RNA interference (RNAi) is a powerful approach for reducing expression of endogenously expressed proteins. It is widely used for biological applications and is being harnessed to silence mRNAs ...encoding pathogenic proteins for therapy. Various methods - including delivering RNA oligonucleotides and expressing RNAi triggers from viral vectors - have been developed for successful RNAi in cell culture and in vivo. Recently, RNAi-based gene silencing approaches have been demonstrated in humans, and ongoing clinical trials hold promise for treating fatal disorders or providing alternatives to traditional small molecule therapies. Here we describe the broad range of approaches to achieve targeted gene silencing for therapy, discuss important considerations when developing RNAi triggers for use in humans, and review the current status of clinical trials.
Cystic fibrosis (CF) is a life-shortening disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Although bacterial lung infection and the resulting ...inflammation cause most of the morbidity and mortality, how the loss of CFTR function first disrupts airway host defence has remained uncertain. To investigate the abnormalities that impair elimination when a bacterium lands on the pristine surface of a newborn CF airway, we interrogated the viability of individual bacteria immobilized on solid grids and placed onto the airway surface. As a model, we studied CF pigs, which spontaneously develop hallmark features of CF lung disease. At birth, their lungs lack infection and inflammation, but have a reduced ability to eradicate bacteria. Here we show that in newborn wild-type pigs, the thin layer of airway surface liquid (ASL) rapidly kills bacteria in vivo, when removed from the lung and in primary epithelial cultures. Lack of CFTR reduces bacterial killing. We found that the ASL pH was more acidic in CF pigs, and reducing pH inhibited the antimicrobial activity of ASL. Reducing ASL pH diminished bacterial killing in wild-type pigs, and, conversely, increasing ASL pH rescued killing in CF pigs. These results directly link the initial host defence defect to the loss of CFTR, an anion channel that facilitates HCO(3)(-) transport. Without CFTR, airway epithelial HCO(3)(-) secretion is defective, the ASL pH falls and inhibits antimicrobial function, and thereby impairs the killing of bacteria that enter the newborn lung. These findings suggest that increasing ASL pH might prevent the initial infection in patients with CF, and that assaying bacterial killing could report on the benefit of therapeutic interventions.
Infection by enveloped coronaviruses (CoVs) initiates with viral spike (S) proteins binding to cellular receptors, and is followed by proteolytic cleavage of receptor-bound S proteins, which prompts ...S protein-mediated virus-cell membrane fusion. Infection therefore requires close proximity of receptors and proteases. We considered whether tetraspanins, scaffolding proteins known to facilitate CoV infections, hold receptors and proteases together on cell membranes. Using knockout cell lines, we found that the tetraspanin CD9, but not the tetraspanin CD81, formed cell-surface complexes of dipeptidyl peptidase 4 (DPP4), the MERS-CoV receptor, and the type II transmembrane serine protease (TTSP) member TMPRSS2, a CoV-activating protease. This CD9-facilitated condensation of receptors and proteases allowed MERS-CoV pseudoviruses to enter cells rapidly and efficiently. Without CD9, MERS-CoV viruses were not activated by TTSPs, and they trafficked into endosomes to be cleaved much later and less efficiently by cathepsins. Thus, we identified DPP4:CD9:TTSP as the protein complexes necessary for early, efficient MERS-CoV entry. To evaluate the importance of these complexes in an in vivo CoV infection model, we used recombinant Adenovirus 5 (rAd5) vectors to express human DPP4 in mouse lungs, thereby sensitizing the animals to MERS-CoV infection. When the rAd5-hDPP4 vectors co-expressed small RNAs silencing Cd9 or Tmprss2, the animals were significantly less susceptible, indicating that CD9 and TMPRSS2 facilitated robust in vivo MERS-CoV infection of mouse lungs. Furthermore, the S proteins of virulent mouse-adapted MERS-CoVs acquired a CD9-dependent cell entry character, suggesting that CD9 is a selective agent in the evolution of CoV virulence.
The airway epithelium is now considered to be central to the orchestration of pulmonary inflammatory and immune responses, and is also key to tissue remodelling. It acts as the first barrier in the ...defence against a wide range of inhaled challenges, and is critically involved in the regulation of both innate and adaptive immune responses to these challenges. Recent progress in our understanding of the developmental regulation of this tissue, the differentiation pathways, recognition of pathogens and antimicrobial responses is now exploited to help understand how epithelial cell function and dysfunction contributes to the pathogenesis of a variety of inflammatory lung diseases. Herein, advances in our knowledge of the biology of airway epithelium, as well as its role and (dys)function in asthma, chronic obstructive pulmonary fibrosis and cystic fibrosis will be discussed.
Measles virus is an aerosol-transmitted virus that affects more than 10 million children each year and accounts for approximately 120,000 deaths. Although it was long believed to replicate in the ...respiratory epithelium before disseminating, it was recently shown to infect initially macrophages and dendritic cells of the airways using signalling lymphocytic activation molecule family member 1 (SLAMF1; also called CD150) as a receptor. These cells then cross the respiratory epithelium and transport the infection to lymphatic organs where measles virus replicates vigorously. How and where the virus crosses back into the airways has remained unknown. On the basis of functional analyses of surface proteins preferentially expressed on virus-permissive human epithelial cell lines, here we identify nectin-4 (ref. 8; also called poliovirus-receptor-like-4 (PVRL4)) as a candidate host exit receptor. This adherens junction protein of the immunoglobulin superfamily interacts with the viral attachment protein with high affinity through its membrane-distal domain. Nectin-4 sustains measles virus entry and non-cytopathic lateral spread in well-differentiated primary human airway epithelial sheets infected basolaterally. It is downregulated in infected epithelial cells, including those of macaque tracheae. Although other viruses use receptors to enter hosts or transit through their epithelial barriers, we suggest that measles virus targets nectin-4 to emerge in the airways. Nectin-4 is a cellular marker of several types of cancer, which has implications for ongoing measles-virus-based clinical trials of oncolysis.
Type 1 IFNs (IFN-I) generally protect mammalian hosts from virus infections, but in some cases, IFN-I is pathogenic. Because IFN-I is protective, it is commonly used to treat virus infections for ...which no specific approved drug or vaccine is available. The Middle East respiratory syndrome-coronavirus (MERS-CoV) is such an infection, yet little is known about the role of IFN-I in this setting. Here, we show that IFN-I signaling is protective during MERS-CoV infection. Blocking IFN-I signaling resulted in delayed virus clearance, enhanced neutrophil infiltration, and impaired MERS-CoV-specific T cell responses. Notably, IFN-I administration within 1 day after infection (before virus titers peak) protected mice from lethal infection, despite a decrease in IFN-stimulated gene (ISG) and inflammatory cytokine gene expression. In contrast, delayed IFN-β treatment failed to effectively inhibit virus replication, increased infiltration and activation of monocytes, macrophages, and neutrophils in the lungs, and enhanced proinflammatory cytokine expression, resulting in fatal pneumonia in an otherwise sublethal infection. Together, these results suggest that the relative timing of the IFN-I response and maximal virus replication is key in determining outcomes, at least in infected mice. By extension, IFN-αβ or combination therapy may need to be used cautiously to treat viral infections in clinical settings.
Middle East respiratory syndrome coronavirus (MERS-CoV) infects humans from zoonotic sources and causes severe pulmonary disease. Virions require spike (S) glycoproteins for binding to cell receptors ...and for catalyzing virus–cell membrane fusion. Fusion occurs only after S proteins are cleaved sequentially, first during their secretion through the exocytic organelles of virus-producing cells, and second after virus binding to target-cell receptors. To more precisely determine how sequential proteolysis contributes to CoV infection, we introduced S mutations obstructing the first cleavages. These mutations severely compromised MERS-CoV infection into human lung-derived cells, but had little effect on infection into several other cell types. These cell type-specific requirements for proteolysis correlated with S conformations during cell entry. Without the first cleavages, S proteins resisted cell receptor-induced conformational changes, which restricted the second, fusion-activating cleavages. Consistent with these findings, precleaved MERS viruses used receptor-proximal, cell-surface proteases to effect the second fusion-activating cleavages during cell entry, whereas the more rigid uncleaved MERS viruses trafficked past these cell-surface proteases and into endosomes. Uncleaved viruses were less infectious to human airway epithelial and Calu3 cell cultures because they lacked sufficient endosomal fusion-activating proteases. Thus, by sensitizing viruses to receptor-induced conformational changes, the first S cleavages expand virus tropism to cell types that are relevant to lung infection, and therefore may be significant determinants of MERS-CoV virulence.
Dipeptidyl peptidase 4 (DPP4, CD26), a type II transmembrane ectopeptidase, is the receptor for the Middle Eastern respiratory syndrome coronavirus (MERS-CoV). MERS emerged in 2012 and has a high ...mortality associated with severe lung disease. A lack of autopsy studies from MERS fatalities has hindered understanding of MERS-CoV pathogenesis. We investigated the spatial and cellular localization of DPP4 to evaluate an association MERS clinical disease. DPP4 was rarely detected in the surface epithelium from nasal cavity to conducting airways with a slightly increased incidence in distal airways. DPP4 was also found in a subset of mononuclear leukocytes and in serous cells of submucosal glands. In the parenchyma, DPP4 was found principally in type I and II cells and alveolar macrophages and was also detected in vascular endothelium (eg, lymphatics) and pleural mesothelia. Patients with chronic lung disease, such as chronic obstructive pulmonary disease and cystic fibrosis, exhibited increased DPP4 immunostaining in alveolar epithelia (type I and II cells) and alveolar macrophages with similar trends in reactive mesothelia. This finding suggests that preexisting pulmonary disease could increase MERS-CoV receptor abundance and predispose individuals to MERS morbidity and mortality, which is consistent with current clinical observations. We speculate that the preferential spatial localization of DPP4 in alveolar regions may explain why MERS is characterized by lower respiratory tract disease.
The coronavirus disease 2019 (COVID-19) pandemic has caused significant morbidity and mortality on a global scale. The etiologic agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), ...initiates host cell entry when its spike protein (S) binds to its receptor, angiotensin-converting enzyme 2 (ACE2). In airway epithelia, the spike protein is cleaved by the cell surface protease TMPRSS2, facilitating membrane fusion and entry at the cell surface. This dependence on TMPRSS2 and related proteases suggests that protease inhibitors might limit SARS-CoV-2 infection in the respiratory tract. Here, we tested two serine protease inhibitors, camostat mesylate and nafamostat mesylate, for their ability to inhibit entry of SARS-CoV-2 and that of a second pathogenic coronavirus, Middle East respiratory syndrome coronavirus (MERS-CoV). Both camostat and nafamostat reduced infection in primary human airway epithelia and in the Calu-3 2B4 cell line, with nafamostat exhibiting greater potency. We then assessed whether nafamostat was protective against SARS-CoV-2
using two mouse models. In mice sensitized to SARS-CoV-2 infection by transduction with human
, intranasal nafamostat treatment prior to or shortly after SARS-CoV-2 infection significantly reduced weight loss and lung tissue titers. Similarly, prophylactic intranasal treatment with nafamostat reduced weight loss, viral burden, and mortality in K18-
transgenic mice. These findings establish nafamostat as a candidate for the prevention or treatment of SARS-CoV-2 infection and disease pathogenesis.
The causative agent of COVID-19, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), requires host cell surface proteases for membrane fusion and entry into airway epithelia. We tested the hypothesis that inhibitors of these proteases, the serine protease inhibitors camostat and nafamostat, block infection by SARS-CoV-2. We found that both camostat and nafamostat reduce infection in human airway epithelia, with nafamostat showing greater potency. We then asked whether nafamostat protects mice against SARS-CoV-2 infection and subsequent COVID-19 lung disease. We performed infections in mice made susceptible to SARS-CoV-2 infection by introducing the human version of ACE2, the SARS-CoV-2 receptor, into their airway epithelia. We observed that pretreating these mice with nafamostat prior to SARS-CoV-2 infection resulted in better outcomes, in the form of less virus-induced weight loss, viral replication, and mortality than that observed in the untreated control mice. These results provide preclinical evidence for the efficacy of nafamostat in treating and/or preventing COVID-19.
Abstract
Cystic fibrosis (CF) is a multiorgan recessive genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Gene therapy efforts have focused ...on treating the lung, since it manifests the most significant life-threatening disease. Over two decades have past since the first CF lung gene therapy trials and significant advances in the therapeutic implementation of pharmacologic CFTR modulators have renewed the field's focus on developing gene therapies for the 10% of CF patients these modulators cannot help. This review summarizes recent progress made in developing vectors for airway transduction and CF animal models required for understanding the relevant cellular targets in the lung and testing the efficacy of gene therapy approaches. We also highlight future opportunities in emerging gene editing strategies that may offer advantages for treating diseases like CF where the gene target is highly regulated at the cellular level. The outcomes of CF lung gene therapy trials will likely inform productive paths toward gene therapy for other complex genetic disorders, while also advancing treatments for all CF patients.
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