Chronic rhinosinusitis engenders enormous morbidity in the general population, and is often refractory to medical intervention. Compounds that augment mucociliary clearance in airway epithelia ...represent a novel treatment strategy for diseases of mucus stasis. A dominant fluid and electrolyte secretory pathway in the nasal airways is governed by the cystic fibrosis transmembrane conductance regulator (CFTR). The objectives of the present study were to test resveratrol, a strong potentiator of CFTR channel open probability, in preparation for a clinical trial of mucociliary activators in human sinus disease.
Primary sinonasal epithelial cells, immortalized bronchoepithelial cells (wild type and F508del CFTR), and HEK293 cells expressing exogenous human CFTR were investigated by Ussing chamber as well as patch clamp technique under non-phosphorylating conditions. Effects on airway surface liquid depth were measured using confocal laser scanning microscopy. Impact on CFTR gene expression was measured by quantitative reverse transcriptase polymerase chain reaction.
Resveratrol is a robust CFTR channel potentiator in numerous mammalian species. The compound also activated temperature corrected F508del CFTR and enhanced CFTR-dependent chloride secretion in human sinus epithelium ex vivo to an extent comparable to the recently approved CFTR potentiator, ivacaftor. Using inside out patches from apical membranes of murine cells, resveratrol stimulated an ~8 picosiemens chloride channel consistent with CFTR. This observation was confirmed in HEK293 cells expressing exogenous CFTR. Treatment of sinonasal epithelium resulted in a significant increase in airway surface liquid depth (in µm: 8.08+/-1.68 vs. 6.11+/-0.47,control,p<0.05). There was no increase CFTR mRNA.
Resveratrol is a potent chloride secretagogue from the mucosal surface of sinonasal epithelium, and hydrates airway surface liquid by increasing CFTR channel open probability. The foundation for a clinical trial utilizing resveratrol as a therapeutic intervention to increase mucociliary transport and airway surface liquid hydration in sinus disease is strongly supported by these findings.
The most common cystic fibrosis (CF) causing mutation, deletion of phenylalanine 508 (ΔF508 or Phe508del), results in functional expression defect of the CF transmembrane conductance regulator (CFTR) ...at the apical plasma membrane (PM) of secretory epithelia, which is attributed to the degradation of the misfolded channel at the endoplasmic reticulum (ER). Deletion of phenylalanine 670 (ΔF670) in the yeast oligomycin resistance 1 gene (YOR1, an ABC transporter) of Saccharomyces cerevisiae phenocopies the ΔF508-CFTR folding and trafficking defects. Genome-wide phenotypic (phenomic) analysis of the Yor1-ΔF670 biogenesis identified several modifier genes of mRNA processing and translation, which conferred oligomycin resistance to yeast. Silencing of orthologues of these candidate genes enhanced the ΔF508-CFTR functional expression at the apical PM in human CF bronchial epithelia. Although knockdown of RPL12, a component of the ribosomal stalk, attenuated the translational elongation rate, it increased the folding efficiency as well as the conformational stability of the ΔF508-CFTR, manifesting in 3-fold augmented PM density and function of the mutant. Combination of RPL12 knockdown with the corrector drug, VX-809 (lumacaftor) restored the mutant function to ~50% of the wild-type channel in primary CFTRΔF508/ΔF508 human bronchial epithelia. These results and the observation that silencing of other ribosomal stalk proteins partially rescue the loss-of-function phenotype of ΔF508-CFTR suggest that the ribosomal stalk modulates the folding efficiency of the mutant and is a potential therapeutic target for correction of the ΔF508-CFTR folding defect.
Lipid nanoparticles (LNPs) are a clinically relevant way to deliver therapeutic mRNA to hepatocytes in patients. However, LNP-mRNA delivery to end-stage solid tumors such as head and neck squamous ...cell carcinoma (HNSCC) remains more challenging. While scientists have used in vitro assays to evaluate potential nanoparticles for HNSCC delivery, high-throughput delivery assays performed directly in vivo have not been reported. Here we use a high-throughput LNP assay to evaluate how 94 chemically distinct nanoparticles delivered nucleic acids to HNSCC solid tumors in vivo. DNA barcodes were used to identify LNPHNSCC, a novel LNP for systemic delivery to HNSCC solid tumors. Importantly, LNPHNSCC retains tropism to HNSCC solid tumors while minimizing off-target delivery to the liver.
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Cystic Fibrosis (CF) is caused by a diverse set of mutations distributed across the approximately 250 thousand base pairs of the
gene locus, of which at least 382 are disease-causing (CFTR2.org). ...Although a variety of editing tools are now available for correction of individual mutations, a strong justification can be made for a more universal gene insertion approach, in principle capable of correcting virtually all
mutations. Provided that such a methodology is capable of efficiently correcting relevant stem cells of the airway epithelium, this could potentially provide life-long correction for the lung. In this Perspective we highlight several requirements for efficient gene insertion into airway epithelial stem cells. In addition, we focus on specific features of the transgene construct and the endogenous
locus that influence whether the inserted gene sequences will give rise to robust and physiologically relevant levels of CFTR function in airway epithelium. Finally, we consider how
gene insertion methodologies may be adapted for direct
editing.
Formation of a novel structure, the aggresome, has been proposed to represent a general cellular response to the presence of misfolded proteins (Johnston, J.A., C.L. Ward, and R.R. Kopito. 1998. J. ...Cell Biol. 143:1883-1898; Wigley, W.C., R.P. Fabunmi, M.G. Lee, C.R. Marino, S. Muallem, G.N. DeMartino, and P.J. Thomas. 1999. J. Cell Biol. 145:481-490). To test the generality of this finding and characterize aspects of aggresome composition and its formation, we investigated the effects of overexpressing a cytosolic protein chimera (GFP-250) in cells. Overexpression of GFP-250 caused formation of aggresomes and was paralleled by the redistribution of the intermediate filament protein vimentin as well as by the recruitment of the proteasome, and the Hsp70 and the chaperonin systems of chaperones. Interestingly, GFP-250 within the aggresome appeared not to be ubiquitinated. In vivo time-lapse analysis of aggresome dynamics showed that small aggregates form within the periphery of the cell and travel on microtubules to the MTOC region where they remain as distinct but closely apposed particulate structures. Overexpression of p50/dynamitin, which causes the dissociation of the dynactin complex, significantly inhibited the formation of aggresomes, suggesting that the minus-end-directed motor activities of cytoplasmic dynein are required for aggresome formation. Perinuclear aggresomes interfered with correct Golgi localization and disrupted the normal astral distribution of microtubules. However, ER-to-Golgi protein transport occurred normally in aggresome containing cells. Our results suggest that aggresomes can be formed by soluble, nonubiquitinated proteins as well as by integral transmembrane ubiquitinated ones, supporting the hypothesis that aggresome formation might be a general cellular response to the presence of misfolded proteins.
Small molecules that correct the folding defects and enhance surface localization of the F508del mutation in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) comprise an important ...therapeutic strategy for cystic fibrosis lung disease. However, compounds that rescue the F508del mutant protein to wild type (WT) levels have not been identified. In this report, we consider obstacles to obtaining robust and therapeutically relevant levels of F508del CFTR. For example, markedly diminished steady state amounts of F508del CFTR compared to WT CFTR are present in recombinant bronchial epithelial cell lines, even when much higher levels of mutant transcript are present. In human primary airway cells, the paucity of Band B F508del is even more pronounced, although F508del and WT mRNA concentrations are comparable. Therefore, to augment levels of "repairable" F508del CFTR and identify small molecules that then correct this pool, we developed compound library screening protocols based on automated protein detection. First, cell-based imaging measurements were used to semi-quantitatively estimate distribution of F508del CFTR by high content analysis of two-dimensional images. We evaluated ~2,000 known bioactive compounds from the NIH Roadmap Molecular Libraries Small Molecule Repository in a pilot screen and identified agents that increase the F508del protein pool. Second, we analyzed ~10,000 compounds representing diverse chemical scaffolds for effects on total CFTR expression using a multi-plate fluorescence protocol and describe compounds that promote F508del maturation. Together, our findings demonstrate proof of principle that agents identified in this fashion can augment the level of endoplasmic reticulum (ER) resident "Band B" F508del CFTR suitable for pharmacologic correction. As further evidence in support of this strategy, PYR-41-a compound that inhibits the E1 ubiquitin activating enzyme-was shown to synergistically enhance F508del rescue by C18, a small molecule corrector. Our combined results indicate that increasing the levels of ER-localized CFTR available for repair provides a novel route to correct F508del CFTR.
Objectives/Hypothesis:
Although the pathophysiology leading to rhinosinusitis is complex, evidence indicates that decreased mucociliary clearance (MCC) is a major contributing feature. Normal ...respiratory epithelial MCC is an important host defense mechanism that is dependent on proper ciliary beating and the biological properties of the airway surface liquid (ASL). The role that tobacco smoke exposure plays as an inhibitor of MCC has yet to be elucidated. The present study investigates the consequences of cigarette smoke exposure on ciliary function and transepithelial chloride (Cl−) secretion, a major determinant of ASL.
Study Design:
In vitro investigation.
Methods:
Well‐characterized primary murine nasal septal epithelial (MNSE) and human sinonasal epithelial (HSNE) cultures were exposed to cigarette smoke condensate (CSC) and compared to control cultures. Effects on ciliary beat frequency (CBF) and Cl− secretion were investigated using pharmacologic manipulation.
Results:
Change in forskolin‐stimulated current (ΔISC), representing transepithelial Cl− secretion, was significantly decreased in CSC exposed MNSE (14.97 ± 1.2 μA/cm2 vs. control, 19.1 ± 1.56 μA/cm2 P = .04) and HSNE (2.68 ± 0.79 μA/cm2 vs. control, 10.8 ± 1.73 μA/cm2) cultures (P = .001). Forskolin‐stimulated CBF was also significantly reduced when acutely exposed to CSC (5.64 ± 0.06 Hz vs. control 7.15 ± 0.18 Hz).
Conclusions:
The present study provides direct evidence that tobacco smoke diminishes two major components of MCC. This links tobacco smoke as a potential contributing and/or exacerbating factor in exposed individuals with chronic rhinosinusitis. Laryngoscope, 2009
VX-770 (ivacaftor) is approved for clinical use in CF patients bearing multiple CFTR mutations. VX-770 potentiated wildtype CFTR and several disease mutants expressed in oocytes in a manner modulated ...by PKA-mediated phosphorylation. Potentiation of some other mutants, including G551D-CFTR, was less dependent upon the level of phosphorylation, likely related to the severe gating defects in these mutants exhibited in part by a shift in PKA sensitivity to activation, possibly due to an electrostatic interaction of D551 with K1250. Phosphorylation-dependent potentiation of wildtype CFTR and other variants also was observed in epithelial cells. Hence, the efficacy of potentiators may be obscured by a ceiling effect when drug screening is performed under strongly phosphorylating conditions. These results should be considered in campaigns for CFTR potentiator discovery, and may enable the expansion of VX-770 to CF patients bearing ultra-orphan CFTR mutations.
Epistasis refers to the dependence of a mutation on other mutation(s) and the genetic context in general. In the context of human disorders, epistasis complicates the spectrum of disease symptoms and ...has been proposed as a major contributor to variations in disease outcome. The nonadditive relationship between mutations and the lack of complete understanding of the underlying physiological effects limit our ability to predict phenotypic outcome. Here, we report positive epistasis between intragenic mutations in the cystic fibrosis transmembrane conductance regulator (CFTR)-the gene responsible for cystic fibrosis (CF) pathology. We identified a synonymous single-nucleotide polymorphism (sSNP) that is invariant for the CFTR amino acid sequence but inverts translation speed at the affected codon. This sSNP in
exhibits positive epistatic effects on some CF disease-causing missense mutations. Individually, both mutations alter CFTR structure and function, yet when combined, they lead to enhanced protein expression and activity. The most robust effect was observed when the sSNP was present in combination with missense mutations that, along with the primary amino acid change, also alter the speed of translation at the affected codon. Functional studies revealed that synergistic alteration in ribosomal velocity is the underlying mechanism; alteration of translation speed likely increases the time window for establishing crucial domain-domain interactions that are otherwise perturbed by each individual mutation.
Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR), with approximately 90% of patients harboring at least one copy of the disease-associated variant ...F508del. We utilized a yeast phenomic system to identify genetic modifiers of F508del-CFTR biogenesis, from which ribosomal protein L12 (RPL12/uL11) emerged as a molecular target. In the present study, we investigated mechanism(s) by which suppression of RPL12 rescues F508del protein synthesis and activity. Using ribosome profiling, we found that rates of translation initiation and elongation were markedly slowed by RPL12 silencing. However, proteolytic stability and patch-clamp assays revealed RPL12 depletion significantly increased F508del-CFTR steady-state expression, interdomain assembly, and baseline open-channel probability. We next evaluated whether Rpl12-corrected F508del-CFTR could be further enhanced with concomitant pharmacologic repair (e.g., using clinically approved modulators lumacaftor and tezacaftor) and demonstrated additivity of these treatments. Rpl12 knockdown also partially restored maturation of specific CFTR variants in addition to F508del, and WT Cftr biogenesis was enhanced in the pancreas, colon, and ileum of Rpl12 haplosufficient mice. Modulation of ribosome velocity therefore represents a robust method for understanding both CF pathogenesis and therapeutic response.
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