Functional rescue of c.3846G&gt;A (W1282X) in patient-derived nasal cultures achieved by inhibition of nonsense mediated decay and protein modulators with complementary mechanisms of action

Laselva, Onofrio; Eckford, Paul D. W.; Bartlett, Claire; Ouyang, Hong; Gunawardena, Tarini; Gonska, Tanja; Moraes, Theo J.; Bear, Christine E.

doi:10.1016/j.jcf.2019.12.001

Cited by 59 publications

(83 citation statements)

References 63 publications

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“…The nasal brushing and subsequent cell culture was performed as previously described [ 20 , 21 , 22 ]. Briefly, the brushing was performed by a research nurse (JA) and epithelial cells from the inferior turbinate were obtained.…”

Section: Methodsmentioning

confidence: 99%

“…CFTR function was determined after inhibition of the epithelial sodium channel (ENaC) with amiloride (30 µM, Spectrum Chemical, Gardena, CA, USA) and following cAMP activation with forskolin (10 µM, Sigma-Aldrich, St. Louis, MO, USA). CFTR activity was confirmed as Ieq difference following CFTR inhibition with CFTRInh -172 (10 µM, EMD Millipore Corp, Burlington, MA, USA) [ 22 ].…”

Section: Methodsmentioning

confidence: 99%

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The CFTR Mutation c.3453G > C (D1152H) Confers an Anion Selectivity Defect in Primary Airway Tissue that Can be Rescued by Ivacaftor

Laselva

Moraes

et al. 2020

JPM

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The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene variant, c.3453G > C (D1152H), is associated with mild Cystic Fibrosis (CF) disease, though there is considerable clinical variability ranging from no detectable symptoms to lung disease with early acquisition of Pseudomonas aeruginosa. The approval extension of ivacaftor, the first CFTR modulator drug approved, to include D1152H was based on a positive drug response of defective CFTR-D1152H chloride channel function when expressed in FRT cells. Functional analyses of primary human nasal epithelial cells (HNE) from an individual homozygous for D1152H now revealed that while CFTR-D1152H demonstrated normal, wild-type level chloride conductance, its bicarbonate-selective conductance was impaired. Treatment with ivacaftor increased this bicarbonate-selective conductance. Extensive genetic, protein and functional analysis of the nasal cells of this D1152H/D1152H patient revealed a 90% reduction of CFTR transcripts due to the homozygous presence of the 5T polymorphism in the poly-T tract forming a complex allele with D1152H. Thus, we confirm previous observation in patient-derived tissue that 10% normal CFTR transcripts confer normal, wild-type level chloride channel activity. Together, this study highlights the benefit of patient-derived tissues to study the functional expression and pharmacological modulation of CF-causing mutations, in order to understand pathogenesis and therapeutic responses.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

The CFTR Mutation c.3453G > C (D1152H) Confers an Anion Selectivity Defect in Primary Airway Tissue that Can be Rescued by Ivacaftor

Laselva

Moraes

et al. 2020

JPM

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“…The blots were developed with ECL (Amersham) using the Li-Cor Odyssey Fc (LI-COR Biosciences, Lincoln, NE) in a linear range of exposure (2-45 min). [72][73] Relative levels of CFTR proteins were quantitated by densitometry of immunoblots using ImageStudioLite (LI-COR Biosciences, Lincoln, NE).…”

Section: (13 Of 15)mentioning

confidence: 99%

Photochemically Activated Notch Signaling Hydrogel Preferentially Differentiates Human Derived Hepatoblasts to Cholangiocytes

Rizwan

Fokina

Kivijärvi

et al. 2020

Adv Funct Materials

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Cholangiocytes form an intricate network of bile ducts to enable proper liver function; yet, recapitulating human stem cell differentiation to cholangiocytes in vitro requires Notch signaling and soluble ligands do not activate the Notch pathway. To overcome these limitations, jagged1 is immobilized on a chemically defined hyaluronan to specifically differentiate human embryonic stem cell‐derived hepatoblasts to cholangiocytes. Hepatoblasts cultured on the jagged1‐hydrogels upregulate Notch target genes and express key cholangiocyte markers including cystic fibrosis transmembrane conductance regulator. Moreover, cholangiocytes adopt morphological changes that resemble liver biliary structures. To emulate natural biliary system development, a new strategy is developed to achieve spatiotemporal control over the Jagged1–Notch2 interaction: jagged1 is first caged with a photocleavable streptavidin and then it is uncaged photochemically to restore the biological function of Jagged1, which is confirmed with Notch2 activation in a fluorescent reporter cell line. Moreover, the differentiation of human embryonic stem cell‐derived hepatoblasts to cholangiocytes is temporally controlled with photochemical uncaging of this streptavidin‐Jagged1‐immobilized hyaluronan hydrogel. This strategy defines a framework to control protein signaling in time and space and specifically for Notch signaling for ultimate use in regenerative medicine strategies of the liver.

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“…16HBE14o-cells were genome-edited to produce the homozygous CFF-16HBEge CFTR F508del cell line were obtained from the Cystic Fibrosis Foundation [33]. WT and F508del-HBE cells were grown at 37 • C for 5 days post-confluence submerged on 96-well black well, clear bottom culture plates (Costar, Amsterdam, The Netherlands) in Eagle's minimal essential medium (EMEM) (Wisent BioProducts, Saint-Jean-Baptiste, QC, Canada) with 10% Fetal Bovine Serum (Wisent BioProducts, Quebec, Canada) and 1% Penicillin/Streptomycin (Wisent BioProducts, QC, Canada) [34].…”

Section: Human Cell Linementioning

confidence: 99%

Anti-Infectives Restore ORKAMBI® Rescue of F508del-CFTR Function in Human Bronchial Epithelial Cells Infected with Clinical Strains of P. aeruginosa

et al. 2020

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Chronic infection and inflammation are the primary causes of declining lung function in Cystic Fibrosis (CF) patients. ORKAMBI® (Lumacaftor-Ivacaftor) is an approved combination therapy for Cystic Fibrosis (CF) patients bearing the most common mutation, F508del, in the cystic fibrosis conductance regulator (CFTR) protein. It has been previously shown that ORKAMBI®-mediated rescue of CFTR is reduced by a pre-existing Pseudomonas aeruginosa infection. Here, we show that the infection of F508del-CFTR human bronchial epithelial (HBE) cells with lab strain and four different clinical strains of P. aeruginosa, isolated from the lung sputum of CF patients, decreases CFTR function in a strain-specific manner by 48 to 88%. The treatment of infected cells with antibiotic tobramycin or cationic antimicrobial peptide 6K-F17 was found to decrease clinical strain bacterial growth on HBE cells and restore ORKAMBI®-mediated rescue of F508del-CFTR function. Further, 6K-F17 was found to downregulate the expression of pro-inflammatory cytokines, interleukin (IL)-8, IL-6, and tumor necrosis factor-α in infected HBE cells. The results provide strong evidence for a combination therapy approach involving CFTR modulators and anti-infectives (i.e., tobramycin and/or 6K-F17) to improve their overall efficacy in CF patients.

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Functional rescue of c.3846G>A (W1282X) in patient-derived nasal cultures achieved by inhibition of nonsense mediated decay and protein modulators with complementary mechanisms of action

Abstract: Functional rescue of c.3846G > A (W1282X) in patient-derived nasal cultures achieved by inhibition of nonsense me diate d decay and protein modulators with complementary mechanisms of action

Cited by 59 publications

References 63 publications

The CFTR Mutation c.3453G > C (D1152H) Confers an Anion Selectivity Defect in Primary Airway Tissue that Can be Rescued by Ivacaftor

The CFTR Mutation c.3453G > C (D1152H) Confers an Anion Selectivity Defect in Primary Airway Tissue that Can be Rescued by Ivacaftor

Photochemically Activated Notch Signaling Hydrogel Preferentially Differentiates Human Derived Hepatoblasts to Cholangiocytes

Anti-Infectives Restore ORKAMBI® Rescue of F508del-CFTR Function in Human Bronchial Epithelial Cells Infected with Clinical Strains of P. aeruginosa

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