Lentiviral small hairpin RNA delivery reduces apical sodium channel activity in differentiated human airway epithelial cells

Aarbiou, Jamil; Copreni, Elena; Buijs-Offerman, Ruvalic M.; Wegen, Pascal van der; Castellani, Stefano; Carbone, Annalucia; Tilesi, Francesca; Fradiani, Piera Assunta; Hiemstra, Pieter S.; Yueksekdag, Guelnihal; Diana, Anna; Rosenecker, Joseph; Ascenzioni, Fiorentina; Conese, Massimo; Scholte, Bob J.

doi:10.1002/jgm.2672

Cited by 14 publications

(16 citation statements)

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“… 13 Finally, an shRNA approach following lentiviral transduction of immortalised and primary cell lines used αENaC as their target. 12 The authors transduced primary cells before the formation of tight junctions, unlike our fully differentiated cell approach, and showed up to 60% silencing. Similar to our findings, they have shown that reducing mRNA results in proportional changes in the short circuit current responses and also reduced net apical to basal fluid flux.…”

Section: Discussionmentioning

confidence: 98%

“… 11 Silencing of ENaC expression, by short interfering RNA (siRNA)-mediated RNA interference, offers a more promising therapeutic route. 12–14 Potential advantages of nanoparticle-mediated siRNA therapy include its potency, specificity, duration 15 and restriction to the airways to prevent nephrotoxicity. Transfection of the CF airway epithelium requires protection of siRNA from nucleases, penetration of the mucus and periciliary liquid layer (PCL) and targeted uptake by the epithelial cells 16 with purpose-designed nanoparticles.…”

Section: Introductionmentioning

confidence: 99%

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

Tagalakis

Munye

Ivanova

et al. 2018

Thorax

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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.

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Section: Discussionmentioning

confidence: 98%

Section: Introductionmentioning

confidence: 99%

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

Tagalakis

Munye

Ivanova

et al. 2018

Thorax

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“…The self-inactivating pRRLsin18.cPPT.CMV.eGFP.Wpre and pCCL.PPT.U1aENaCshRNA.hPGKΔNGFR.Wpre constructs were used to generate vesicular stomatitis virus (VSV)-pseudotyped LV stocks for green fluorescent protein (GFP)—LV–GFP and nerve growth factor receptor (NGFR) —LV–ΔNGFR, respectively—as previously described [14,15,16]. The viral titer was determined by HeLa cell (ATCC ® CCL-2™) infection and subsequent flow cytometry analysis, as previously described [14].…”

Section: Methodsmentioning

confidence: 99%

“…Ten thousand cells were examined in each experiment. Analysis of ΔNGFR + cells was performed by plotting the R-PE channel (FLH-2) against the FLH-3 channel, as previously done [16]. For the evaluation of GFP expression in domes, cells were infected with LV–GFP at 2000 MOI preincubated with 2 μL of VM in presence of magnetic field for 20 min.…”

Section: Methodsmentioning

confidence: 99%

Magnetofection Enhances Lentiviral-Mediated Transduction of Airway Epithelial Cells through Extracellular and Cellular Barriers

Castellani

Orlando

Carbone

et al. 2016

Genes

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Gene transfer to airway epithelial cells is hampered by extracellular (mainly mucus) and cellular (tight junctions) barriers. Magnetofection has been used to increase retention time of lentiviral vectors (LV) on the cellular surface. In this study, magnetofection was investigated in airway epithelial cell models mimicking extracellular and cellular barriers. Bronchiolar epithelial cells (H441 line) were evaluated for LV-mediated transduction after polarization onto filters and dexamethasone (dex) treatment, which induced hemicyst formation, with or without magnetofection. Sputum from cystic fibrosis (CF) patients was overlaid onto cells, and LV-mediated transduction was evaluated in the absence or presence of magnetofection. Magnetofection of unpolarized H441 cells increased the transduction with 50 MOI (multiplicity of infection, i.e., transducing units/cell) up to the transduction obtained with 500 MOI in the absence of magnetofection. Magnetofection well-enhanced LV-mediated transduction in mucus-layered cells by 20.3-fold. LV-mediated transduction efficiency decreased in dex-induced hemicysts in a time-dependent fashion. In dome-forming cells, zonula occludens-1 (ZO-1) localization at the cell borders was increased by dex treatment. Under these experimental conditions, magnetofection significantly increased LV transduction by 5.3-fold. In conclusion, these results show that magnetofection can enhance LV-mediated gene transfer into airway epithelial cells in the presence of extracellular (sputum) and cellular (tight junctions) barriers, representing CF-like conditions.

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“…The molecular tools currently available to perform functional studies in fully differentiated ciliated bronchial epithelial cells are still very limited. However, silencing of PRRs using lentivirus-mediated short hairpin RNA (shRNA) transduction has been described without apparent artefactual activation of the innate immune responses [45], opening the path to studies of PRR function in this model. Although restricted to healthy adult volunteers, controlled human challenge studies, which consist of carefully monitored and controlled infection, are beginning to provide unique insights into RSV pathogenesis in humans [46].…”

Section: Prr Function: Moving From Experimental Models To Natural Hummentioning

confidence: 99%

Pathogen recognition receptor crosstalk in respiratory syncytial virus sensing: a host and cell type perspective

Marr

Turvey

Grandvaux

2013

Trends in Microbiology

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Human respiratory syncytial virus (RSV) is a major cause of acute lower respiratory tract infection in young children, immunocompromised adults, and the elderly. The innate immune response plays a pivotal role in host defense against RSV, but whether severe outcomes following RSV infection result from excessive or poor innate immune recognition remains unclear. Recent research suggests a situation in which crosstalk between families of pattern recognition receptors (PRRs) occurs in a cell type-dependent manner. The current challenge to empower novel therapeutic approaches and vaccine development is to confirm the role of the individual receptors in RSV pathogenesis in humans.

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Lentiviral small hairpin RNA delivery reduces apical sodium channel activity in differentiated human airway epithelial cells

Cited by 14 publications

References 61 publications

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

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

Magnetofection Enhances Lentiviral-Mediated Transduction of Airway Epithelial Cells through Extracellular and Cellular Barriers

Pathogen recognition receptor crosstalk in respiratory syncytial virus sensing: a host and cell type perspective

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