scholarly journals 2058 miRNA manipulation to improve CFTR correction in cystic fibrosis

2018 ◽  
Vol 2 (S1) ◽  
pp. 20-20
Author(s):  
William Thomas Harris ◽  
Farruk Kabir
Keyword(s):  
Cystic Fibrosis ◽  
Binding Site ◽  
Adenoviral Vector ◽  
Short Circuit ◽  
Protein Translation ◽  
Strong Increase ◽  
Post Inoculation ◽  
Tgf Beta ◽  
Tracheal Explants

OBJECTIVES/SPECIFIC AIMS: CFTR is the mutant protein that causes cystic fibrosis (CF), a fatal respiratory diseases affecting 1 in 3500 children. CFTR modulators are small molecules that directly address mutant CFTR function. Improving correction of the F508del CFTR mutation (affecting 90% of CF patients) is one of the most pressing unmet needs in CF. Currently available F508del therapeutics only marginally improve CF, In vitro, we have identified a miRNA that impairs utility of CFTR directed therapies. miR-145 is upregulated by TGF-β (a genetic modifier of CF lung disease) with a direct binding site on the 3’-untranslated region of CFTR mRNA. Binding of miR-145 to CFTR destabilizes mRNA transcript and impedes protein translation. Overexpression of miR-145 abolishes benefit of F508del CFTR correction. Antagonists to miR-145 block TGF-β suppression of CFTR function and augment response to CFTR correction. This project evaluate in vivo impact of TGF-beta and miRNA manipulation on CFTR functional readouts including nasal potential difference (NPD) and short circuit current (Isc) across tracheal explants in addition to standard biochemical measures. METHODS/STUDY POPULATION: Wild-type Sprague-Dawley rats were inoculated with an adenoviral vector containing bioactive TGF-beta or sham at 1×109 pfu/animal placed in the left nares. Seven days post-inoculation, functional, and biochemical measures were conducted. NPD was measured with a microelectrode placed in the left nare and grounded the tail. The nare was sequentially perfused with standard Ringer’s solution, amiloride (to block the ENaC sodium channel), low chloride Ringer’s (to stimulate chloride efflux), forskolin (to open the CFTR channel) and CFTRinh-172 (to block the CFTR channel. Tracheal explants were harvested, microdissected, and placed on modified Ussing chambers. RESULTS/ANTICIPATED RESULTS: We have inoculated WT rats with bioactive TGF-β Versus sham delivered by intranasal inoculation of an adenoviral vector. Functional readout of CFTR function is by Isc across tracheal epithelia and NPD. Lung homogenates are analyzed for TGF-β signaling, miRNA expression, and CFTR transcripts. Both tracheal explants and NPD indicate TGF-β stimulation diminishes CFTR function in vivo. In tracheal explants, TGF-β exposure diminishes CFTR response to forskolin-stimulation by 75%. Loss of current after CFTR inhibition (CFTRinh-172) is halved. By nasal PD, TGF-β inoculation similarly halves the bioelectric response to low chloride and forskolin stimulation. Evaluation by qPCR reveals a strong increase in TGF-β signaling demarcated by PAI-1, prompting a reduction in CFTR mRNA. miR-145 is expressed highly in rat pulmonary tissue, but no change in overall miR-145 levels was detected between TGF-β and sham exposed rats. This finding reflects what we have observed in human lungs, with a localized increased miR-145 expression in CF epithelia, but similarly high levels of miR-145 in both CF and non-CF whole lung homogenates. Although expressed at lower levels than miR-145, we did find increased expression in TGF-β relevant miR-101, miR-494, and miR-144 that have a predicted binding site on rat 3’-UTR in TGF-β exposed Versus sham lungs. DISCUSSION/SIGNIFICANCE OF IMPACT: Our data indicate the relevance of TGF-β stimulation to suppress CFTR synthesis and function in vivo. Future work will evaluate whether these additional miRNA with CFTR binding sites may mediate TGF-β suppression of CFTR in the rat model, and the utility of miRNA manipulation to augment F508del CFTR correction.

scholarly journals Decreased electrogenic anionic secretory response in the porcine colon following in vivo challenge withBrachyspiraspp. supports an altered mucin environment

2019 ◽  
Vol 316 (4) ◽  
pp. G495-G508 ◽  
Author(s):  
Cole B. Enns ◽  
John C. S. Harding ◽  
Matthew E. Loewen
Keyword(s):  
Cystic Fibrosis ◽  
Mrna Expression ◽  
Rheological Properties ◽  
Diarrheal Disease ◽  
Short Circuit ◽  
Anion Channel ◽  
Secretory Response ◽  
Secretory Function ◽  
Ussing Chambers

Brachyspira spp. cause diarrheal disease in multiple animal species by colonization of the colon, resulting in colitis, mucus induction, and disrupted ion transport. Unique to spirochete pathogenesis is the immense production of mucus, resulting in a niche mucin environment likely favoring spirochete colonization. Mucin rheological properties are heavily influenced by anionic secretion, and loss of secretory function has been implicated in diseases such as cystic fibrosis. Here, the effects on the agonist-induced electrogenic anionic secretory response by infectious colonic spirochete bacteria Brachyspira hyodysenteriae and Brachyspira hampsonii were assessed in the proximal, apex, and distal sections of colon in Ussing chambers. Activation of secretion via isoproterenol, carbachol, and forskolin/3-isobutyl-1-methylxanthine demonstrated a significantly decreased change in short-circuit current ( Isc) in Brachyspira-infected pigs in all sections. Tissue resistances did not account for this difference, rather, it was attributed to a decrease in anionic secretion as indicated by a decrease in bumetanide inhibitable Isc. Quantitative RT-PCR and Western blot analyses determined that the major anionic channels of the epithelium were downregulated in diarrheic pigs paired with altered mucin gene expression. The investigated cytokines were not responsible for the downregulation of anion channel gene transcripts. Although IL-1α was upregulated in all segments, it did not alter cystic fibrosis transmembrane conductance regulator (CFTR) mRNA expression in Caco-2 monolayers. However, a whole cell Brachyspira hampsonii lysate significantly reduced CFTR mRNA expression in Caco-2 monolayers. Together, these findings indicate that these two Brachyspira spp. may directly cause a decreased anionic secretory response in the porcine colon, supporting an altered mucin environment likely favoring spirochete colonization.NEW & NOTEWORTHY This research demonstrates for the first time that the niche mucin environment produced by two infectious spirochete spp. is supported by a decrease in the electrogenic anionic secretory response throughout the porcine colon. Our findings suggest that the host’s cytokine response is not likely responsible for the decrease in anionic secretory function. Rather, it appears that Brachyspira spp. directly impede ion channel transcription and translation, potentially altering colonic mucin rheological properties, which may favor spirochete colonization.

scholarly journals A Novel G542X CFTR Rat Model of Cystic Fibrosis Is Sensitive to Nonsense Mediated Decay

2020 ◽  
Vol 11 ◽  
Author(s):  
Jyoti Sharma ◽  
Joseph Abbott ◽  
Lauren Klaskala ◽  
Guojun Zhao ◽  
Susan E. Birket ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Rat Model ◽  
Tooth Enamel ◽  
Short Circuit ◽  
Functional Restoration ◽  
Mrna Levels ◽  
Nonsense Mutations ◽  
Nonsense Mediated Decay ◽  
Rat Models

Nonsense mutations that lead to the insertion of a premature termination codon (PTC) in the cystic fibrosis transmembrane conductance regulator (CFTR) transcript affect 11% of patients with cystic fibrosis (CF) worldwide and are associated with severe disease phenotype. While CF rat models have contributed significantly to our understanding of CF disease pathogenesis, there are currently no rat models available for studying CF nonsense mutations. Here we created and characterized the first homozygous CF rat model that bears the CFTR G542X nonsense mutation in the endogenous locus using CRISPR/Cas9 gene editing. In addition to displaying severe CF manifestations and developmental defects such as reduced growth, abnormal tooth enamel, and intestinal obstruction, CFTR G542X knockin rats demonstrated an absence of CFTR function in tracheal and intestinal sections as assessed by nasal potential difference and transepithelial short-circuit current measurements. Reduced CFTR mRNA levels in the model further suggested sensitivity to nonsense-mediated decay, a pathway elicited by the presence of PTCs that degrades the PTC-bearing transcripts and thus further diminishes the level of CFTR protein. Although functional restoration of CFTR was observed in G542X rat tracheal epithelial cells in response to single readthrough agent therapy, therapeutic efficacy was not observed in G542X knockin rats in vivo. The G542X rat model provides an invaluable tool for the identification and in vivo validation of potential therapies for CFTR nonsense mutations.

scholarly journals The Ubiquitin Ligase Nedd4L Regulates the Na/K/2Cl Co-transporter NKCC1/SLC12A2 in the Colon

2017 ◽  
Vol 292 (8) ◽  
pp. 3137-3145 ◽  
Author(s):  
Chong Jiang ◽  
Hiroshi Kawabe ◽  
Daniela Rotin
Keyword(s):  
Ubiquitin Ligase ◽  
Fluid Transport ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Distal Colon ◽  
Strong Increase ◽  
Colon Tissue ◽  
Knock Out ◽  
Novel Target

The ubiquitin ligase Nedd4-like (Nedd4L, or Nedd4-2) binds to and regulates stability of the epithelial Na+ channel (ENaC) in salt-absorbing epithelia in the kidney, lung, and other tissues. Its role in the distal colon, which also absorbs salt and fluid and expresses ENaC, is unknown. Using a conditional knock-out approach to knock out Nedd4L in mice intestinal epithelium (Nedd4Lf/f;Vil-CreERT2) we show here that Nedd4L depletion leads to a higher steady-state short circuit current (Isc) in mouse distal colon tissue relative to controls. This higher Isc was partially reduced by the addition of apical amiloride and strongly reduced by basolateral bumetanide as well as by depletion of basolateral Cl−, suggesting that Na+/K+/2Cl− (NKCC1/SLC12A2) co-transporter and ENaC are targets of Nedd4L in the colon. In accordance, NKCC1 (and γENaC) protein abundance in the colon of the Nedd4L knock-out animals was increased, indicating that Nedd4L normally suppresses these proteins. However, we did not observe co-immunoprecipitation between Nedd4L and NKCC1, suggesting that Nedd4L indirectly suppresses NKCC1 expression. Low salt diet resulted in a strong increase in β and γ (but not α) ENaC mRNA and protein expression and ENaC activity. Although salt restriction also increased NKCC1 protein and mRNA abundance, it did not lead to its elevated activity (Isc). These results identify NKCC1 as a novel target for Nedd4L-mediated down-regulation in vivo, which modulates ion and fluid transport in the distal colon together with ENaC.

Bioelectric properties of human cystic fibrosis and non-cystic fibrosis fetal tracheal xenografts in SCID mice

1998 ◽  
Vol 274 (4) ◽  
pp. C875-C882 ◽  
Author(s):  
Rabindra Tirouvanziam ◽  
Mama Desternes ◽  
Anouar Saari ◽  
Edith Puchelle ◽  
Bruno Péault ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Short Circuit ◽  
Severe Combined Immunodeficiency ◽  
Short Circuit Current ◽  
Transport Processes ◽  
Scid Mice ◽  
Tracheal Mucosa ◽  
Induced Changes

We measured the bioelectric properties of 14 cystic fibrosis (CF) and 33 non-CF human fetal tracheal xenografts in severe combined immunodeficiency (SCID) mice. All xenografts exhibited a mature airway-type epithelium irrespective of their gestational age, duration of engraftment, and genotype. The in vivo potential difference and the in vitro baseline short-circuit current ( I sc) were significantly higher in non-CF than in CF xenografts. In non-CF xenografts, sequential addition of amiloride, forskolin, and ATP resulted in a 39.4% decrease, a 24.1% increase, and a 43.6% increase in I sc, respectively. In CF xenografts, forskolin had no significant effect on I sc, whereas amiloride- and ATP-induced changes in I sc were proportionally higher than in non-CF xenografts (−60.0 and +68.8%, respectively). These results indicate that the bioelectric properties of non-CF xenografts are similar to those of postnatal airways and that CF xenografts exhibit lower baseline electrogenic activity than non-CF xenografts but similar regulation of ion transport processes to postnatal CF airways. This model of mature human fetal tracheal mucosa may help gain insight into early CF airway pathogenesis.

scholarly journals Effective silencing of ENaC by siRNA delivered with epithelial-targeted nanocomplexes in human cystic fibrosis cells and in mouse lung

Thorax ◽  
2018 ◽  
Vol 73 (9) ◽  
pp. 847-856 ◽  
Author(s):  
Aristides D Tagalakis ◽  
Mustafa M Munye ◽  
Rositsa Ivanova ◽  
Hanpeng Chen ◽  
Claire M Smith ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Fluid Transport ◽  
Transfection Efficiency ◽  
Short Circuit ◽  
Beat Frequency ◽  
Short Circuit Current ◽  
Mouse Lung

IntroductionLoss of the cystic fibrosis transmembrane conductance regulator in cystic fibrosis (CF) leads to hyperabsorption of sodium and fluid from the airway due to upregulation of the epithelial sodium channel (ENaC). Thickened mucus and depleted airway surface liquid (ASL) then lead to impaired mucociliary clearance. ENaC regulation is thus a promising target for CF therapy. Our aim was to develop siRNA nanocomplexes that mediate effective silencing of airway epithelial ENaC in vitro and in vivo with functional correction of epithelial ion and fluid transport.MethodsWe investigated translocation of nanocomplexes through mucus and their transfection efficiency in primary CF epithelial cells grown at air–liquid interface (ALI).Short interfering RNA (SiRNA)-mediated silencing was examined by quantitative RT-PCR and western analysis of ENaC. Transepithelial potential (Vt), short circuit current (Isc), ASL depth and ciliary beat frequency (CBF) were measured for functional analysis. Inflammation was analysed by histological analysis of normal mouse lung tissue sections.ResultsNanocomplexes translocated more rapidly than siRNA alone through mucus. Transfections of primary CF epithelial cells with nanocomplexes targeting αENaC siRNA, reduced αENaC and βENaC mRNA by 30%. Transfections reduced Vt, the amiloride-sensitive Isc and mucus protein concentration while increasing ASL depth and CBF to normal levels. A single dose of siRNA in mouse lung silenced ENaC by approximately 30%, which persisted for at least 7 days. Three doses of siRNA increased silencing to approximately 50%.ConclusionNanoparticle-mediated delivery of ENaCsiRNA to ALI cultures corrected aspects of the mucociliary defect in human CF cells and offers effective delivery and silencing in vivo.

Hyperabsorption of Na+ and raised Ca(2+)-mediated Cl- secretion in nasal epithelia of CF mice

1994 ◽  
Vol 266 (5) ◽  
pp. C1478-C1483 ◽  
Author(s):  
B. R. Grubb ◽  
R. N. Vick ◽  
R. C. Boucher
Keyword(s):  
Cystic Fibrosis ◽  
Secretory Pathway ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Airway Disease ◽  
Na Channel ◽  
Cl Secretion ◽  
Regulatory Link

We investigated the effect of homozygous genetic disruption of the murine cystic fibrosis transmembrane regulator (CFTR) gene on regulation of the rates of Na+ absorption and Cl- secretion by nasal epithelia in cystic fibrosis (CF) mice. The basal in vivo nasal potential difference (PD; -28.8 +/- 1.8 mV, n = 10) and amiloride-sensitive PD (delta 13.8 +/- 1.0 mV, n = 10) were raised in CF mice compared with controls [-7.8 +/- 0.8 mV, n = 14 (basal); delta 4.5 +/- 0.7 mV, n = 14 (amiloride)], consistent with raised Na+ transport. In vitro studies of freshly excised nasal epithelia confirmed that CF epithelia exhibited a greater basal equivalent short-circuit current (Ieq; 63.5 +/- 12 microA/cm2, n = 15) vs. control (30.2 +/- 7.2 microA/cm2, n = 16) and amiloride-sensitive Ieq (delta 46.2 +/- 12.5 microA/cm2) vs. control (delta 11.3 +/- 4.5 microA/cm2). Tissue from normal mice failed to secrete Cl- in response to ionomycin (delta Ieq: -1.2 +/- 1.9 microA/cm2, n = 18), whereas CF murine tissue responded with a large rise in Ieq (delta 55.1 +/- 19.1 microA/cm2, n = 13). We conclude that CF murine nasal epithelia exhibit Na+ hyperabsorption, providing strong evidence for a regulatory link between CFTR and Na+ channel activity in airway epithelia. We speculate that upregulation of the Ca(2+)-mediated Cl- secretory pathway buffers the severity of airway disease in the CF mouse.

scholarly journals Aqueous cigarette smoke extract induces a voltage-dependent inhibition of CFTR expressed in Xenopus oocytes

2014 ◽  
Vol 306 (3) ◽  
pp. L284-L291 ◽  
Author(s):  
A. R. Moran ◽  
Y. Norimatsu ◽  
D. C. Dawson ◽  
K. D. MacDonald
Keyword(s):  
Cystic Fibrosis ◽  
Cigarette Smoke ◽  
Xenopus Oocytes ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Chronic Obstructive ◽  
Voltage Dependent ◽  
Dependent Inhibition

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel inhabits the apical membrane of airway epithelia, where its function is essential for mucus hydration, mucociliary clearance, and airway defense. Chronic obstructive pulmonary disease (COPD), most often a consequence of cigarette smoke (CS) exposure, affects 15 million persons in the US. Clinically, COPD is characterized by many of the salient features of cystic fibrosis lung disease, where CFTR is either absent or reduced in function. CS is an acidic aerosol (pH 5.3 to 6.3) reported to contain over 4,000 constituents. Acute CS exposure has been reported to decrease airway transepithelial voltage in vivo and short-circuit current in vitro; however, the mechanistic basis of these effects is uncertain. The goal of the studies described here was to develop a bioassay to characterize the effects of aqueous CS preparations on the channel function of CFTR. We studied aqueous CS extract (CSE) prepared in our laboratory, as well as commercial cigarette smoke condensate (CSC) in Xenopus oocytes expressing human CFTR. Application of CSE at pH 5.3 produced a reversible, voltage-dependent inhibition of CFTR conductance. CSE neutralized to pH 7.3 produced less inhibition of CFTR conductance. Serial dilution of CSE revealed a dose-dependent effect at acidic and neutral pH. In contrast, CSC did not inhibit CFTR conductance in oocytes. We conclude that one or more components of CSE inhibits CFTR in a manner similar to diphenylamine-2-carboxylate, a negatively charged, open-channel blocker.

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