In the clinically successful era of CFTR modulators and Theratyping, 10–20% of individuals with cystic fibrosis (CF) may develop disease due to CFTR mutations that remain undruggable. These ...individuals produce low levels of CFTR mRNA and/or not enough protein to be rescued with modulator drugs. Alternative therapeutic approaches to correct the CFTR defect at the mRNA level using nucleic acid technologies are currently feasible; e.g., oligonucleotides platforms, which are being rapidly developed to correct genetic disorders. Drug-like properties, great specificity, and predictable off-target effects by design make oligonucleotides a valuable approach with fewer clinical and ethical challenges than genomic editing strategies. Together with personalized and precision medicine approaches, oligonucleotides are ideal therapeutics to target CF-causing mutations that affect only a few individuals resilient to modulator therapies.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Modulation of airway surface liquid (ASL) pH has been proposed as a therapy for cystic fibrosis (CF). However, evidence that ASL pH is reduced in CF is limited and conflicting. The technical ...challenges associated with measuring ASL pH in vivo have precluded accurate measurements in humans. In order to address this deficiency, ASL pH was measured in vivo in children using a novel luminescent technology integrated with fibre-optic probes. Here we show that ASL pH in children with CF is similar to that of children without CF. Findings were supported by highly controlled direct pH measurements in primary human airway epithelial cell culture models, which also suggest that the potential ASL pH gradient produced by defective apical ion transport is balanced out by paracellular shunting of acid/base. Thus, reduced baseline ASL pH is unlikely to be an important pathobiological factor in early CF lung disease.
ATP released from airway epithelial cells promotes purinergic receptor-regulated mucociliary clearance activities necessary for innate lung defense. Cell swelling-induced membrane stretch/strain is a ...common stimulus that promotes airway epithelial ATP release, but the mechanisms transducing cell swelling into ATP release are incompletely understood. Using knockdown and knockout approaches, we tested the hypothesis that pannexin 1 mediates ATP release from hypotonically swollen airway epithelia and investigated mechanisms regulating this activity. Well differentiated primary cultures of human bronchial epithelial cells subjected to hypotonic challenge exhibited enhanced ATP release, which was paralleled by the uptake of the pannexin probe propidium iodide. Both responses were reduced by pannexin 1 inhibitors and by knocking down pannexin 1. Importantly, hypotonicity-evoked ATP release from freshly excised tracheas and dye uptake in primary tracheal epithelial cells were impaired in pannexin 1 knockout mice. Hypotonicity-promoted ATP release and dye uptake in primary well differentiated human bronchial epithelial cells was accompanied by RhoA activation and myosin light chain phosphorylation and was reduced by the RhoA dominant negative mutant RhoA(T19N) and Rho and myosin light chain kinase inhibitors. ATP release and Rho activation were reduced by highly selective inhibitors of transient receptor potential vanilloid 4 (TRPV4). Lastly, knocking down TRPV4 impaired hypotonicity-evoked airway epithelial ATP release. Our data suggest that TRPV4 and Rho transduce cell membrane stretch/strain into pannexin 1-mediated ATP release in airway epithelia.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
ABSTRACT
Cigarette smoke (CS) exposure induces mucus obstruction and the development of chronic bronchitis (CB). While many of these responses are determined genetically, little is known about the ...effects CS can exert on pulmonary epithelia at the protein level. We, therefore, tested the hypothesis that CS exerts direct effects on the CFTR protein, which could impair airway hydration, leading to the mucus stasis characteristic of both cystic fibrosis and CB. In vivo and in vitro studies demonstrated that CS rapidly decreased CFTR activity, leading to airway surface liquid (ASL) volume depletion (i.e., dehydration). Further studies revealed that CS induced internalization of CFTR. Surprisingly, CS‐internalized CFTR did not colocalize with lysosomal proteins. Instead, the bulk of CFTR shifted to a detergent‐resistant fraction within the cell and colocalized with the intermediate filament vimentin, suggesting that CS induced CFTR movement into an aggresome‐like, perinuclear compartment. To test whether airway dehydration could be reversed, we used hypertonic saline (HS) as an osmolyte to rehydrate ASL. HS restored ASL height in CS‐exposed, dehydrated airway cultures. Similarly, inhaled HS restored mucus transport and increased clearance in patients with CB. Thus, we propose that CS exposure rapidly impairs CFTR function by internalizing CFTR, leading to ASL dehydration, which promotes mucus stasis and a failure of mucus clearance, leaving smokers at risk for developing CB. Furthermore, our data suggest that strategies to rehydrate airway surfaces may provide a novel form of therapy for patients with CB.—Clunes, L. A., Davies, C. M., Coakley, R. D., Aleksandrov, A. A., Henderson, A. G., Zeman, K. L., Worthington, E. N., Gentzsch, M., Kreda, S. M., Cholon, D., Bennett, W. D., Riordan, J. R., Boucher, R. C., Tarran, R. Cigarette smoke exposure induces CFTR internalization and insolubility, leading to airway surface liquid dehydration. FASEB J. 26, 533–545 (2012). www.fasebj.org
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Abstract
Pulmonary diseases offer many targets for oligonucleotide therapeutics. However, effective delivery of oligonucleotides to the lung is challenging. For example, splicing mutations in the ...cystic fibrosis transmembrane conductance regulator (CFTR) affect a significant cohort of Cystic Fibrosis (CF) patients. These individuals could potentially benefit from treatment with splice switching oligonucleotides (SSOs) that can modulate splicing of CFTR and restore its activity. However, previous studies in cell culture used oligonucleotide transfection methods that cannot be safely translated in vivo. In this report, we demonstrate effective correction of a splicing mutation in the lung of a mouse model using SSOs. Moreover, we also demonstrate effective correction of a CFTR splicing mutation in a pre-clinical CF patient-derived cell model. We utilized a highly effective delivery strategy for oligonucleotides by combining peptide-morpholino (PPMO) SSOs with small molecules termed OECs. PPMOs distribute broadly into the lung and other tissues while OECs potentiate the effects of oligonucleotides by releasing them from endosomal entrapment. The combined PPMO plus OEC approach proved to be effective both in CF patient cells and in vivo in the mouse lung and thus may offer a path to the development of novel therapeutics for splicing mutations in CF and other lung diseases.
Mucus pathology in cystic fibrosis (CF) has been known for as long as the disease has been recognized and is sometimes called mucoviscidosis. The disease is marked by mucus hyperproduction and ...plugging in many organs, which are usually most fatal in the airways of CF patients, once the problem of meconium ileus at birth is resolved. After the CF gene, CFTR, was cloned and its protein product identified as a cAMP-regulated Cl(-) channel, causal mechanisms underlying the strong mucus phenotype of the disease became obscure. Here we focus on mucin genes and polymeric mucin glycoproteins, examining their regulation and potential relationships to a dysfunctional cystic fibrosis transmembrane conductance regulator (CFTR). Detailed examination of CFTR expression in organs and different cell types indicates that changes in CFTR expression do not always correlate with the severity of CF disease or mucus accumulation. Thus, the mucus hyperproduction that typifies CF does not appear to be a direct cause of a defective CFTR but, rather, to be a downstream consequence. In organs like the lung, up-regulation of mucin gene expression by inflammation results from chronic infection; however, in other instances and organs, the inflammation may have a non-infectious origin. The mucus plugging phenotype of the β-subunit of the epithelial Na(+) channel (βENaC)-overexpressing mouse is proving to be an archetypal example of this kind of inflammation, with a dehydrated airway surface/concentrated mucus gel apparently providing the inflammatory stimulus. Data indicate that the luminal HCO(3)(-) deficiency recently described for CF epithelia may also provide such a stimulus, perhaps by causing a mal-maturation of mucins as they are released onto luminal surfaces. In any event, the path between CFTR dysfunction and mucus hyperproduction has proven tortuous, and its unraveling continues to offer its own twists and turns, along with fascinating glimpses into biology.
Given the widespread importance of purinergic receptor-evoked signaling, understanding how ATP and other nucleotides are released from cells in a regulated manner is an essential physiological ...question. Nonlytic release of ATP, UTP, UDP-glucose, and other nucleotides occurs in all cell types and tissues via both constitutive mechanisms, that is, in the absence of external stimuli, and to a greater extent in response to biochemical or mechanical/physical stimuli. However, a molecular understanding of the processes regulating nucleotide release has only recently begun to emerge. It is generally accepted that nucleotide release occurs in two different scenarios, exocytotic release from the secretory pathway or via conductive/transport mechanisms, and a critical review of our current understanding of these mechanisms is presented in this chapter.
Addition of small molecule Retro-1 has been described to enhance antisense and splice switching oligonucleotides. With the aim of assessing the effect of covalently linking Retro-1 to the ...biologically active oligonucleotide, three different derivatives of Retro-1 were prepared that incorporated a phosphoramidite group, a thiol or a 1,3-diene, respectively. Retro-1⁻oligonucleotide conjugates were assembled both on-resin (coupling of the phosphoramidite) and from reactions in solution (Michael-type thiol-maleimide reaction and Diels-Alder cycloaddition). Splice switching assays with the resulting conjugates showed that they were active but that they provided little advantage over the unconjugated oligonucleotide in the well-known HeLa Luc705 reporter system.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Extracellular ATP and its metabolite adenosine regulate mucociliary clearance in airway epithelia. Little has been known, however, regarding the actual ATP and adenosine concentrations in the thin ...(∼7 μm) liquid layer lining native airway surfaces and the link between ATP release/metabolism and autocrine/paracrine regulation of epithelial function. In this study, chimeric Staphylococcus aureus protein A-luciferase (SPA-luc) was bound to endogenous antigens on primary human bronchial epithelial (HBE) cell surface and ATP concentrations assessed in real-time in the thin airway surface liquid (ASL). ATP concentrations on resting cells were 1-10 nm. Inhibition of ecto-nucleotidases resulted in ATP accumulation at a rate of ∼250 fmol/min/cm2, reflecting the basal ATP release rate. Following hypotonic challenge to promote cell swelling, cell-surface ATP concentration measured by SPA-luc transiently reached ∼1 μm independent of ASL volume, reflecting a transient 3-log increase in ATP release rates. In contrast, peak ATP concentrations measured in bulk ASL by soluble luciferase inversely correlated with volume. ATP release rates were intracellular calcium-independent, suggesting that non-exocytotic ATP release from ciliated cells, which dominate our cultures, mediated hypotonicity-induced nucleotide release. However, the cystic fibrosis transmembrane conductance regulator (CFTR) did not participate in this function. Following the acute swelling phase, HBE cells exhibited regulatory volume decrease which was impaired by apyrase and facilitated by ATP or UTP. Our data provide the first evidence that ATP concentrations at the airway epithelial surface reach the range for P2Y2 receptor activation by physiological stimuli and identify a role for mucosal ATP release in airway epithelial cell volume regulation.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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