Age-specific nasal epithelial responses to SARS-CoV-2 infection.

Children infected with SARS-CoV-2 rarely progress to respiratory failure. However, the risk of mortality in infected people over 85 years of age remains high. Here we investigate differences in the cellular landscape and function of paediatric (<12 years), adult (30-50 years) and older adult (>70 years) ex vivo cultured nasal epithelial cells in response to infection with SARS-CoV-2. We show that cell tropism of SARS-CoV-2, and expression of ACE2 and TMPRSS2 in nasal epithelial cell subtypes, differ between age groups. While ciliated cells are viral replication centres across all age groups, a distinct goblet inflammatory subtype emerges in infected paediatric cultures and shows high expression of interferon-stimulated genes and incomplete viral replication. In contrast, older adult cultures infected with SARS-CoV-2 show a proportional increase in basaloid-like cells, which facilitate viral spread and are associated with altered epithelial repair pathways. We confirm age-specific induction of these cell types by integrating data from in vivo COVID-19 studies and validate that our in vitro model recapitulates early epithelial responses to SARS-CoV-2 infection.

Intratumoural delivery of TRAIL mRNA induces colon cancer cell apoptosis.

Novel strategies in intratumoral injection and emerging immunotherapies have heralded a new era of precise cancer treatments. The affinity of SARS-CoV-2 to ACE2 receptors, a feature which facilitates virulent human infection, is leveraged in this research. Colon cancer cells, with their high ACE2 expression, provide a potentially strategic target for using this SARS-CoV-2 feature. While the highly expression of ACE2 is observed in several cancer types, the idea of using the viral spike protein for targeting colon cancer cells offers a novel approach in cancer treatment. Intratumoral delivery of nucleic acid-based drugs is a promising alternative to overcoming the limitations of existing therapies. The increasing importance of nucleic acids in this realm, and the use of Lipid Nanoparticles (LNPs) for local delivery of nucleic acid therapeutics, are important breakthroughs. LNPs protect nucleic acid drugs from degradation and enhance cellular uptake, making them a rapidly evolving nano delivery system with high precision and adaptability. Our study leveraged a tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) combined with a receptor-binding domain from the SARS-CoV-2 spike protein, encapsulated in LNPs, to target colon cancer cells. Our results indicated that the TRAIL fusion-mRNA induced apoptosis in vitro and in vivo. Collectively, our findings highlight LNP-encapsulated TRAIL fusion-mRNA as a potential colon cancer therapy.

Transcriptional regulation of SARS-CoV-2 receptor ACE2 by SP1.

Angiotensin-converting enzyme 2 (ACE2) is a major cell entry receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The induction of ACE2 expression may serve as a strategy by SARS-CoV-2 to facilitate its propagation. However, the regulatory mechanisms of ACE2 expression after viral infection remain largely unknown. Using 45 different luciferase reporters, the transcription factors SP1 and HNF4α were found to positively and negatively regulate ACE2 expression, respectively, at the transcriptional level in human lung epithelial cells (HPAEpiCs). SARS-CoV-2 infection increased the transcriptional activity of SP1 while inhibiting that of HNF4α. The PI3K/AKT signaling pathway, activated by SARS-CoV-2 infection, served as a crucial regulatory node, inducing ACE2 expression by enhancing SP1 phosphorylation-a marker of its activity-and reducing the nuclear localization of HNF4α. However, colchicine treatment inhibited the PI3K/AKT signaling pathway, thereby suppressing ACE2 expression. In Syrian hamsters (Mesocricetus auratus) infected with SARS-CoV-2, inhibition of SP1 by either mithramycin A or colchicine resulted in reduced viral replication and tissue injury. In summary, our study uncovers a novel function of SP1 in the regulation of ACE2 expression and identifies SP1 as a potential target to reduce SARS-CoV-2 infection.

MiR-145-5p reduced ANG II-induced ACE2 shedding and the inflammatory response in alveolar epithelial cells by targeting ADAM17 and inhibiting the AT1R/ADAM17 pathway.

The excessive elevation of angiotensin II (ANG II) is closely associated with the occurrence and development of aortic dissection (AD)-related acute lung injury (ALI), through its binding to angiotensin II receptor type I (AT1R). MiR-145-5p is a noncoding RNA that can be involved in a variety of cellular physiopathological processes. Transfection with miR-145-5p was found to downregulated the expression of A disintegrin and metalloprotease 17 (ADAM17) and reduced the levels of angiotensin-converting enzyme 2 (ACE2) in lung tissue, while concurrently increasing plasma ACE2 levels in the AD combined with ALI mice. ADAM17 was proved to be a target of miR-145-5p. Transfection with miR-145-5p decreased the shedding of ACE2 and alleviated the inflammatory response induced by ANG II through targeting ADAM17 and inhibiting the AT1R/ADAM17 pathway in A549 cells. In conclusion, our present study demonstrates the role and mechanism of miR-145-5p in alleviating ANG II-induced acute lung injury, providing a new insight into miRNA therapy for reducing lung injury in patients with aortic dissection.

Quercetin inhibits SARS-CoV-2 infection and prevents syncytium formation by cells co-expressing the viral spike protein and human ACE2.

BACKGROUND: Several in silico studies have determined that quercetin, a plant flavonol, could bind with strong affinity and low free energy to SARS-CoV-2 proteins involved in viral entry and replication, suggesting it could block infection of human cells by the virus. In the present study, we examined the ex vivo ability of quercetin to inhibit of SARS-CoV-2 replication and explored the mechanisms of this inhibition. METHODS: Green monkey kidney Vero E6 cells and in human colon carcinoma Caco-2 cells were infected with SARS-CoV-2 and incubated in presence of quercetin; the amount of replicated viral RNA was measured in spent media by RT-qPCR. Since the formation of syncytia is a mechanism of SARS-CoV-2 propagation, a syncytialization model was set up using human embryonic kidney HEK293 co-expressing SARS-CoV-2 Spike (S) protein and human angiotensin converting enzyme 2 (ACE2), [HEK293(S + ACE2) cells], to assess the effect of quercetin on this cytopathic event by microscopic imaging and protein immunoblotting. RESULTS: Quercetin inhibited SARS-CoV-2 replication in Vero E6 cells and Caco-2 cells in a concentration-dependent manner with a half inhibitory concentration (IC50) of 166.6 and 145.2 µM, respectively. It also inhibited syncytialization of HEK293(S + ACE2) cells with an IC50 of 156.7 µM. Spike and ACE2 co-expression was associated with decreased expression, increased proteolytic processing of the S protein, and diminished production of the fusogenic S2' fragment of S. Furin, a proposed protease for this processing, was inhibited by quercetin in vitro with an IC50 of 116 µM. CONCLUSION: These findings suggest that at low 3-digit micromolar concentrations of quercetin could impair SARS-CoV-2 infection of human cells partly by blocking the fusion process that promotes its propagation.

Alterations in the gene expression of SARS-COV-2 entry receptors and enzymes in lungs and hearts of controlled and uncontrolled diabetic mice.

BACKGROUND: The entry of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into the host cell is carried out by specific receptors and enzymes, including human angiotensin-converting enzyme 2 receptor (ACE2), transmembrane serine protease 2 (TMPRSS2), and cathepsin-L (CTSL). COVID-19 patients with comorbidities, such as diabetes mellitus (DM), are more prone to severe symptoms and have a higher risk of mortality. AIMS: The present study aimed to investigate the impact of controlled and uncontrolled type 1 DM (T1DM) on the gene expression of mouse Ace2, Tmprss2, and Ctsl and correlate it with the pathological alterations in the lungs and the heart of DM mice. METHODS: Balb/c mice were administered a single dose of 240 mg/kg streptozocin to induce T1DM. The blood glucose level was measured to confirm the induction of DM. Normalization of blood glucose levels in T1DM mice was achieved using 0.1 mL/kg Mixtard® insulin therapy. The mice's lungs and hearts were harvested, and the mRNA was extracted and converted to cDNA. The gene expression of Ace2, Tmprss2, Ctsl, Cyp4a11, and Adrb1 genes, which play a role in the homeostasis of lungs and hearts, were measured using quantitative real-time polymerase chain reaction (RT-PCR). The pathological alterations in the hearts and lungs induced by T1DM were evaluated using the relative heart and lung weights, in addition to the pathohistological examination. RESULTS: After inducing T1DM for 14 days, we observed a significant reduction in the total weight of uncontrolled DM (UDM) mice (P < 0.05). Pathohistological examination of UDM lung tissues revealed thickening of the alveolar walls with narrowing of the surface of the alveolar sacs. Additionally, we found that UDM mice exhibited downregulation of Ace2 gene expression (P < 0.05) in their lungs, while both UDM and control DM (CDM) mice showed upregulation of Ctsl gene expression in their hearts (P < 0.05). Notably, Cyp4a12 gene expression was significantly downregulated (P < 0.05) in UDM mice but returned to normal levels in CDM mice. CONCLUSIONS: We conclude from this study that T1DM downregulates Ace2 receptor and Cyp4a12 gene expression, which is correlated with the thickening of alveolar walls and narrowing of the surface of alveolar sacs in the lungs. Insulin administration for controlling T1DM ameliorated these pathological alterations. These results can help increase our understanding of the impact of controlled and uncontrolled T1DM on the lungs and may explain, at least in part, why DM patients with COVID-19 experience exacerbation of symptoms.

Angiotensin converting enzyme 2 does not facilitate porcine epidemic diarrhea virus entry into porcine intestinal epithelial cells and inhibits it-induced inflammatory injury by promoting STAT1 phosphorylation.

ACE2 has been confirmed to be a functional receptor for SARS-CoV and SARS-CoV-2, but research on animal coronaviruses, especially PEDV, are still unknown. The present study investigated whether ACE2 plays a role in receptor recognition and subsequent infection during PEDV invasion of host cells. IPEC-J2 cells stably expressing porcine ACE2 did not increase the production of PEDV-N but inhibited its expression. Porcine ACE2 knockout cells was generated by CRISPR/Cas9 genome editing in IPEC-J2 cells. The expression of PEDV-N did not decrease but slightly increased. The Co-IP results showed that there was no significant association between ACE2 and PEDV-S. There were no obvious interaction between PEDV-S, PEDV-E, PEDV-M and porcine ACE2 promoters, but PEDV-N could inhibit the activity of ACE2 promoters. PEDV-N degraded STAT1 and prevented its phosphorylation, thereby inhibiting the expression of interferon-stimulated genes. Repeated infection of PEDV further confirmed the above results. PEDV activated ACE-Ang II-AT1R axis, while ACE2-Ang (1-7)-MasR axis activity was decreased and inflammatory response was intensified. However, excess ACE2 can reverse this reaction. These results reveal that ACE2 does not facilitate PEDV entry into cells, but relieves PEDV-induced inflammation by promoting STAT1 phosphorylation.

Myeloid ACE2 protects against septic hypotension and vascular dysfunction through Ang-(1-7)-Mas-mediated macrophage polarization.

Angiotensin converting enzyme 2 (ACE2) is a new identified member of the renin-angiotensin-aldosterone system (RAAS) that cleaves angiotensin II (Ang II) to Ang (1-7), which exerts anti-inflammatory and antioxidative activities via binding with Mas receptor (MasR). However, the functional role of ACE2 in sepsis-related hypotension remains unknown. Our results indicated that sepsis significantly reduced blood pressure and led to disruption between ACE-Ang II and ACE2-Ang (1-7) balance. ACE2 knock-in mice exhibited improved sepsis-induced mortality, hypotension and vascular dysfunction, while ACE2 knockout mice exhibited the opposite effects. Bone marrow transplantation and in vitro experiments confirmed that myeloid ACE2 exerted a protective role by suppressing oxidative stress, NO production and macrophage polarization via the Ang (1-7)-MasR-NF-κB and STAT1 pathways. Thus, ACE2 on myeloid cells could protect against sepsis-mediated hypotension and vascular dysfunction, and upregulating ACE2 may represent a promising therapeutic option for septic patients with hypotension.

Eye Washing Downregulated Angiotensin-Converting Enzyme 2 in Conjunctival Tissue Samples from Smokers.

This study aimed to (1) determine whether the expression of angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine 2 is increased in tobacco smokers, which potentially increases their susceptibility to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, and (2) assess whether eye rinsing can reduce susceptibility. This prospective study included 20 eyes of 10 smokers and 18 eyes of nine healthy non-smokers (control) for reverse-transcription polymerase chain reaction. This study also included 28 eyes of 14 smokers and 16 eyes of eight healthy non-smokers (control) for enzyme-linked immunosorbent assay. Tear and impression cytology samples were collected from the right eye of each patient. The left eye was then rinsed for 30 s, and after 5 min, the tear and impression cytology samples were collected in the same manner. The expression of the ACE2 gene was significantly higher in the conjunctiva of smokers (n = 17; median 3.07 copies/ng of total RNA) than in those of non-smokers (n = 17; median 1.92 copies/ng of total RNA, p = 0.003). Further, mRNA expression and protein levels of ACE2 were weakly correlated in smokers (r = 0.49). ACE2 protein levels in Schirmer's strip samples were significantly reduced from 5051 to 3202 pg/mL after eye washing (n = 10; p = 0.001). Ocular surface cells are susceptible to SARS-CoV-2 infection. Smoking may be a risk factor for SARS-CoV-2 infection, and eye rinsing may reduce the risk of infection.

Mouse genome rewriting and tailoring of three important disease loci.

Genetically engineered mouse models (GEMMs) help us to understand human pathologies and develop new therapies, yet faithfully recapitulating human diseases in mice is challenging. Advances in genomics have highlighted the importance of non-coding regulatory genome sequences, which control spatiotemporal gene expression patterns and splicing in many human diseases1,2. Including regulatory extensive genomic regions, which requires large-scale genome engineering, should enhance the quality of disease modelling. Existing methods set limits on the size and efficiency of DNA delivery, hampering the routine creation of highly informative models that we call genomically rewritten and tailored GEMMs (GREAT-GEMMs). Here we describe 'mammalian switching antibiotic resistance markers progressively for integration' (mSwAP-In), a method for efficient genome rewriting in mouse embryonic stem cells. We demonstrate the use of mSwAP-In for iterative genome rewriting of up to 115 kb of a tailored Trp53 locus, as well as for humanization of mice using 116 kb and 180 kb human ACE2 loci. The ACE2 model recapitulated human ACE2 expression patterns and splicing, and notably, presented milder symptoms when challenged with SARS-CoV-2 compared with the existing K18-hACE2 model, thus representing a more human-like model of infection. Finally, we demonstrated serial genome writing by humanizing mouse Tmprss2 biallelically in the ACE2 GREAT-GEMM, highlighting the versatility of mSwAP-In in genome writing.

Expression of key SARS-CoV-2 entry molecules in surgically obtained human retinal biopsies.

PURPOSE: To investigate the presence of ACE2, TMPRSS2 and Furin, i.e., a key player in the ocular infection with SARS-COV-2, in surgically obtained human retinal tissue samples from SARS-CoV-2-negative patients, using gene expression analysis. METHODS: The mechanisms and entry paths of ocular infections have been ill-defined so far. To better understand the possible entry routes, we used surgically explanted retinal tissue from nine patients that were not infected with SARS-CoV-2 and analyzed the message expression of the three key molecules that confer viral entry into cells using polymerase chain reaction. RESULTS: The median age of the patients (n = 9) included in the study was 52 years (IQR 48, 55). Eight patients underwent surgery for rhegmatogenous retinal detachment and one patient for tractional retinal detachment. Gene expression for the proteins studied was detected in all nine patients. The results of analysis by Livak's method (2001) demonstrated a median TMPRSS2 gene expression value of 20.9 (IQR 11.7, 33.7), a median ACE2 gene expression value of 2.09 (IQR 1.14, 2.79) and a median Furin gene expression value of 8.33 (IQR 5.90, 11.8). CONCLUSION: In conclusion, TMPRSS2, Furin and ACE2 are expressed in the retina and may contribute to the retinal involvement in COVID-19 patients. Expression may vary among individuals, which may explain why some patients may be more prone to retinal involvement during SARS-CoV-2 infection COVID-19 patients than others. Variability in the expression of TMPRSS2, Furin and ACE2 proteins themselves may also explain the presence or development of retinal symptoms of varying severity.

SARS-CoV-2 virus associated angiotensin converting enzyme 2 expression modulation in colorectal cancer: Insights from mRNA and protein analysis COVID-19 associated (ACE2) expression in colorectal cancer.

The SARS-CoV-2 virus gains entry into human cells by exploiting the angiotensin-converting enzyme 2 (ACE2), a key component known as the spike protein (S), as a point of entry. Initially, SARS-CoV-2 suppresses the natural function of ACE2, leading to a gradual decline in cell health. Additionally, individuals with cancer are considered more susceptible to COVID-19. This study investigates the expression patterns of ACE2 in colorectal cancer (CRC) patients with and without a history of COVID-19 infection. RT-PCR was used to analyze samples from both cancerous and adjacent non-affected colorectal tissues of 47 CRC patients, comprising two groups: 24 CRC patients with no history of COVID-19 and 23 CRC patients with a recent history of COVID-19 infection. Epithelial CR cells were isolated from both types of tissues and cultured to evaluate cell adhesion. Immunohistochemistry analyses were conducted to examine ACE2 protein expression using various ACE2 antibodies for both cell types. The study revealed ACE2 mRNA expression in all CRC tissues of patients with and without a history of COVID-19. ACE2 expression was significantly higher in CRC patients without a history of COVID-19. Notably, the non-affected colorectal cancer (NACRC) tissues of patients without a history of COVID-19 also showed ACE2 expression, whereas no ACE2 expression was detected in the biopsies of CRC patients with a positive COVID-19 history. ACE2 antibodies were employed to validate ACE2 protein expression at the mRNA level. COVID-19 appears to downregulate ACE2 expression in both CRC and NACRC tissues of CRC patients with a positive history of COVID-19 infection.

Hypercapnia increases ACE2 expression and pseudo-SARS-CoV-2 entry in bronchial epithelial cells by augmenting cellular cholesterol.

Patients with chronic lung disease, obesity, and other co-morbid conditions are at increased risk of severe illness and death when infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Hypercapnia, the elevation of CO2 in blood and tissue, commonly occurs in patients with severe acute and chronic lung disease, including those with pulmonary infections, and is also associated with high mortality risk. We previously reported that hypercapnia increases viral replication and mortality of influenza A virus infection in mice. We have also shown that culture in elevated CO2 upregulates expression of cholesterol synthesis genes in primary human bronchial epithelial cells. Interestingly, factors that increase the cholesterol content of lipid rafts and lipid droplets, platforms for viral entry and assembly, enhance SARS-CoV-2 infection. In the current study, we investigated the effects of hypercapnia on ACE2 expression and entry of SARS-CoV-2 pseudovirus (p-SARS-CoV-2) into airway epithelial cells. We found that hypercapnia increased ACE2 expression and p-SARS-CoV-2 uptake by airway epithelium in mice, and in cultured VERO and human bronchial epithelial cells. Hypercapnia also increased total cellular and lipid raft-associated cholesterol in epithelial cells. Moreover, reducing cholesterol synthesis with inhibitors of sterol regulatory element binding protein 2 (SREBP2) or statins, and depletion of cellular cholesterol, each blocked the hypercapnia-induced increases in ACE2 expression and p-SARS-CoV-2 entry into epithelial cells. Cigarette smoke extract (CSE) also increased ACE2 expression, p-SARS-CoV-2 entry and cholesterol accumulation in epithelial cells, an effect not additive to that of hypercapnia, but also inhibited by statins. These findings reveal a mechanism that may account, in part, for poor clinical outcomes of SARS-CoV-2 infection in patients with advanced lung disease and hypercapnia, and in those who smoke cigarettes. Further, our results suggest the possibility that cholesterol-lowering therapies may be of particular benefit in patients with hypercapnia when exposed to or infected with SARS-CoV-2.

The Influence of KE and EW Dipeptides in the Composition of the Thymalin Drug on Gene Expression and Protein Synthesis Involved in the Pathogenesis of COVID-19.

Thymalin is an immunomodulatory drug containing a polypeptide extract of thymus that has demonstrated efficacy in the therapy of acute respiratory distress syndrome and chronic obstructive pulmonary disease, as well as in complex therapy related to severe COVID-19 in middle-aged and elderly patients.. KE and EW dipeptides are active substances of Thymalin. There is evidence that KE stimulates cellular immunity and nonspecific resistance in organisms, exerting an activating effect on macrophages, blood lymphocytes, thymocytes, and neutrophils, while EW reduces angiotensin-induced vasoconstriction and preserves endothelium-dependent vascular relaxation by inhibiting ACE2, the target protein of SARS-CoV-2. However, the mechanism of the immunomodulatory action of Thymalin, KE, and EW during COVID-19 remains unclear. To identify the potential mechanism of action underlying the immunomodulatory activity of Thymalin and its active components, EW and KE dipeptides, we assessed inflammatory response in the context of COVID-19. Interactions between EW and KE dipeptides and double-stranded DNA (dsDNA) were investigated by molecular modeling and docking using ICM-Pro. Analysis of the possible effect of EW and KE dipeptides on gene expression and protein synthesis involved in the pathogenesis of COVID-19 was conducted through the use of bioinformatics methods, including a search for promoter sequences in the Eukaryotic Promoter Database, the determination of genes associated with the development of COVID-19 using the PathCards database of human biological pathways (pathway unification database), identification of the relationship between proteins through cluster analysis in the STRING database ('Search Tool for Retrieval of Interacting Genes/Proteins'), and assessment of the functional enrichment of protein-protein interaction (PPI) using the terms of gene ontology (GO) and the Markov cluster algorithm (MCL). After that, in vitro studying of a lipopolysaccharide (LPS)-induced model of inflammation using human peripheral blood mononuclear cells was performed. ELISA was applied to assess the level of cytokines (IL-1ß, IL-6, TNFα) in the supernatant of cells with or without the impact of EW and KE peptides. Blood samples were obtained from four donors; for each cytokine, ELISA was performed 2-4 times, with two parallel experimental or control samples for each experiment (experiments to assess the effects of peptides on LPS-stimulated cells were repeated four times, while additional experiments with unstimulated cells were performed two times). Using molecular docking, GGAG was found to be the best dsDNA sequence in the classical B-form for binding the EW dipeptide, while GCGC is the preferred dsDNA sequence in the curved nucleosomal form for the KE dipeptide. Cluster analysis revealed that potential target genes for the EW and KE peptides encode the AKT1 and AKT2 proteins involved in the development of the cytokine storm. The specific targets for the EW peptide are the ACE2 and CYSLTR1 genes, and specific target for the KE peptide is the CHUK gene. Protein products of the ACE2, CYSLTR1, and CHUK genes are functionally associated with IL-1ß, IL-6, TNF-α, IL-4, and IL-10 cytokines. An in vitro model of an inflammatory reaction demonstrated that Thymalin and EW and KE dipeptides reduced the synthesis of IL-1ß, IL-6, and TNF-α cytokines in human peripheral blood mononuclear cells by 1.4-6.0 times. The immunomodulatory effect of Thymalin under the inflammatory response conditions in COVID-19 is based on the potential ability of its active components, EW and KE dipeptides, to regulate protein synthesis involved in the development of the cytokine storm.

Genome-wide loss-of-function screen using human pluripotent stem cells to study virus-host interactions for SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019, has become a global health concern. Therefore, there is an immense need to understand the network of virus-host interactions by using human disease-relevant cells. We have thus conducted a loss-of-function genome-wide screen using haploid human embryonic stem cells (hESCs) to identify genes involved in SARS-CoV-2 infection. Although the undifferentiated hESCs are resistant to SARS-CoV-2, their differentiated definitive endoderm (DE) progenies, which express high levels of ACE2, are highly sensitive to the virus. Our genetic screening was able to identify the well-established entry receptor ACE2 as a host factor, along with additional potential novel modulators of SARS-CoV-2. Two such novel screen hits, the transcription factor MAFG and the transmembrane protein TMEM86A, were further validated as conferring resistance against SARS-CoV-2 by using CRISPR-mediated mutagenesis in hESCs, followed by differentiation of mutant lines into DE cells and infection by SARS-CoV-2. Our genome-wide genetic screening investigated SARS-CoV-2 host factors in non-cancerous human cells with endogenous ACE2 expression, providing a unique platform to identify novel modulators of SARS-CoV-2 cytopathology in human cells.

Development of AAV-delivered broadly neutralizing anti-human ACE2 antibodies against SARS-CoV-2 variants.

The ongoing evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), resulting in the emergence of new variants that are resistant to existing vaccines and therapeutic antibodies, has raised the need for novel strategies to combat the persistent global COVID-19 epidemic. In this study, a monoclonal anti-human angiotensin-converting enzyme 2 (hACE2) antibody, ch2H2, was isolated and humanized to block the viral receptor-binding domain (RBD) binding to hACE2, the major entry receptor of SARS-CoV-2. This antibody targets the RBD-binding site on the N terminus of hACE2 and has a high binding affinity to outcompete the RBD. In vitro, ch2H2 antibody showed potent inhibitory activity against multiple SARS-CoV-2 variants, including the most antigenically drifted and immune-evading variant Omicron. In vivo, adeno-associated virus (AAV)-mediated delivery enabled a sustained expression of monoclonal antibody (mAb) ch2H2, generating a high concentration of antibodies in mice. A single administration of AAV-delivered mAb ch2H2 significantly reduced viral RNA load and infectious virions and mitigated pulmonary pathological changes in mice challenged with SARS-CoV-2 Omicron BA.5 subvariant. Collectively, the results suggest that AAV-delivered hACE2-blocking antibody provides a promising approach for developing broad-spectrum antivirals against SARS-CoV-2 and potentially other hACE2-dependent pathogens that may emerge in the future.

Cell free ACE2 RNA: A potential biomarker of COVID-19 severity.

Despite the downward trend of COVID-19 pandemic and increased immunity of the general population, COVID-19 is still an elusive disease with risks due to emerging variants. Fast and reliable diagnosis of COVID-19 disease would allow better therapeutic interventions for patients at risk to develop more severe outcomes. Cell-free RNAs (cfRNAs) have been proven to be an effective biomarker in cancer and infectious diseases. It has been reported that cfRNAs are amplified in the bloodstream of these patients and at earlier stages of the disease, reflecting tissue damage. Hence, we hypothesize that cfRNAs may serve as a potential indicator of COVID-19 disease severity. To our knowledge, this is the first report to display a significant link between COVID-19 severity and cfRNA of angiotensin converting enzyme-2 (ACE2), the receptor for SARS-CoV-2 virus. qRT-PCR analysis of liquid biopsies from COVID-19 patients (n = 82) displayed a significant increase in ACE2-cfRNA levels in patients with severe manifestations. This finding correlated with blood biomarkers (ANC, WBC, and Creatinine) that were also significantly increased in these patients. We previously showed that bronchial cells from obese subjects express higher ACE2 levels, hence, we further analysed the involvement of obesity as a main contributor to severe outcomes. We confirm a significant increase of ACE2-cfRNA in the plasma of obese/overweight (Ob/Ov) COVID-19 patients compared to lean subjects, with no observed significant change in blood biomarkers. These findings suggest that monitoring ACE2-cfRNAs, as a biomarker, during COVID-19 infection may allow for better disease management, specifically for severe-COVID-19 patients.

Comparison of SARS-CoV-2 entry inhibitors based on ACE2 receptor or engineered Spike-binding peptides.

With increasing resistance of SARS-CoV-2 variants to antibodies, there is interest in developing entry inhibitors that target essential receptor-binding regions of the viral Spike protein and thereby present a high bar for viral resistance. Such inhibitors could be derivatives of the viral receptor, ACE2, or peptides engineered to interact specifically with the Spike receptor-binding pocket. We compared the efficacy of a series of both types of entry inhibitors, constructed as fusions to an antibody Fc domain. Such a design can increase protein stability and act to both neutralize free virus and recruit effector functions to clear infected cells. We tested the reagents against prototype variants of SARS-CoV-2, using both Spike pseudotyped vesicular stomatitis virus vectors and replication-competent viruses. These analyses revealed that an optimized ACE2 derivative could neutralize all variants we tested with high efficacy. In contrast, the Spike-binding peptides had varying activities against different variants, with resistance observed in the Spike proteins from Beta, Gamma, and Omicron (BA.1 and BA.5). The resistance mapped to mutations at Spike residues K417 and N501 and could be overcome for one of the peptides by linking two copies in tandem, effectively creating a tetrameric reagent in the Fc fusion. Finally, both the optimized ACE2 and tetrameric peptide inhibitors provided some protection to human ACE2 transgenic mice challenged with the SARS-CoV-2 Delta variant, which typically causes death in this model within 7-9 days. IMPORTANCE The increasing resistance of SARS-CoV-2 variants to therapeutic antibodies has highlighted the need for new treatment options, especially in individuals who do not respond to vaccination. Receptor decoys that block viral entry are an attractive approach because of the presumed high bar to developing viral resistance. Here, we compare two entry inhibitors based on derivatives of the ACE2 receptor, or engineered peptides that bind to the receptor-binding pocket of the SARS-CoV-2 Spike protein. In each case, the inhibitors were fused to immunoglobulin Fc domains, which can further enhance therapeutic properties, and compared for activity against different SARS-CoV-2 variants. Potent inhibition against multiple SARS-CoV-2 variants was demonstrated in vitro, and even relatively low single doses of optimized reagents provided some protection in a mouse model, confirming their potential as an alternative to antibody therapies.

TGF-ß1 Inhibition of ACE2 Mediated by miRNA Uncovers Novel Mechanism of SARS-CoV-2 Pathogenesis.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for COVID-19, utilizes receptor binding domain (RBD) of spike glycoprotein to interact with angiotensin (Ang)-converting enzyme 2 (ACE2). Altering ACE2 levels may affect entry of SARS-CoV-2 and recovery from COVID-19. Decreased cell surface density of ACE2 leads to increased local levels of Ang II and may contribute to mortality resulting from acute lung injury and fibrosis during COVID-19. Studies published early during the COVID-19 pandemic reported that people with cystic fibrosis (PwCF) had milder symptoms, compared to people without CF. This finding was attributed to elevated ACE2 levels and/or treatment with the high efficiency CFTR modulators. Subsequent studies did not confirm these findings reporting variable effects of CFTR gene mutations on ACE2 levels. Transforming growth factor (TGF)-ß signaling is essential during SARS-CoV-2 infection and dominates the chronic immune response in severe COVID-19, leading to pulmonary fibrosis. TGF-ß1 is a gene modifier associated with more severe lung disease in PwCF but its effects on the COVID-19 course in PwCF is unknown. To understand whether TGF-ß1 affects ACE2 levels in the airway, we examined miRNAs and their gene targets affecting SARS-CoV-2 pathogenesis in response to TGF-ß1. Small RNAseq and micro(mi)RNA profiling identified pathways uniquely affected by TGF-ß1, including those associated with SARS-CoV-2 invasion, replication, and the host immune responses. TGF-ß1 inhibited ACE2 expression by miR-136-3p and miR-369-5p mediated mechanism in CF and non-CF bronchial epithelial cells. ACE2 levels were higher in two bronchial epithelial cell models expressing the most common CF-causing mutation in CFTR gene F508del, compared to controls without the mutation. After TGF-ß1 treatment, ACE2 protein levels were still higher in CF, compared to non-CF cells. TGF-ß1 prevented the modulator-mediated rescue of F508del-CFTR function while the modulators did not prevent the TGF-ß1 inhibition of ACE2 levels. Finally, TGF-ß1 reduced the interaction between ACE2 and the recombinant spike RBD by lowering ACE2 levels and its binding to RBD. Our data demonstrate novel mechanism whereby TGF-ß1 inhibition of ACE2 in CF and non-CF bronchial epithelial cells may modulate SARS-CoV-2 pathogenicity and COVID-19 severity. By reducing ACE2 levels, TGF-ß1 may decrease entry of SARS-CoV-2 into the host cells while hindering the recovery from COVID-19 due to loss of the anti-inflammatory and regenerative effects of ACE2. The above outcomes may be modulated by other, miRNA-mediated effects exerted by TGF-ß1 on the host immune responses, leading to a complex and yet incompletely understood circuitry.

Demographic, clinical and genetic factors associated with COVID-19 disease susceptibility and mortality in a Kurdish population.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is a devastating pandemic that causes disease with a variability in susceptibility and mortality based on variants of various clinical and demographic factors, including particular genes among populations. OBJECTIVES: Determine associations of demographic, clinical, laboratory, and single nucleotide polymorphisms in the ACE2, TMPRSS2, TNF-α, and IFN-γ genes to the incidence of infection and mortality in COVID-19 patients. DESIGN: Prospective cohort study SETTINGS: Various cities in the Kurdistan Region of Iraq. PATIENTS AND METHODS: This prospective cohort study compared laboratory markers (D-dimer, tumor necrosis factor-alpha [TNF-α], interferon-gamma [IFN-γ], C-reactive protein [CRP], lymphocyte and neutrophil counts) between COVID-19 patients and healthy controls. DNA was extracted from blood, and genotypes were done by Sanger sequencing. MAIN OUTCOME MEASURES: Single nucleotide polymorphisms of the ACE2, TMPRSS2, TNF-α, and IFN-γ genes and demographic characteristics and laboratory markers for predicting mortality in COVID-19. SAMPLE SIZE: 203 (153 COVID-19 patients, 50 health control subjects). RESULTS: Forty-eight (31.4%) of the COVID-19 patients died. Age over 40 and comorbidities were risk factors for mortality, but the strongest associations were with serum IFN-γ, the neutrophil-to-lymphocyte ratio (NLR), and serum TNF-α. The AA genotype and A allele of TMPRSS2 rs2070788 decreased while the GA genotype and A allele of TNF-α increased susceptibility to COVID-19. Patients with the GA genotype of TNF-α rs1800629 had shorter survival times (9.9 days) than those carrying the GG genotype (18.3 days) (P<.0001 by log-rank test). The GA genotype versus the GG genotype was associated with higher levels of serum TNF-α. The GA genotype increased mortality rates by up to 3.8 fold. The survival rate for COVID-19 patients carrying the IFN-γ rs2430561 TT genotype (58.5%) was lower than in patients with the TA and AA genotypes (80.3%). The TT genotype increased the risk of death (HR=3.664, P<.0001) and was linked to high serum IFN-γ production. Olfactory dysfunction was a predictor of survival among COVID-19 patients. CONCLUSIONS: Age older than 40, comorbidities, the NLR and particular genotypes for and the IFN-γ and TNF-α genes were risk factors for death. Larger studies in different populations must be conducted to validate the possible role of particular SNPs as genetic markers for disease severity and mortality in COVID-19 disease. LIMITATIONS: Small sample size. CONFLICT OF INTEREST: None.