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Background: Next-generation SARS-CoV-2 vaccines demonstrated that nanoparticle messenger ribonucleic acid (mRNA) delivery is effective and safe for in vivo delivery in humans. Current treatments for cystic fibrosis (CF) primarily focus on modulator drug therapies designed to correct malfunctioning CF transmembrane conductance regulator (CFTR) protein, but these modulators are ineffective for the 10% of people with CF with variants that do not allow protein production. Among these is the splice variant 3120 + 1G >A, the most common CF-causing mutation in native Africans. Gene editing would allow production of CFTR protein and enhancement of function using available CFTR modulators. We have demonstrated that electroporation of a modified CRISPR-Cas9 base editor to primary human bronchial epithelial cells carrying 3120 + 1G >A and F508del mutant alleles achieved 75% genome editing of the splice variant, resulting in approximately 40% wild-type (WT) CFTR function [1]. Here,we evaluate the effectiveness of several new nanoparticle formulations at delivering green fluorescent protein (GFP) mRNA to CF bronchial epithelial (CFBE41o-) cells. Using the optimal formulation,we then tested the efficacy correction of the 3120 + 1G >Avariant in a CFTR expression minigene (EMG) integrated into the genome of isogenic CFBE cells using mRNA and plasmid deoxyribonucleic acid (DNA) encoding adenine base editor (ABE) and guide (g)RNA. Method(s): GFP served as a reporter to evaluate transfection efficiency, cell viability, and mean fluorescence intensity (MFI) for three dosages (150, 75, 32.5 ng of mRNA), four polymer-to-mRNA to weight (w/w) ratios (60, 40, 30, 20), and four polymers (R, Y, G, B). 7-AAD served as a live/dead stain to quantify viability, with flow cytometry results analyzed using FlowJo software. CFBE cells stably expressing the 3120 + 1G >A EMG were transfected with the optimized nanoparticle formulation to deliver ABE and gRNA at two dosages (150, 75 ng) of mRNA and DNA. CFTR function in CFBE cellswas measured by short circuit current, forskolin stimulation, and inh-172 inhibition as a measure of editing efficiency. Result(s): Flow cytometry showed that polymer R achieved more than 85% GFP transfection, compared with a maximum of approximately 35% for the other three polymers at the maximum 150-ng dose, with approximately 80% viability normalized to untreated cells. In addition, polymer R achieved GFP MFI more than one order of magnitude as high as other formulations (~30 000 vs 2700 MFI) for the other three polymers at 150-ng dose and 40 w/w ratio. CFBE cells transfected with polymer R nanoparticles containing ABE and guide RNA at 75 ng and 150 ng showed mean CFTR function increase to 10 muA 6 (standard error of the mean [SEM] 1.1 muA) (~10% of WT) and 6.3 muA (SEM 0.9 muA) (~6% of WT), respectively. Greater toxicity at the higher dose could explain the larger increase in CFTR current at the lower dose. DNA-encoded ABE plasmid and gRNA showed a less robust increase in CFTR function (2.9 muA [SEM 0.4 muA] for 75-ng dose;3.0 muA [SEM 0.4 muA] for 150-ng dose), which was probably a result of the nanoparticle formulation being optimized for RNA instead of DNA cargo or the additional intracellular barriers that must be overcome for successful DNA delivery. Conclusion(s): We demonstrated that an optimized nanoparticle formulation containing ABE and gRNA can correct splicing of isogenic cells bearing the 3120 + 1G >A CFTR variant, resulting in recovery of CFTR function. In ongoing work, we are adapting these nanoparticles for RNA- and DNAencoded ABE and gRNA delivery to primary human bronchial epithelial cells.Copyright © 2022, European Cystic Fibrosis Society. All rights reserved
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Introduction/Aim: As the causative agent of COVID-19, SARS-CoV-2 remains a global cause for concern. Compared to other highly pathogenic coronaviruses (SARS-CoV and MERS-CoV), SARS-CoV-2 exhibits stronger transmissibility but less lethality, indicating that SARS-CoV-2 displays unique characteristics, despite the partial genomic proximity. Thus, we aim to employ RNA sequencing to define transcriptional differences in epithelial responses following infection with SARS-CoV-2 compared to pathogenic SARS-CoV and MERS-CoV, and low pathogenic HCoV-229E. Method(s): Primary human bronchial epithelial cells (PBEC) were differentiated for 6 weeks at the air-liquid interface (ALI) before parallel infection by the 4 different coronaviruses (n = 4). After infection following apical application of coronaviruses at low dose (MOI 0.1), cells were harvested for bulk RNA sequencing. Gene were considered significant with a fold change (FC) > 2 and false discovery rate of FDR < 0.05. Inhibitor experiments were conducted on CALU-3 cells using DIM-C-pPhOH 10 muM (NR4A1 antagonist), Sp600125 10 muM (JNK inhibitor), T-5224 10 muM (AP-1 transcription factor inhibitor) and Cytosporone B (CsB 5 muM;NR4A1 agonist) preincubated for 1 h with these compounds and subsequently infected with SARS-CoV-2 or MERS-CoV (MOI of 1). Samples were collect 24 h later for PCR. Result(s): PCR and RNA-Seq demonstrated that all tested coronaviruses efficiently infected ALI-PBEC and replicated over 72 h (p < 0.05). RNA sequencing analysis revealed that infection with SARS-CoV, MERS-CoV and HCoV-229E resulted in largely similar transcriptional responses by the epithelial cells. However, whereas infection with these viruses was accompanied by an increased expression of genes associated with JNK/AP-1 signalling, including FOS, FOSB and NR4A1 (FC > 1, FDR < 0.05), no such increase was observed following SARS-CoV-2 infection. Further, we found that an NR4A1 antagonist reduced viral replication of MERS and SARs-CoV-2 100-fold in Calu-3 cells. Conclusion(s): In conclusion, these data suggest that SARS-CoV-2-infected ALI-PBEC exhibit a unique transcriptional response compared to other coronaviruses, which might relate to the pathogenicity of the virus.
ABSTRACT
As the causative agent of COVID-19, SARS-CoV-2 remains a global cause for concern. Compared to other highly pathogenic coronaviruses (SARS-CoV and MERS-CoV), SARS-CoV-2 exhibits stronger transmissibility but less lethality, indicating that SARS-CoV-2 displays unique characteristics, despite the partial genomic proximity. Thus, we aim to employ RNA sequencing to define transcriptional differences in epithelial responses following infection with SARS-CoV-2 compared to pathogenic SARS-CoV and MERS-CoV, and low pathogenic HCoV-229E. Primary human bronchial epithelial cells (PBEC) were differentiated for 6 weeks at the air-liquid interface (ALI) before parallel infection by the 4 different coronaviruses. After infection following apical application of coronaviruses at low dose, cells were harvested for bulk RNA sequencing. Results demonstrated that all tested coronaviruses efficiently infected ALI-PBEC. RNA sequencing analysis revealed that infection with SARS-CoV, MERS-CoV and HCoV-229E resulted in largely similar transcriptional responses by the epithelial cells. However, whereas infection with these viruses was accompanied by an increased expression of genes associated with JNK/AP-1 signalling, including FOS, FOSB and NR4A1, no such increase was observed following SARS-CoV-2 infection. Further, preliminary experiments indicated that an NR4A1 antagonist reduced viral replication in Calu-3 cells. In conclusion, these data suggest that SARS-CoV2-infected ALI-PBEC exhibit a unique transcriptional response compared to other coronaviruses, which might relate to the pathogenicity of the virus.
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Background: TGF-beta signaling plays an essential role in tissue fibrosis and mediates profibrotic programs after SARS-CoV-2 infection in the kidney and lung. SARS-CoV-2 also induces humoral immune responses controlled by cytokines, including TGF-beta. Studies have found that the incidence of SARS-CoV-2 infection and the severity of Covid-19 in cystic fibrosis (CF) patients is lower than the general population. We studied how SARS-CoV-2 regulates TGF-beta-mediated gene expression in the CF airway. Method(s): Small RNAseq was performed in human bronchial epithelial cells CFBE41o-from a patient homozygous for the F508del mutation in the CFTR gene on Illumina NextSeq 500's. Pathway analysis was done by Ingenuity Pathway Analysis (IPA) software (QIAGEN) and miRNet browser. IPA was used for analyzing coronavirus associated pathways affected by differentially regulated miRNAs. miRNAs predicted to target the coronavirus associated genes were collected from TargetScan Human release 7.2, miRmap, Diana-TarBase v.8, and miRBase bioinformatics tools. Anti-miRNA oligonucleotide miRCURY LNATM Power Inhibitors or control (Exiqon) were used. Cells expressing F508del or wild type CFTR were used to compare the results in CF and non-CF models. Findings were validated in primary human bronchial epithelial (HBE) cells. Result(s): Compared to vehicle control, TGF-beta1 dysregulated 48 miRNAs;38 and 19 pathways were uniquely affected by the upregulated and downregulated miRNAs, respectively. We found 43 miRNAs targeting 119 different mRNA of the proteins associated with coronavirus pathogenesis pathway and 21 miRNAs targeting 21 different mRNA of the proteins associated with coronavirus replication pathway. Two miRNAs upregulated by TGF-beta1 target the host receptor for SARS-CoV-2 invasion, angiotensin converting enzyme 2 (ACE2). We confirmed the results by qRT-PCR that TGF-beta1 increased expression of specific miRNAs targeting ACE2 mRNA. Upregulation of the miRNAs was followed by inhibition of ACE2 mRNA and protein levels and the effect was blocked by specific anti-miRNA oligonucleotides. The above results differed between the CF and non-CF cells. Conclusion(s): miRNAs may be important effectors of TGF-beta modulating SARSCoV-2 pathogenicity and replication in the CF airway. Ongoing studies focus on elucidating the mechanisms of SARS-CoV-2 invasion of kidney cells.
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RATIONALE: Treatments for the coronavirus disease of 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), are urgently needed but remain limited. SARS-CoV-2 infects cells through the interactions of its spike (S) protein with ACE2 and TMPRSS2 on host cells. Multiple cells and organs are targeted, particularly airway epithelial cells. OM-85, a standardized lysate of human airway bacteria with strong immunomodulating properties and an impeccable safety profile, is widely used to prevent recurrent respiratory infections. Our finding that the airway administration of OM-85 inhibits Ace2 and Tmprss2 transcription in mouse lungs prompted us to investigate whether and how OM-85 may protect non-human primate and human epithelial cells against SARS-CoV-2 infection. METHODS: ACE2 and TMPRSS2 mRNA and protein expression, cell binding of SARS-CoV-2 S1 protein, cell entry of SARS-CoV-2 S protein-pseudotyped lentiviral particles, and SARS-CoV-2 cell infection were measured in kidney, lung and intestinal epithelial cell lines, primary human bronchial epithelial cells, and ACE2- transfected HEK293T cells treated with OM-85 in vitro. RESULTS: OM-85 significantly downregulated ACE2 and TMPRSS2 mRNA in epithelial cell lines and primary bronchial epithelial cells, and strongly inhibited SARS-CoV-2 S protein binding to, SARS-CoV-2 S proteinpseudotyped lentivirus entry into, and SARS-CoV-2 infection of epithelial cells. These effects of OM-85 appeared to depend on the downregulation of SARS-CoV-2 receptor expression. CONCLUSIONS: OM-85 inhibits SARS-CoV-2 epithelial cell infection in vitro by downregulating SARS-CoV-2 receptor expression. Further studies are warranted to assess whether OM-85 may prevent and/or reduce the severity of COVID-19.
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Rationale: Individuals with COPD who develop COVID-19 are at increased risk of hospitalization, ICU admission and death. COPD is associated with increased airway epithelial expression of ACE2, the receptor mediating SARS-CoV-2 entry into cells. Hypercapnia commonly develops in advanced COPD and is associated with frequent and potentially fatal pulmonary infections. We previously reported that hypercapnia increases viral replication, lung injury and mortality in mice infected with influenza A virus. Also, global gene expression profiling of primary human bronchial epithelial (HBE) cells showed that elevated CO2 upregulates expression of cholesterol biosynthesis genes, including HMGCS1, and downregulates ATP-binding cassette (ABC) transporters that promote cholesterol efflux. Given that cellular cholesterol is important for entry of viruses into cells, in the current study we assessed the impact of hypercapnia on regulation of cellular cholesterol levels, and resultant effects on expression of ACE2 and entry of Pseudo-SARS-CoV-2 in cultured HBE, BEAS-2B and VERO cells, and airway epithelium of mice. Methods: Differentiated HBE, BEAS-2B or VERO cells were pre-incubated in normocapnia (5% CO2, PCO2 36 mmHg) or hypercapnia (15% CO2, PCO2 108 mmHg), both with normoxia, for 4 days. Expression of ACE2 and sterol regulatory element binding protein 2 (SREPB2), the master regulator of cholesterol synthesis, was assessed by immunoblot or immunofluorescence. Cholesterol was measured in cell lysates by Amplex red assay. Cells cultured in normocapnia or hypercapnia were also infected with Pseudo SARS-CoV-2, a Neon Green reporter baculovirus. For in vivo studies, C57BL/6 mice were exposed to normoxic hypercapnia (10% CO2/21% O2) for 7 days, or air as control, and airway epithelial expression of ACE2, SREBP2, ABCA1, ABCG1 and HMGCS1 was assessed by immunofluorescence. SREBP2 was blocked using the small molecules betulin or AM580, and cellular cholesterol was disrupted using MβCD. Results: Hypercapnia increased expression and activation of SREBP2 and decreased expression of ABC transporters, thereby augmenting epithelial cholesterol levels. Elevated CO2 also augmented ACE2 expression and Pseudo-SARSCoV- 2 entry into epithelial cells in vitro and in vivo. These effects were all reversed by blocking SREBP2 or disrupting cellular cholesterol. Conclusion: Hypercapnia augments cellular cholesterol levels by altering expression of cholesterol biosynthetic enzymes and efflux transporters, leading to increased epithelial expression of ACE2 and entry of Pseudo-SARS-CoV-2 into cells. These findings suggest that ventilatory support to limit hypercapnia or pharmacologic interventions to decrease cellular cholesterol might reduce viral burden and improve clinical outcomes of SARSCoV- 2 infection in advanced COPD and other severe lung diseases.
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Background: Humoral innate immunity consists of a limited, but diverse, set of humoral fluid phase pattern recognition molecules (PRMs) that represent a first line of resistance against microbial invaders by promoting pathogen disposal by phagocytosis, complement activation and inflammation. These factors encompass complement, ficolin, collectin and pentraxin family of proteins. Methods: We have analyzed the activity of PRMs for their potential capacity of inhibiting SARS-CoV-2 entry and replication into epithelial cells by a microneutralization assay based on a lentiviral particles pseudotyped with the SARS-CoV-2 spike protein in HEK293T cells overexpressing the angiotensin converting enzyme 2 (ACE2). Either SARS-CoV-2 or target cells were incubated with Mannose Binding Lectin (MBL, concentration range: 1-50 μ g/ml) to further characterize its anti-viral activity for 1 h prior to infection in both human Calu-3 cells and air-liquid interface cultures of human bronchial epithelial cells (HBEC). Binding experiments were carried out with SARS-CoV-2 Spike protein and recombinant MBL to further investigate its antiviral action. Results: Among 12 PRMs tested, only MBL inhibited viral entry in the pseudotyped neutralization assay. Furthermore, MBL protein inhibited SARS-CoV-2 viral replication in Calu-3 and HBEC by ca. one log10 at the top concentration (10 μ g/ml and 50 μ g/ml, respectively). MBL antiviral activity was confirmed also against alpha, beta and gamma SARS-CoV-2 variants of concern. Binding experiments showed that MBL specifically interacts with the trimeric form of SARS-CoV-2 spike. Conclusion: MBL binds to the Spike protein in its active trimeric conformation leading to the inhibition of SARS-CoV-2 infection and replication in vitro. These results suggest that MBL possesses an antiviral activity against SARS-CoV-2 that could bear therapeutic potential.
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Background: Severe acute respiratory coronavirus 2 (SARS-CoV-2) is the infectious agent of the current COVID-19 pandemic and is responsible for more than 2 million deaths worldwide. The virus utilizes the surface receptor angiotensin-converting enzyme 2 (ACE2) to infiltrate its target cells. It has been postulated that ACE2 is a human interferon-stimulated gene (ISG) and is upregulated by interferon (IFN) and virus stimulation. However, previous studies showed that not ACE2, moreover a novel short isoform of the enzyme is mainly expressed after IFN stimulation. Method: In this study, we aimed to investigate the impact of IFN stimulation on ACE2 expression in human bronchial epithelial cells. We measured the expression levels of two ACE2 isoforms, namely the full-length isoform and the IFN-induced truncated isoform proposed by Onabajo et al. (truncACE2) at the transcription and protein level following stimulation of Calu-3 and primary human bronchial epithelial cells (NHBE) with type-I,-II and-III IFNs. Specific primers along the ACE2 coding sequence were used for quantitative and semi-quantitative analysis of the transcript isoforms. Induced ACE2 protein isoforms were additionally analyzed using immunoblotting. Results: In both, the Calu3 cell line and the NHBE, truncACE2 was dose-dependently upregulated at the transcription level after 6h of IFNγ stimulation, whereas the full-length transcript levels did not change. IFNs of type-I and type-III stimulation induced a dosedependent upregulation of both ACE2 versions at the mRNA level, although the truncACE2 showed a higher expression level. In the immunoblotting analysis, neither the full-length nor the truncated ACE2 version showed a difference after IFNγ stimulation of NHBE. Conclusion: This study is in line with previous findings that only a truncated ACE2 isoform acts as an ISG. However, our data additionally show that not only type-II, but also type-I and type-III interferons induce this truncated version of ACE2. This is especially important, as type-I and type-III IFNs are secreted during the initial epithelial host response to the virus. It remains to be uncovered whether SARS-CoV-2 can utilize also truncACE2 to enter cells thereby using the initial host response to increase truncACE2 and facilitate viral spread.
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Background/Introduction: SARS-CoV-2 causes life threatening COVID- 19 complications including acute coronary syndrome, venous thromboembolism, hyperinflammation and damage in multiple tissues. The SARSCoV- 2 spike protein binds cell surface receptors including angiotensinconverting enzyme 2 (ACE2) for entry into host cells to initiate infection. Host cell dipeptidyl peptidase-4 (DPP4 / CD26) is implicated as a cofactor in uptake. Recent evidence indicates expression of factors involved in SARS-CoV-2 uptake into host cells is regulated by BET proteins, epigenetic readers modulating gene expression. Apabetalone, the most clinically advanced BET inhibitor (BETi), is in phase 3 trials for cardiovascular disease (CVD) (a, b). In cultured human cardiomyocytes, apabetalone suppressed infection with SARS-CoV-2 and prevented dysfunction of cardiac organoids induced by the cytokine-storm that arises in patients with severe symptoms (c). However, anti-viral properties of apabetalone in other cell types are not known. Purpose: To examine effects of apabetalone on SARS-CoV-2 infection in cell culture via downregulated expression of cell surface receptors involved in viral entry. Cell systems used mimic initial sites of infection in the lung as well as cell types contributing to complications in late stages of infection. Methods: Gene expression was measured by real-time PCR, protein levels by immunoblot or flow cytometry, and binding of recombinant SARSCoV- 2 spike protein by flow cytometry. Infection with SARS-CoV-2 was determined in a BSL3 facility. Infectivity was quantified by determining levels of viral spike protein amongst total cells via imaging on an Operetta CLS. Results: In Calu-3, a human bronchial epithelial cell line, apabetalone dose-dependently downregulated ACE2 gene expression (up to 98%), reduced ACE2 protein levels (up to 84%) and diminished binding of SARSCoV- 2 spike protein (up to 77%, p<0.001 for all parameters). Further, apabetalone abolished infection of Calu-3 cells with live SARS-CoV-2, which was comparable to other antiviral agents. Apabetalone-driven ACE2 downregulation was also observed in extrapulmonary cell types including HepG2, Huh-7 or primary hepatocytes (up to 90%, p<0.001 for all cell types), and Vero E6, a monkey kidney epithelial cell line (up to 38%, p<0.05). DPP4/CD26, a potential cofactor for SARS-CoV-2 uptake, was also downregulated by apabetalone in Calu-3 cells (mRNA ∼65% and protein ∼40%, p<0.001), which may be synergistic with ACE2 reductions to impede SARS-CoV-2 infection. Conclusions: Apabetalone, an investigational drug for CVD, reduced cell surface receptors (ACE2 and DPP4) involved in SARS-CoV-2 uptake into host cells and dramatically attenuated SARS-CoV-2 infection/propagation in vitro. Our results suggest apabetalone can mitigate SARS-CoV-2 replication in multiple organs, which together with an established safety profile supports clinical evaluation of apabetalone to treat.