlined in red. (C) Expression of Ace2 in multiple tissues, as measured by real-time qPCR in mAce2^F83Y,H353K mice and wild type littermates (WT) and normalized to beta actin, showing that gene editing did not significantly alter the expression level. The ratio of mAce2^F83Y,H353K to endogenous (WT) mAce2 level is plotted in various tissues. Data shown as mean +/− SD, n=3–4 per tissue.

Figure 2.. SARS-CoV-2 infection in mAce2^F83Y,H353K mouse model.

(A) Weight has not changed significantly over 6 days post infection. Data presented as proportion of starting weight (weight at day 0). (B) Measurement of viral load in mutant mice lungs indicated successful inoculation in 4/5 mice (measurement was below limit of detection in 1/5 mice). Data presented as viral titer per gram lung tissue. Limit of detection (LOD) for tissue viral titer is Log₁₀ 1.69 TCID₅₀/gm.

Figure 3.. Targeting the hACE2 cDNA to the Rosa26 locus to generate a conditional Rosa26^hACE2 mouse model.

(A) Schematic presentation of the approach to generate the targeted Rosa26 allele via CRISPR/Cas9 gene editing. Human ACE2 cDNA was cloned into the Rosa26-DEST vector downstream of a loxP-polyA-loxP stop cassette. (B) Expression of the hACE2 allele will be activated upon Cre-mediated removal of the loxP cassette. (C) Expression of hACE2 in kidney, liver and lung tissues, as measured by real-time qPCR in Rosa26^hACE2;CMV-Cre mice. Graph presents the ratio of hACE2 to endogenous (WT) mAce2 level in various tissues. Data shown as mean +/− SD, n=2 per tissue.

Figure 4.. SARS-CoV-2 infection leads to significant morbidity and mortality in Rosa26^hACE2;CMV-Cre mouse model.

Outcome of viral infection was assessed daily up to 6 days post infection, using (A) Clinical scoring system, ranging in severity from 1 (well-appearing) to 4 (severely affected), (B) Change in body weight post infection, presented as proportion of starting weight (weight at day 0).; and (C) Kaplan-Meier survival curve, demonstrating morbidity and lethality in 4 out of 5 Rosa26^hACE2;CMV-Cre mice by day 4–6 post infection. (D) Viral titers measured in lung and brain tissue from Rosa26^hACE2;CMV-Cre mice on day 3 post infection (data presented as viral titer per gram lung tissue, n= 4). Limit of detection (LOD) for tissue viral titer is Log₁₀ 1.69 TCID₅₀/gm. (E) Hematoxylin-stained histological lung sections of SARS-CoV-2 treated Rosa26^hACE2;CMV-Cre mice on day 3 post infection (x20, Scale bar 200uM). Bottom panel: Magnification of the insert area (marked in red), showing mild alveolar wall thickening (red arrows) in the mutant mice.

Figure 4.. SARS-CoV-2 infection leads to significant morbidity and mortality in Rosa26^hACE2;CMV-Cre mouse model.

Outcome of viral infection was assessed daily up to 6 days post infection, using (A) Clinical scoring system, ranging in severity from 1 (well-appearing) to 4 (severely affected), (B) Change in body weight post infection, presented as proportion of starting weight (weight at day 0).; and (C) Kaplan-Meier survival curve, demonstrating morbidity and lethality in 4 out of 5 Rosa26^hACE2;CMV-Cre mice by day 4–6 post infection. (D) Viral titers measured in lung and brain tissue from Rosa26^hACE2;CMV-Cre mice on day 3 post infection (data presented as viral titer per gram lung tissue, n= 4). Limit of detection (LOD) for tissue viral titer is Log₁₀ 1.69 TCID₅₀/gm. (E) Hematoxylin-stained histological lung sections of SARS-CoV-2 treated Rosa26^hACE2;CMV-Cre mice on day 3 post infection (x20, Scale bar 200uM). Bottom panel: Magnification of the insert area (marked in red), showing mild alveolar wall thickening (red arrows) in the mutant mice.

Figure 4.. SARS-CoV-2 infection leads to significant morbidity and mortality in Rosa26^hACE2;CMV-Cre mouse model.

Outcome of viral infection was assessed daily up to 6 days post infection, using (A) Clinical scoring system, ranging in severity from 1 (well-appearing) to 4 (severely affected), (B) Change in body weight post infection, presented as proportion of starting weight (weight at day 0).; and (C) Kaplan-Meier survival curve, demonstrating morbidity and lethality in 4 out of 5 Rosa26^hACE2;CMV-Cre mice by day 4–6 post infection. (D) Viral titers measured in lung and brain tissue from Rosa26^hACE2;CMV-Cre mice on day 3 post infection (data presented as viral titer per gram lung tissue, n= 4). Limit of detection (LOD) for tissue viral titer is Log₁₀ 1.69 TCID₅₀/gm. (E) Hematoxylin-stained histological lung sections of SARS-CoV-2 treated Rosa26^hACE2;CMV-Cre mice on day 3 post infection (x20, Scale bar 200uM). Bottom panel: Magnification of the insert area (marked in red), showing mild alveolar wall thickening (red arrows) in the mutant mice.