TMEM16A Mediated Mucus Production in Human Airway Epithelial Cells.
Am J Respir Cell Mol Biol. 2020 Oct 07;:
Authors: Cabrita I, Benedetto R, Wanitchakool P, Lerias J, Centeio R, Ousingsawat J, Schreiber R, Kunzelmann K
TMEM16A is a Ca2+ activated chloride channel that was shown to enhance production and secretion of mucus in inflamed airways. It is, however, not clear whether TMEM16A is truly responsible for mucus production, or whether mucin and TMEM16A are upregulated independently during inflammatory airway diseases such as asthma and cystic fibrosis (CF). We examined this question using BCi-NS1 cells, a human airway basal cell line that maintains multipotent differentiation capacity, and the two human airway epithelial cell lines, Calu-3 and CFBE. The data demonstrate that exposure of airway epithelial cells to IL-8 and IL-13, two cytokines known to be enhanced in CF and asthma, respectively, leads to increase in mucus production. Expression of MUC5AC was fully dependent on expression of TMEM16A, as shown by siRNA-knockdown of TMEM16A. In addition, different inhibitors of TMEM16A attenuated IL-13 induced mucus production. Interestingly, in CFBE cells expressing F508delCFTR, IL-13 was unable to upregulate membrane expression of TMEM16A or Ca2+-activated whole cell currents. The regulator of TMEM16A, CLCA1, strongly augmented both Ca2+- and cAMP-activated Cl- currents in cells expressing wtCFTR, but failed to augment membrane expression of TMEM16A in F508delCFTR-expressing CFBE cells. The data confirm the functional relationship between CFTR and TMEM16A and suggest an impaired upregulation of TMEM16A by IL-13 or CLCA1 in cells expressing the most frequent CF-causing mutation F508delCFTR.
PMID: 33026825 [PubMed – as supplied by publisher]
Detargeting Lentiviral-Mediated CFTR Expression in Airway Basal Cells Using miR-106b.
Genes (Basel). 2020 Oct 06;11(10):
Authors: Choi SH, Reeves RE, Romano Ibarra GS, Lynch TJ, Shahin WS, Feng Z, Gasser GN, Winter MC, Evans TIA, Liu X, Luo M, Zhang Y, Stoltz DA, Devor EJ, Yan Z, Engelhardt JF
Lentiviral-mediated integration of a CFTR transgene cassette into airway basal cells is a strategy being considered for cystic fibrosis (CF) cell-based therapies. However, CFTR expression is highly regulated in differentiated airway cell types and a subset of intermediate basal cells destined to differentiate. Since basal stem cells typically do not express CFTR, suppressing the CFTR expression from the lentiviral vector in airway basal cells may be beneficial for maintaining their proliferative capacity and multipotency. We identified miR-106b as highly expressed in proliferating airway basal cells and extinguished in differentiated columnar cells. Herein, we developed lentiviral vectors with the miR-106b-target sequence (miRT) to both study miR-106b regulation during basal cell differentiation and detarget CFTR expression in basal cells. Given that miR-106b is expressed in the 293T cells used for viral production, obstacles of viral genome integrity and titers were overcome by creating a 293T-B2 cell line that inducibly expresses the RNAi suppressor B2 protein from flock house virus. While miR-106b vectors effectively detargeted reporter gene expression in proliferating basal cells and following differentiation in the air-liquid interface and organoid cultures, the CFTR-miRT vector produced significantly less CFTR-mediated current than the non-miR-targeted CFTR vector following transduction and differentiation of CF basal cells. These findings suggest that miR-106b is expressed in certain airway cell types that contribute to the majority of CFTR anion transport in airway epithelium.
PMID: 33036232 [PubMed – as supplied by publisher]
Long-term docosahexaenoic acid (DHA) supplementation in cystic fibrosis patients: a randomized, multi-center, double-blind, placebo-controlled trial.
Prostaglandins Leukot Essent Fatty Acids. 2020 Oct 01;162:102186
Authors: López-Neyra A, Suárez L, Muñoz M, de Blas A, Ruiz de Valbuena M, Garriga M, Calvo J, Ribes C, Girón Moreno R, Máiz L, González D, Bousoño C, Manzanares J, Pastor Ó, Martínez-Botas J, Del Campo R, Cantón R, Roy G, Menacho M, Arroyo D, Zamora J, Soriano JB, Lamas A
BACKGROUND: Cystic fibrosis (CF) patients have an alteration in fatty acid (FA) metabolism, associated with increased omega-6 and low omega-3 FA. Previous studies on supplementation with omega-3 FA in CF had contradictory results, and to date there is no evidence to recommend routine use of omega-3 supplements in CF patients. We hypothesized that long-term supplementation with docosahexaenoic acid (DHA) will have beneficial effects in these patients, by reducing pulmonary, systemic and intestinal inflammation.
METHODS: This was a randomized, double-blind, parallel, placebo-controlled trial. CF patients (age >2 months) were randomized to receive a seaweed DHA oil solution (50 mg/Kg/day) or matching placebo for 48 weeks. Primary outcomes were pulmonary (interleukin [IL]-8), systemic (IL-8) and intestinal (calprotectin) inflammatory biomarkers. Secondary outcomes included other pulmonary (IL-1β, IL-6, neutrophil elastase, lactate and calprotectin) and systemic (serum-IL-1β, IL-6) inflammatory biomarkers, as well as clinical outcomes (FEV1, pulmonary exacerbations, antibiotic use, nutritional status and quality of life).
RESULTS: Ninety six CF patients, 44 female, age 14.6±11.9 years (48 DHA and 48 placebo) were included. At trial completion, there were no differences in all primary outcomes [serum-IL-8 (p=0.909), respiratory-IL-8 (p=0.384) or fecal calprotectin (p=0.948)], all secondary inflammatory biomarkers, or in any of the clinical outcomes evaluated. There were few adverse events, with similar incidence in both study groups.
CONCLUSION: In this study, long-term DHA supplementation in CF patients was safe, but did not offer any benefit on inflammatory biomarkers, or in clinical outcomes compared with placebo. (NCT01783613).
PMID: 33038833 [PubMed – as supplied by publisher]
ELX-02: an investigational read-through agent for the treatment of nonsense mutation related genetic disease.
Expert Opin Investig Drugs. 2020 Sep 24;:
Authors: Kerem E
INTRODUCTION: ELX-02, an investigational compound that is structurally an aminoglycoside analogue, induces read-through of nonsense mutations through interaction with the ribosome, through which full-length functional proteins can be produced. It is being developed as a therapy for genetic diseases caused by nonsense mutations such as cystic fibrosis (CF) and nephropathic cystinosis. In Phase 1 clinical trials, 105 volunteers were exposed to ELX-02. To date, ELX-02 is well tolerated and there has been no reported treatment-related serious adverse events or deaths. Areas Covered: The development of this molecule, from its pharmacology to the ongoing Phase 2 clinical trials is discussed. Expert Opinion: Globally, nonsense mutations account for ~11% of all described gene lesions causing inherited monogenetic diseases. In CF and nephropathic cystinosis, they comprise from 10-12% of the disease-causative alleles. ELX-02 is in development as a therapeutic for patients with these alleles as in vitro and in vivo data demonstrated dose-dependent read-through of nonsense mutations to produce full-length, functional proteins. Since read-through efficiency varies between alleles, and mRNA context, careful consideration of target patient populations is required. The results to date support the on-going Phase 2 clinical evaluations of ELX-02 as a read-through agent.
PMID: 32972261 [PubMed – as supplied by publisher]
Treating cystic fibrosis with mRNA and CRISPR.
Hum Gene Ther. 2020 Aug 16;:
Authors: Sanchez ADS, Paunovska K, Cristian A, Dahlman J
Less than 20% of the protein coding genome is thought to be targetable using small molecules. mRNA therapies are not limited in the same way since in theory they can silence or edit any gene by encoding CRISPR nucleases, or alternatively, produce any missing protein. Yet not all mRNA therapies are equally likely to succeed. Over the past several years, an increasing number of clinical trials with siRNA- and ASO-based drugs have revealed three key concepts that will likely extend to mRNA therapies delivered by non-viral systems. First, scientists have come to understand that some genes make better targets for RNA therapies than others. Second, scientists have learned that the type and position of chemical modifications made to an RNA drug can alter its therapeutic window, toxicity and bioavailability. Third, scientists have found that safe and targeted drug delivery vehicles are required to ferry mRNA therapies into diseased cells. Here, we apply these learnings to cystic fibrosis (CF). We also describe lessons learned from a subset of CF gene therapies that have already been tested in patients. Finally, we highlight the scientific advances that are still required for non-viral mRNA or CRISPR-based drugs to treat CF successfully in patients.
PMID: 32799680 [PubMed – as supplied by publisher]
Novel lung tropic AAV capsids for therapeutic gene delivery.
Hum Gene Ther. 2020 Aug 16;:
Authors: Carneiro A, Lee HC, Lin L, van Haasteren J, Schaffer D
Efforts to identify mutations that underlie inherited genetic diseases combined with strides in the development of gene therapy vectors over the last three decades have culminated in the approval of several adeno-associated virus (AAV)-based gene therapies. Genetic diseases that manifest in the lung such as cystic fibrosis and surfactant deficiencies, however, have so far proven to be elusive targets. Early clinical trials in CF using AAV serotype 2 (AAV2) achieved safety but not efficacy endpoints, but importantly these studies provided critical information on barriers that need to be surmounted to translate AAV lung gene therapy towards clinical success. Bolstered with an improved understanding of AAV biology and more clinically relevant lung models, next generation molecular biology and bioinformatics approaches have given rise to novel AAV capsid variants that offer improvements in transduction efficiency, immunological profile, and the ability to circumvent physical barriers in the lung such as mucus. This review discusses the principal limiting barriers to clinical success in lung gene therapy and focuses on novel engineered AAV capsid variants that have been developed to overcome those challenges.
PMID: 32799685 [PubMed – as supplied by publisher]
Front. Pharmacol., 13 August 2020 | https://doi.org/10.3389/fphar.2020.01219
CFTR Modulator Therapy Enhances Peripheral Blood Monocyte Contributions to Immune Responses in People With Cystic Fibrosis
Non-viral mediated gene therapy in human cystic fibrosis airway epithelial cells recovers chloride channel functionality.
Int J Pharm. 2020 Aug 10;:119757
Authors: Sainz-Ramos M, Villate-Beitia I, Gallego I, Qtaish NAL, Lopez-Mendez TB, Eritja R, Grijalvo S, Puras G, Pedraz JL
Gene therapy strategies based on non-viral vectors are currently considered as a promising therapeutic option for the treatment of cystic fibrosis (CF), being liposomes the most commonly used gene carriers. Niosomes offer a powerful alternative to liposomes due to their higher stability and lower cytotoxicity, provided by their non-ionic surfactant and helper components. In this work, a three-formulation screening ois performed, in terms of physicochemical and biological behavior, in CF patient derived airway epithelial cells. The most efficient niosome formulation reaches 28% of EGFP expressing live cells and follows caveolae-mediated endocytosis. Transfection with therapeutic cystic fibrosis transmembrane conductance regulator (CFTR) gene results in 5-fold increase of CFTR protein expression in transfected versus non-transfected cells, which leads to 1.5-fold increment of the chloride channel functionality. These findings highlight the relevance of niosome-based systems as an encouraging non-viral gene therapy platform with potential therapeutic benefits for CF.
PMID: 32791297 [PubMed – as supplied by publisher]
Synthesis and biological evaluation of thiazole derivatives on basic defects underlying cystic fibrosis.
Bioorg Med Chem Lett. 2020 Aug 09;:127473
Authors: Pesce E, Pedemonte N, Leoni A, Locatelli A, Morigi R
Cystic fibrosis is a genetic disease caused by loss-of-function mutations in the cystic fibrosis transmembrane conductance regulator gene, encoding for CFTR protein. The most frequent mutation is the deletion of phenylalanine at position 508 (F508del), which leads to distinct defects in channel gating and cellular processing. In last years, several thiazole containing small molecules, endowed with dual F508del-CFTR modulator activity, proved to be able to target these defects. In search of new chemical entities able to restore CFTR function, we designed and synthesized a small series of sixteen thiazole derivatives. The designed compounds were studied as correctors and potentiators of F508del-CFTR. Although none of the molecules showed significant corrector activity, compounds 10 and 11 exhibited potentiator effects, thus allowing to determine some basic structural features which enable to obtain F508del-CFTR potentiator activity. In silico ADME studies showed that these derivatives obey Lipinski’s rule of five and are expected to be orally bioavailable. Therefore, these molecules may represent a good starting point for the design of analogues endowed with improved CFTR potentiator activity and a good pharmacokinetic profile.
PMID: 32784089 [PubMed – as supplied by publisher]
Gene Therapy in Rare Respiratory Diseases: What Have We Learned So Far?
J Clin Med. 2020 Aug 08;9(8):
Authors: Bañuls L, Pellicer D, Castillo S, Navarro-García MM, Magallón M, González C, Dasí F
Gene therapy is an alternative therapy in many respiratory diseases with genetic origin and currently without curative treatment. After five decades of progress, many different vectors and gene editing tools for genetic engineering are now available. However, we are still a long way from achieving a safe and efficient approach to gene therapy application in clinical practice. Here, we review three of the most common rare respiratory conditions-cystic fibrosis (CF), alpha-1 antitrypsin deficiency (AATD), and primary ciliary dyskinesia (PCD)-alongside attempts to develop genetic treatment for these diseases. Since the 1990s, gene augmentation therapy has been applied in multiple clinical trials targeting CF and AATD, especially using adeno-associated viral vectors, resulting in a good safety profile but with low efficacy in protein expression. Other strategies, such as non-viral vectors and more recently gene editing tools, have also been used to address these diseases in pre-clinical studies. The first gene therapy approach in PCD was in 2009 when a lentiviral transduction was performed to restore gene expression in vitro; since then, transcription activator-like effector nucleases (TALEN) technology has also been applied in primary cell culture. Gene therapy is an encouraging alternative treatment for these respiratory diseases; however, more research is needed to ensure treatment safety and efficacy.
PMID: 32784514 [PubMed]
Diversified Synthetic Pathway of 1, 4-Dihydropyridines: A Class of Pharmacologically Important Molecules.
Mini Rev Med Chem. 2020 Aug 07;:
Authors: Khot S, Auti PB, Khedkar SA
The current review discusses the different synthetic pathways for one of the most important and interesting heterocyclic ring systems 1,4-dihydropyridine. This cyclic system depicts diverse pharmacological action at several receptors, channels, and enzymes. Dihydropyridine moiety plays an important role in several calcium-channel blockers. Moreover, it has been exploited for the treatment of a variety of cardiovascular diseases due to its potential antihypertensive, anti-angina, vasodilator, and cardiac depressant activities. Furthermore, it also shows antibacterial, anticancer, antileishmanial, anticoagulant, anticonvulsant, anti-tubercular, antioxidant, antiulcer, and neuroprotective properties. Several reports have demonstrated dihydropyridine derivatives as a potentiator of cystic fibrosis transmembrane conductance regulator protein, potent antimalarial agent and HIV-1 protease inhibitor. Herein, we have briefly reviewed different novel chemistry and synthesis of 1,4-dihydropyridine.
PMID: 32767934 [PubMed – as supplied by publisher]
Cystic Fibrosis Defective Response to Infection Involves Autophagy and Lipid Metabolism.
Cells. 2020 Aug 06;9(8):
Authors: Mingione A, Ottaviano E, Barcella M, Merelli I, Rosso L, Armeni T, Cirilli N, Ghidoni R, Borghi E, Signorelli P
Cystic fibrosis (CF) is a hereditary disease, with 70% of patients developing a proteinopathy related to the deletion of phenylalanine 508. CF is associated with multiple organ dysfunction, chronic inflammation, and recurrent lung infections. CF is characterized by defective autophagy, lipid metabolism, and immune response. Intracellular lipid accumulation favors microbial infection, and autophagy deficiency impairs internalized pathogen clearance. Myriocin, an inhibitor of sphingolipid synthesis, significantly reduces inflammation, promotes microbial clearance in the lungs, and induces autophagy and lipid oxidation. RNA-seq was performed in Aspergillusfumigatus-infected and myriocin-treated CF patients’ derived monocytes and in a CF bronchial epithelial cell line. Fungal clearance was also evaluated in CF monocytes. Myriocin enhanced CF patients’ monocytes killing of A. fumigatus. CF patients’ monocytes and cell line responded to infection with a profound transcriptional change; myriocin regulates genes that are involved in inflammation, autophagy, lipid storage, and metabolism, including histones and heat shock proteins whose activity is related to the response to infection. We conclude that the regulation of sphingolipid synthesis induces a metabolism drift by promoting autophagy and lipid consumption. This process is driven by a transcriptional program that corrects part of the differences between CF and control samples, therefore ameliorating the infection response and pathogen clearance in the CF cell line and in CF peripheral blood monocytes.
PMID: 32781626 [PubMed – in process]
Pancreatic enzyme replacement therapy for people with cystic fibrosis.
Cochrane Database Syst Rev. 2020 Aug 05;8:CD008227
Authors: Somaraju URR, Solis-Moya A
BACKGROUND: Most people with cystic fibrosis (CF) (80% to 90%) need pancreatic enzyme replacement therapy (PERT) to prevent malnutrition. Enzyme preparations need to be taken whenever food is taken, and the dose needs to be adjusted according to the food consumed. A systematic review on the efficacy and safety of PERT is needed to guide clinical practice, as there is variability between centres with respect to assessment of pancreatic function, time of commencing treatment, dose and choice of supplements. This is an updated version of a published review.
OBJECTIVES: To evaluate the efficacy and safety of PERT in children and adults with CF and to compare the efficacy and safety of different formulations of PERT and their appropriateness in different age groups. Also, to compare the effects of PERT in CF according to different diagnostic subgroups (e.g. different ages at introduction of therapy and different categories of pancreatic function).
SEARCH METHODS: We searched the Cochrane Cystic Fibrosis and Genetic Disorders Group Trials Register comprising references identified from comprehensive electronic database searches and handsearches of relevant journals and abstract books of conference proceedings. Most recent search: 07 November 2019. We also searched an ongoing trials website and the websites of the pharmaceutical companies who manufacture pancreatic enzyme replacements for any additional trials. Most recent search: 26 December 2019.
SELECTION CRITERIA: Randomised and quasi-randomised controlled trials in people of any age, with CF and receiving PERT, at any dosage and in any formulation, for a period of not less than four weeks, compared to placebo or other PERT preparations.
DATA COLLECTION AND ANALYSIS: Two authors independently assessed trials and extracted outcome data. They also assessed the risk of bias and quality of the evidence (GRADE) of the trials included in the review.
MAIN RESULTS: 14 trials were included in the review (641 children and adults with CF), two of these were parallel trials and 12 were cross-over trials. Interventions included different enteric and non-enteric-coated preparations of varying formulations in comparison to each other. The number of participants in each trial varied between 14 and 129. 13 trials were for a duration of four weeks and one trial lasted seven weeks. The majority of the trials had an unclear risk of bias from the randomisation process as the details of this were not given; they also had a high risk of attrition bias and reporting bias. The quality of the evidence ranged from moderate to very low. We mostly could not combine data from the trials as they compared different formulations and the findings from individual trials provided insufficient evidence to determine the size and precision of the effects of different formulations.
AUTHORS’ CONCLUSIONS: There is limited evidence of benefit from enteric-coated microspheres when compared to non-enteric coated pancreatic enzyme preparations up to one month. In the only comparison where we could combine any data, the fact that these were cross-over trials is likely to underestimate the level of inconsistency between the results of the trials due to over-inflation of CIs from the individual trials.There is no evidence on the long-term effectiveness and risks associated with PERT. There is also no evidence on the relative dosages of enzymes needed for people with different levels of severity of pancreatic insufficiency, optimum time to start treatment and variations based on differences in meals and meal sizes. There is a need for a properly designed trial that can answer these questions.
PMID: 32761612 [PubMed – in process]
Correction of CFTR function in intestinal organoids to guide treatment of Cystic Fibrosis.
Eur Respir J. 2020 Aug 03;:
Authors: Ramalho AS, Fürstová E, Vonk AM, Ferrante M, Verfaillie C, Dupont L, Boon M, Proesmans M, Beekman JM, Sarouk I, Cordero CV, Vermeulen F, De Boeck K, Belgian Organoid Project
RATIONALE: Given the vast number of CFTR mutations, biomarkers predicting benefit from CFTR modulator therapies are needed for subjects with cystic fibrosis (CF).
OBJECTIVES: To study CFTR function in organoids of subjects with common and rare CFTR mutations and evaluate correlations between CFTR function and clinical data.
METHODS: Intestinal organoids were grown from rectal biopsies in a cohort of 97 subjects with CF. Residual CFTR function was measured by quantifying organoid swelling induced by forskolin and response to modulators by quantifying organoid swelling induced by CFTR correctors, potentiator and their combination. Organoid data were correlated with clinical data from literature.
MEASUREMENTS AND MAIN RESULTS: Across 28 genotypes, residual CFTR function correlated tightly (r2=0.87) with sweat chloride values. When studying the same genotypes, CFTR function rescue by CFTR modulators in organoids correlated tightly with mean improvement in lung function (r2=0.90) and sweat chloride (r2=0.95) reported in clinical trials. We identified candidate genotypes for modulator therapy, like E92K, Q237E, R334W and L159S. Based on organoid results, two subjects started modulator treatment: one homozygous for complex allele Q359K_T360K, and the second with mutation E60K. Both subjects had major clinical benefit.
CONCLUSIONS: Measurements of residual CFTR function and rescue of function by CFTR modulators in intestinal organoids correlate closely with clinical data. Our results for reference genotypes concur with previous results. CFTR function measured in organoids can be used to guide precision medicine in patients with CF, positioning organoids as a potential in vitro model to bring treatment to patients carrying rare CFTR mutations.
PMID: 32747394 [PubMed – as supplied by publisher]
Proteomics and Metabolomics for Cystic Fibrosis Research.
Int J Mol Sci. 2020 Jul 30;21(15):
Authors: Liessi N, Pedemonte N, Armirotti A, Braccia C
The aim of this review article is to introduce the reader to the state-of-the-art of the contribution that proteomics and metabolomics sciences are currently providing for cystic fibrosis (CF) research: from the understanding of cystic fibrosis transmembrane conductance regulator (CFTR) biology to biomarker discovery for CF diagnosis. Our work particularly focuses on CFTR post-translational modifications and their role in cellular trafficking as well as on studies that allowed the identification of CFTR molecular interactors. We also show how metabolomics is currently helping biomarker discovery in CF. The most recent advances in these fields are covered by this review, as well as some considerations on possible future scenarios for new applications.
PMID: 32751630 [PubMed – in process]
CFTR Modulators Dampen Aspergillus-Induced Reactive Oxygen Species Production by Cystic Fibrosis Phagocytes.
Front Cell Infect Microbiol. 2020;10:372
Authors: Currie AJ, Main ET, Wilson HM, Armstrong-James D, Warris A
Excessive inflammation by phagocytes during Aspergillus fumigatus infection is thought to promote lung function decline in CF patients. CFTR modulators have been shown to reduce A. fumigatus colonization in vivo, however, their antifungal and anti-inflammatory mechanisms are unclear. Other treatments including azithromycin and acebilustat may dampen Aspergillus-induced inflammation due to their immunomodulatory properties. Therefore, we set out in this study to determine the effects of current CF therapies on ROS production and fungal killing, either direct or indirect by enhancing antifungal immune mechanisms in peripheral blood immune cells from CF patients upon A. fumigatus infection. Isolated peripheral blood mononuclear cells (PBMCs) and polymorphonuclear cells (PMNs) from CF patients and healthy volunteers were challenged with A. fumigatus following pre-treatment with CFTR modulators, azithromycin or acebilustat. Ivacaftor/lumacaftor treated CF and control subject PMNs resulted in a significant reduction (p < 0.05) in Aspergillus-induced ROS. For CF PBMC, Aspergillus-induced ROS was significantly reduced when pre-treated with ivacaftor alone (p < 0.01) or in combination with lumacaftor (p < 0.01), with a comparable significant reduction in control subject PBMC (p < 0.05). Azithromycin and acebilustat had no effect on ROS production by CF or control subject phagocytes. None of the treatments showed an indirect or direct antifungal activity. In summary, CFTR modulators have potential for additional immunomodulatory benefits to prevent or treat Aspergillus-induced inflammation in CF. The comparable effects of CFTR modulators observed in phagocytes from control subjects questions their exact mechanism of action. PMID: 32793514 [PubMed - in process] PubMed:32793514
Pendrin stimulates a chloride absorption pathway to increase CFTR-mediated chloride secretion from Cystic Fibrosis airway epithelia.
FASEB Bioadv. 2020 Sep;2(9):526-537
Authors: Bajko J, Duguid M, Altmann S, Hurlbut GD, Kaczmarek JS
Cystic Fibrosis (CF), an inherited multi-system disease, is caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) that disrupt its ability to secrete anions from epithelia. Recovery of functional anion secretion may be curative for CF, so different components of the ion transport machinery have become attractive therapeutic targets. Several members of the SLC26 ion transporter family have been linked to epithelial ion flux, some through putative functional interactions with CFTR. Using a small-scale qPCR screen, we confirmed that the anion transporter SLC26A4 (pendrin) is downregulated in CF. Upregulation of pendrin using interleukins IL-4 or IL-13 increased Cl- secretion through CFTR in human bronchial epithelial cell (HBEC) derived epithelia differentiated in vitro and measured in the Ussing Chamber. Inhibition or knockdown of pendrin prevented this increased secretion. Increased CFTR activity was not driven by increases in CFTR protein or upstream regulatory pathway components. When basolateral Cl- absorption through NKCC1 was inhibited, a pendrin-dependent Cl- absorption pathway allowing CFTR to continue secreting Cl- from the epithelium was revealed. Although CFTR is often considered the bottleneck in the transepithelial Cl- transport pathway, these studies indicate that basolateral Cl- permeability becomes limiting as CFTR activity increases. Therefore, an increase of epithelial Cl- absorption via pendrin might have additional therapeutic benefit in combination with CFTR modulators.
PMID: 32923987 [PubMed]
GM1 as Adjuvant of Innovative Therapies for Cystic Fibrosis Disease.
Int J Mol Sci. 2020 Jun 24;21(12):
Authors: Mancini G, Loberto N, Olioso D, Dechecchi MC, Cabrini G, Mauri L, Bassi R, Schiumarini D, Chiricozzi E, Lippi G, Pesce E, Sonnino S, Pedemonte N, Tamanini A, Aureli M
Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein is expressed at the apical plasma membrane (PM) of different epithelial cells. The most common mutation responsible for the onset of cystic fibrosis (CF), F508del, inhibits the biosynthesis and transport of the protein at PM, and also presents gating and stability defects of the membrane anion channel upon its rescue by the use of correctors and potentiators. This prompted a multiple drug strategy for F508delCFTR aimed simultaneously at its rescue, functional potentiation and PM stabilization. Since ganglioside GM1 is involved in the functional stabilization of transmembrane proteins, we investigated its role as an adjuvant to increase the effectiveness of CFTR modulators. According to our results, we found that GM1 resides in the same PM microenvironment as CFTR. In CF cells, the expression of the mutated channel is accompanied by a decrease in the PM GM1 content. Interestingly, by the exogenous administration of GM1, it becomes a component of the PM, reducing the destabilizing effect of the potentiator VX-770 on rescued CFTR protein expression/function and improving its stabilization. This evidence could represent a starting point for developing innovative therapeutic strategies based on the co-administration of GM1, correctors and potentiators, with the aim of improving F508del CFTR function.
PMID: 32599772 [PubMed – in process]
Highlights From the 2019 North American Cystic Fibrosis Conference.
Pediatr Pulmonol. 2020 Jun 23;:
Authors: Hoppe JE, Guimbellot J, Martiniano SL, Toprak D, Davis C, Daines CL, Muhlebach MS, Esther CR, Dellon EP
The 33rd annual North American Cystic Fibrosis Conference (NACFC) was held in Nashville, Tennessee on October 31-November 2, 2019. Abstracts of presentations from the conference were published in a supplement to Pediatric Pulmonology 1 . This review briefly summarizes presentations in several major topic areas at the conference: pathophysiology and basic science of cystic fibrosis (CF) lung disease; clinical trials; clinical quality improvement; microbiology and treatment of infection; and transition, advanced lung disease and transplant, mental health and psychosocial concerns. The review is intended to highlight several areas and is not a comprehensive summary of the conference. Citations from the conference are by first author and abstract number or symposium number, as designated in the supplement. This article is protected by copyright. All rights reserved.
PMID: 32573948 [PubMed – as supplied by publisher]
Antisense oligonucleotide-mediated correction of CFTR splicing improves chloride secretion in cystic fibrosis patient-derived bronchial epithelial cells.
Nucleic Acids Res. 2020 Jun 10;:
Authors: Michaels WE, Bridges RJ, Hastings ML
Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, encoding an anion channel that conducts chloride and bicarbonate across epithelial membranes. Mutations that disrupt pre-mRNA splicing occur in >15% of CF cases. One common CFTR splicing mutation is CFTR c.3718-2477C>T (3849+10 kb C>T), which creates a new 5′ splice site, resulting in splicing to a cryptic exon with a premature termination codon. Splice-switching antisense oligonucleotides (ASOs) have emerged as an effective therapeutic strategy to block aberrant splicing. We test an ASO targeting the CFTR c.3718-2477C>T mutation and show that it effectively blocks aberrant splicing in primary bronchial epithelial (hBE) cells from CF patients with the mutation. ASO treatment results in long-term improvement in CFTR activity in hBE cells, as demonstrated by a recovery of chloride secretion and apical membrane conductance. We also show that the ASO is more effective at recovering chloride secretion in our assay than ivacaftor, the potentiator treatment currently available to these patients. Our findings demonstrate the utility of ASOs in correcting CFTR expression and channel activity in a manner expected to be therapeutic in patients.
PMID: 32520327 [PubMed – as supplied by publisher]
TMEM16A Potentiation: A Novel Therapeutic Approach for the Treatment of Cystic Fibrosis
Improving outcomes of infections in cystic fibrosis in the era of CFTR modulator therapy.
Pediatr Pulmonol. 2019 Nov;54 Suppl 3:S18-S26
Authors: Saiman L
Currently, available single and dual-combination cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapies have favorably altered the life course of individuals with cystic fibrosis (CF) by decreasing morbidities and increasing survival. However, even with CFTR modulator use, questions and challenges remain to optimize the management of lung infections. This review (a) identifies these ongoing challenges and discusses the current understanding of the potential impact of CFTR modulator therapy on infections; (b) describes ongoing research to optimize detection, diagnosis, and treatment of CF microorganisms; and (c) discusses strategies to develop new anti-infective therapies. The CF Foundation has launched the Infection Research Initiative to fund research that will improve our understanding of the complex microbial ecology within the CF lung, improve detection of CF pathogens, optimize current treatment, including long-term chronic therapies, and develop new anti-infective therapies. Ongoing clinical trials to determine the optimal duration of treatment of pulmonary exacerbations and to diagnose and treat nontuberculous mycobacteria represent clinical research paradigms that could be used to answer other complex treatment questions. The anti-infective pipeline includes both existing anti-infective and non-anti-infective agents, many of which are proposed to have unique mechanisms of action in CF. Future studies plan to evaluate short- and long-term clinical effectiveness and impact on infections, of the next generation of CFTR modulator therapy, the highly effective triple-combination therapy, for individuals with CF, homozygous or heterozygous for F508del.
PMID: 31715086 [PubMed – in process]
Anti-inflammatories and mucociliary clearance therapies in the age of CFTR modulators.
Pediatr Pulmonol. 2019 Nov;54 Suppl 3:S46-S55
Authors: Perrem L, Ratjen F
Cystic fibrosis (CF) is a genetic and life-limiting disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. This multi-system disease is characterized by progressive lung disease and pancreatic insufficiency amongst other manifestations. CFTR primarily functions as a chloride channel that transports ions across the apical membrane of epithelial cells but has other functions, including bicarbonate secretion and inhibition of sodium transport. Defective CFTR disrupts these functions, causing viscous and dehydrated mucus to accumulate, compromising the airway lumen and contributing to obstructive pulmonary disease. The combination of CFTR dysfunction, mucus obstruction, and infection drive an exaggerated and dysfunctional inflammatory response, which contributes to irreversible airway destruction and fibrosis. CFTR modulators, an exciting new class of drugs, increase the expression and/or function of CFTR variant protein and improve multiple clinical endpoints, such as lung function, pulmonary exacerbation rates, and nutritional status. However, these genotype-specific drugs are not universally available, the clinical response is variable, and lung function still declines over time when bronchiectasis is established. Consequently, even in the age of CFTR modulators, we must target other important aspects of the CF airway disease, such as inflammation and mucociliary clearance. This review highlights the mechanisms of inflammation and mucus accumulation in the CF lung and discusses anti-inflammatory and mucociliary clearance agents that are currently in development focusing on compounds for which clinical trial data have recently become available.
PMID: 31715088 [PubMed – in process]
S-Nitrosylation of CHIP Enhances F508Del CFTR Maturation.
Am J Respir Cell Mol Biol. 2019 Oct 09;:
Authors: Zaman K, Knight J, Hussain F, Cao R, Estabrooks SK, Altawallbeh G, Holloway K, Jafri A, Sawczak V, Li Y, Getsy P, Sun F, Raffay T, Cotton C, Brodsky JL, Periasamy A, Lewis SJ, Gaston B
S-Nitrosothiols (SNOs) are endogenous signaling compounds with a diverse spectrum of beneficial airway effects that are both cGMP-dependent and -independent. SNOs are present in healthy human airways, but levels are low in the airways of cystic fibrosis (CF) patients. Here, we evaluated the interactions of SNOs with molecular co-chaperone C-terminus Hsc70 interacting protein (CHIP)- an E3 ubiquitin ligase that targets improperly folded CFTR for degradation. CHIP was expressed in primary human bronchial epithelial and CFBE41o – cells expressing either wild type or F508del CFTR. Confocal microscopy and immunoprecipitation studies showed the cellular co-localization of CFTR and CHIP and showed that GSNO inhibits the CHIP-CFTR interaction. SNOs significantly reduced both the expression and activity of CHIP, leading to higher levels of both the immature and mature forms of F508del CFTR; in fact, inhibition of the expression and function of CHIP by SNOs not only improved CFTR maturation, but also increased CFTR stability at the cell membrane. GSNO treated cells also had more S-nitrosylated CHIP and less ubiquitinated CFTR than untreated cells, suggesting that S-nitrosylation of CHIP prevents CFTR ubiquitination by inhibiting CHIP’s E3 ubiquitin ligase function. Further, the exogenous SNOs S-nitrosoglutathione diethyl ester (GNODE) and S-nitro-N-acetylcysteine (SNOAC) increased CFTR expression at the cell surface. Following CHIP knockdown with siRNA duplexes specific for CHIP, F508del CFTR expression increased at the cell surface. We conclude that SNOs effectively reduce CHIP-mediated degradation of CFTR, resulting in increased F508del CFTR expression on the surface of airway epithelial cells.
PMID: 31596601 [PubMed – as supplied by publisher]
Transforming Growth Factor-β1 Selectively Recruits microRNAs to the RNA-Induced Silencing Complex and Degrades CFTR mRNA under Permissive Conditions in Human Bronchial Epithelial Cells.
Int J Mol Sci. 2019 Oct 05;20(19):
Authors: Mitash N, Mu F, Donovan JE, Myerburg MM, Ranganathan S, Greene CM, Swiatecka-Urban A
Mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (
PMID: 31590401 [PubMed – in process]
Inflammatory role of extracellular sphingolipids in Cystic Fibrosis.
Int J Biochem Cell Biol. 2019 Sep 26;:105622
Authors: Zulueta A, Peli V, Dei Cas M, Colombo M, Paroni R, Falleni M, Baisi A, Bollati V, Chiaramonte R, Del Favero E, Ghidoni R, Caretti A
Ceramide is emerging as one of the players of inflammation in lung diseases. However, data on its inflammatory role in Cystic Fibrosis (CF) as part of the extracellular machinery driven by lung mesenchymal stem cells (MSCs)-derived extracellular vesicles (EVs) are missing. We obtained an in vitro model of CF-MSC by treating control human lung MSCs with a specific CFTR inhibitor. We characterized EVs populations derived from MSCs (ctr EVs) and CF-MSCs (CF-EVs) and analyzed their sphingolipid profile by LC-MS/MS. To evaluate their immunomodulatory function, we treated an in vitro human model of CF, with both EVs populations. Our data show that the two EVs populations differ for the average size, amount, and rate of uptake. CF-EVs display higher ceramide and dihydroceramide accumulation as compared to control EVs, suggesting the involvement of the de novo biosynthesis pathway in the parental CF-MSCs. Higher sphingomyelinase activity in CF-MSCs, driven by inflammation-induced ceramide accumulation, sustains the exocytosis of vesicles that export new formed pro-inflammatory ceramide. Our results suggest that CFTR dysfunction associates with an enhanced sphingolipid metabolism leading to the release of EVs that export the excess of pro-inflammatory Cer to the recipient cells, thus contributing to maintain the unresolved inflammatory status of CF.
PMID: 31563560 [PubMed – as supplied by publisher]
IL-1β dominates the promucin secretory cytokine profile in cystic fibrosis.
J Clin Invest. 2019 Sep 16;:
Authors: Chen G, Sun L, Kato T, Okuda K, Martino MB, Abzhanova A, Lin JM, Gilmore RC, Batson BD, O’Neal YK, Volmer AS, Dang H, Deng Y, Randell SH, Button B, Livraghi-Butrico A, Kesimer M, Ribeiro CM, O’Neal WK, Boucher RC
Cystic fibrosis (CF) lung disease is characterized by early and persistent mucus accumulation and neutrophilic inflammation in the distal airways. Identification of the factors in CF mucopurulent secretions that perpetuate CF mucoinflammation may provide strategies for novel CF pharmacotherapies. We show that IL-1β, with IL-1α, dominated the mucin prosecretory activities of supernatants of airway mucopurulent secretions (SAMS). Like SAMS, IL-1β alone induced MUC5B and MUC5AC protein secretion and mucus hyperconcentration in CF human bronchial epithelial (HBE) cells. Mechanistically, IL-1β induced the sterile α motif-pointed domain containing ETS transcription factor (SPDEF) and downstream endoplasmic reticulum to nucleus signaling 2 (ERN2) to upregulate mucin gene expression. Increased mRNA levels of IL1B, SPDEF, and ERN2 were associated with increased MUC5B and MUC5AC expression in the distal airways of excised CF lungs. Administration of an IL-1 receptor antagonist (IL-1Ra) blocked SAMS-induced expression of mucins and proinflammatory mediators in CF HBE cells. In conclusion, IL-1α and IL-1β are upstream components of a signaling pathway, including IL-1R1 and downstream SPDEF and ERN2, that generate a positive feedback cycle capable of producing persistent mucus hyperconcentration and IL-1α and/or IL-1β-mediated neutrophilic inflammation in the absence of infection in CF airways. Targeting this pathway therapeutically may ameliorate mucus obstruction and inflammation-induced structural damage in young CF children.
PMID: 31524632 [PubMed – as supplied by publisher]
Revealing the molecular signaling pathways of mucus stasis in cystic fibrosis.
J Clin Invest. 2019 Sep 16;:
Authors: Birket SE, Rowe SM
Mucus obstruction is a hallmark of cystic fibrosis (CF) airway disease, leading to chronic infection, dysregulated inflammation, and progressive lung disease. As mucus hyperexpression is a key component in the initiation and perpetuation of airway obstruction, the triggers underlying mucin release must be identified and understood. In this issue of the JCI, Chen et al. sought to delineate the mechanisms that allow IL-1α/IL-1β to perpetuate the mucoinflammatory environment characteristic of the CF airway. The authors demonstrated that IL-1α and IL-1β stimulated non-CF human bronchial epithelial (HBE) cells to upregulate and secrete both MUC5B and MUC5AC in a dose-dependent manner, an effect that was neutralized by the inhibition of the IL-1α/IL-1β receptor (IL-1R1). Further experiments using mouse models and excised lung tissue identified contributors that drive a vicious feedback cycle of hyperconcentrated mucus secretions and persistent inflammation in the CF airway, factors that are likely at the nidus of progressive lung disease.
PMID: 31524633 [PubMed – as supplied by publisher]
G3BP1, p62 and USP10 could be therapeutic targets for ubiquitinated protein aggregation disorders, including cystic fibrosis (CF)
G3BP1 inhibits ubiquitinated protein aggregations induced by p62 and USP10.
Sci Rep. 2019 Sep 09;9(1):12896
Authors: Anisimov S, Takahashi M, Kakihana T, Katsuragi Y, Kitaura H, Zhang L, Kakita A, Fujii M
The aberrant accumulation of ubiquitinated protein aggregates in cells plays a critical role in the pathogenesis of several degenerative diseases, including Parkinson disease (PD) and cystic fibrosis (CF). In this study, we found that Ras GTPase-activating protein-binding protein 1 (G3BP1) inhibits ubiquitinated protein aggregations induced by p62 and USP10 in cultured cells. p62 is a ubiquitin receptor, and p62 and its binding partner USP10 have been shown to augment ubiquitinated protein aggregation. G3BP1 interacted with p62 and USP10 and inhibited p62/USP10-induced protein aggregation. The G3BP1 inhibition of protein aggregations targeted two aggregation-prone proteins, α-synuclein and CFTR-ΔF508, which are causative factors of PD and CF, respectively. G3BP1 depletion increased the amounts of ubiquitinated α-synuclein and CFTR-ΔF508 protein. A proteasome reporter indicated that G3BP1 depletion inhibits the proteasome activity. We herein present evidence that G3BP1, p62 and USP10 together control ubiquitinated protein toxicity by controlling both ubiquitination and aggregation. Taken together, these results suggest that G3BP1, p62 and USP10 could be therapeutic targets for ubiquitinated protein aggregation disorders, including PD and CF.
PMID: 31501480 [PubMed – in process]
Cystic Fibrosis Lung Disease and Immunometabolism: Targeting the NLRP3 Inflammasome.
Am J Respir Crit Care Med. 2019 Sep 05;:
Authors: Cantin AM
PMID: 31487198 [PubMed – as supplied by publisher]
Whole-blood transcriptomic responses to lumacaftor/ivacaftor therapy in cystic fibrosis.
J Cyst Fibros. 2019 Aug 29;:
Authors: Kopp BT, Fitch J, Jaramillo L, Shrestha CL, Robledo-Avila F, Zhang S, Palacios S, Woodley F, Hayes D, Partida-Sanchez S, Ramilo O, White P, Mejias A
BACKGROUND: Cystic fibrosis (CF) remains without a definitive cure. Novel therapeutics targeting the causative defect in the cystic fibrosis transmembrane conductance regulator (CFTR) gene are in clinical use. Lumacaftor/ivacaftor is a CFTR modulator approved for patients homozygous for the CFTR variant p.Phe508del, but there are wide variations in treatment responses preventing prediction of patient responses. We aimed to determine changes in gene expression related to treatment initiation and response.
METHODS: Whole-blood transcriptomics was performed using RNA-Seq in 20 patients with CF pre- and 6 months post-lumacaftor/ivacaftor (drug) initiation and 20 non-CF healthy controls. Correlation of gene expression with clinical variables was performed by stratification via clinical responses.
RESULTS: We identified 491 genes that were differentially expressed in CF patients (pre-drug) compared with non-CF controls and 36 genes when comparing pre-drug to post-drug profiles. Both pre- and post-drug CF profiles were associated with marked overexpression of inflammation-related genes and apoptosis genes, and significant under-expression of T cell and NK cell-related genes compared to non-CF. CF patients post-drug demonstrated normalized protein synthesis expression, and decreased expression of cell-death genes compared to pre-drug profiles, irrespective of clinical response. However, CF clinical responders demonstrated changes in eIF2 signaling, oxidative phosphorylation, IL-17 signaling, and mitochondrial function compared to non-responders. Top overexpressed genes (MMP9 and SOCS3) that decreased post-drug were validated by qRT-PCR. Functional assays demonstrated that CF monocytes normalized calcium (increases MMP9 expression) concentrations post-drug.
CONCLUSIONS: Transcriptomics revealed differentially regulated pathways in CF patients at baseline compared to non-CF, and in clinical responders to lumacaftor/ivacaftor.
PMID: 31474496 [PubMed – as supplied by publisher]
Thickness of the airway surface liquid layer in the lung is affected in cystic fibrosis by compromised synergistic regulation of the ENaC ion channel.
J R Soc Interface. 2019 Aug 30;16(157):20190187
Authors: Olivença DV, Fonseca LL, Voit EO, Pinto FR
The lung epithelium is lined with a layer of airway surface liquid (ASL) that is crucial for healthy lung function. ASL thickness is controlled by two ion channels: epithelium sodium channel (ENaC) and cystic fibrosis (CF) transmembrane conductance regulator (CFTR). Here, we present a minimal mathematical model of ENaC, CFTR and ASL regulation that sheds light on the control of ENaC by the short palate lung and nasal epithelial clone 1 (SPLUNC1) protein and by phosphatidylinositol 4,5-biphosphate (PI(4,5)P2). The model, despite its simplicity, yields a good fit to experimental observations and is an effective tool for exploring the interplay between ENaC, CFTR and ASL. Steady-state data and dynamic information constrain the model’s parameters without ambiguities. Testing the hypothesis that PI(4,5)P2 protects ENaC from ubiquitination suggests that this protection does not improve the model results and that the control of the ENaC opening probability by PI(4,5)P2 is sufficient to explain all available data. The model analysis further demonstrates that ASL at the steady state is sensitive to small changes in PI(4,5)P2 abundance, particularly in CF conditions, which suggests that manipulation of phosphoinositide metabolism may promote therapeutic benefits for CF patients.
PMID: 31455163 [PubMed – in process]
CK19 stabilizes CFTR at the cell surface by limiting its endocytic pathway degradation.
FASEB J. 2019 Aug 26;:fj201901050R
Authors: Hou X, Wu Q, Rajagopalan C, Zhang C, Bouhamdan M, Wei H, Chen X, Zaman K, Li C, Sun X, Chen S, Frizzell RA, Sun F
Protein interactions that stabilize the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) at the apical membranes of epithelial cells have not yet been fully elucidated. We identified keratin 19 (CK19 or K19) as a novel CFTR-interacting protein. CK19 overexpression stabilized both wild-type (WT)-CFTR and Lumacaftor (VX-809)-rescued F508del-CFTR (where F508del is the deletion of the phenylalanine residue at position 508) at the plasma membrane (PM), promoting Cl- secretion across human bronchial epithelial (HBE) cells. CK19 prevention of Rab7A-mediated lysosomal degradation was a key mechanism in apical CFTR stabilization. Unexpectedly, CK19 expression was decreased by ∼40% in primary HBE cells from homogenous F508del patients with CF relative to non-CF controls. CK19 also positively regulated multidrug resistance-associated protein 4 expression at the PM, suggesting that this keratin may regulate the apical expression of other ATP-binding cassette proteins as well as CFTR.-Hou, X., Wu, Q., Rajagopalan, C., Zhang, C., Bouhamdan, M., Wei, H., Chen, X., Zaman, K., Li, C., Sun, X., Chen, S., Frizzell, R. A., Sun, F. CK19 stabilizes CFTR at the cell surface by limiting its endocytic pathway degradation.
PMID: 31450978 [PubMed – as supplied by publisher]
A new type of mouse has been developed that will advance in vivo testing of new CF therapies
A BAC Transgene Expressing Human CFTR under Control of Its Regulatory Elements Rescues Cftr Knockout Mice.
Sci Rep. 2019 Aug 14;9(1):11828
Authors: Gawenis LR, Hodges CA, McHugh DR, Valerio DM, Miron A, Cotton CU, Liu J, Walker NM, Strubberg AM, Gillen AE, Mutolo MJ, Kotzamanis G, Bosch J, Harris A, Drumm ML, Clarke LL
Small-molecule modulators of cystic fibrosis transmembrane conductance regulator (CFTR) biology show promise in the treatment of cystic fibrosis (CF). A Cftr knockout (Cftr KO) mouse expressing mutants of human CFTR would advance in vivo testing of new modulators. A bacterial artificial chromosome (BAC) carrying the complete hCFTR gene including regulatory elements within 40.1 kb of DNA 5′ and 25 kb of DNA 3′ to the gene was used to generate founder mice expressing hCFTR. Whole genome sequencing indicated a single integration site on mouse chromosome 8 (8qB2) with ~6 gene copies. hCFTR+ offspring were bred to murine Cftr KO mice, producing hCFTR+/mCftr- (H+/m-) mice, which had normal survival, growth and goblet cell function as compared to wild-type (WT) mice. Expression studies showed hCFTR protein and transcripts in tissues typically expressing mCftr. Functionally, nasal potential difference and large intestinal short-circuit (Isc) responses to cAMP stimulation were similar in magnitude to WT mice, whereas small intestinal cAMP ΔIsc responses were reduced. A BAC transgenic mouse with functional hCFTR under control of its regulatory elements has been developed to enable the generation of mouse models of hCFTR mutations by gene editing for in vivo testing of new CF therapies.
PMID: 31413336 [PubMed – in process]
Neutrophil Extracellular Trap Formation: Physiology, Pathology, and Pharmacology.
Biomolecules. 2019 Aug 14;9(8):
Authors: Ravindran M, Khan MA, Palaniyar N
Neutrophil extracellular traps (NETs), a unique DNA framework decorated with antimicrobial peptides, have been in the scientific limelight for their role in a variety of pathologies ranging from cystic fibrosis to cancer. The formation of NETs, as well as relevant regulatory mechanisms, physiological factors, and pharmacological agents have not been systematically discussed in the context of their beneficial and pathological aspects. Novel forms of NET formation including vital NET formation continue to be uncovered, however, there remain fundamental questions around established mechanisms such as NADPH-oxidase (Nox)-dependent and Nox-independent NET formation. Whether NET formation takes place in the tissue versus the bloodstream, internal factors (e.g. reactive oxygen species (ROS) production and transcription factor activation), and external factors (e.g. alkaline pH and hypertonic conditions), have all been demonstrated to influence specific NET pathways. Elements of neutrophil biology such as transcription and mitochondria, which were previously of unknown significance, have been identified as critical mediators of NET formation through facilitating chromatin decondensation and generating ROS, respectively. While promising therapeutics inhibiting ROS, transcription, and gasdermin D are being investigated, neutrophil phagocytosis plays a critical role in host defense and any therapies targeting NET formation must avoid impairing the physiological functions of these cells. This review summarizes what is known in the many domains of NET research, highlights the most relevant challenges in the field, and inspires new questions that can bring us closer to a unified model of NET formation.
PMID: 31416173 [PubMed – in process]
The impact of CFTR modulator therapies on CF airway microbiology.
J Cyst Fibros. 2019 Aug 12;:
Authors: Rogers GB, Taylor SL, Hoffman LR, Burr LD
Major historical advances in cystic fibrosis (CF) respiratory clinical care, including mechanical airway clearance and inhaled medications, have aimed to address the consequences of cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction. In contrast, CFTR modulator therapies instead target the underlying protein defect that leads to CF lung disease. The extent to which these therapies might reduce susceptibility to chronic lung infections remains to be seen. However, by improving airway clearance, reducing the requirement for antibiotics, and in some cases, through direct antimicrobial effects, CFTR modulators are likely to result in substantial changes in CF airway microbiology. These changes could contribute substantially to the clinical benefit associated with modulator therapies, as well as providing an important indicator of treatment efficacy and residual pathophysiology. Indeed, the widespread introduction of modulator therapies might require us to re-consider our models of CF airway microbiology.
PMID: 31416774 [PubMed – as supplied by publisher]
The role of endothelial cells in cystic fibrosis.
J Cyst Fibros. 2019 Aug 07;:
Authors: Declercq M, Treps L, Carmeliet P, Witters P
Cystic fibrosis (CF) is an autosomal recessive disease caused by the loss of function of the cystic fibrosis transmembrane conductance regulator (CFTR) protein which primarily acts as a chloride channel. CFTR has mainly been studied in epithelial cells although it is also functional and expressed in other cell types including endothelial cells. The present review summarizes current knowledge on the role of the endothelium in CF. More specifically, this review highlights the role of endothelial cells in CF in acting as a semipermeable barrier, as a key regulator of angiogenesis, coagulation, the vascular tone and the inflammatory responses. It could contribute to different aspects of the disease including cardiovascular symptoms, excessive blood vessel formation, pulmonary and portal hypertension and CF-related diabetes. Despite the important role of vascular endothelium in many biological processes, it has largely been under investigated in CF.
PMID: 31401006 [PubMed – as supplied by publisher]
Metabolic Reprograming of Cystic Fibrosis Macrophages via the IRE1α Arm of the Unfolded Protein Response Results in Exacerbated Inflammation.
Front Immunol. 2019;10:1789
Authors: Lara-Reyna S, Scambler T, Holbrook J, Wong C, Jarosz-Griffiths HH, Martinon F, Savic S, Peckham D, McDermott MF
Cystic Fibrosis (CF) is a recessive genetic disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR mutations cause dysregulation of channel function with intracellular accumulation of misfolded proteins and endoplasmic reticulum (ER) stress, with activation of the IRE1α-XBP1 pathway that regulates a subset of unfolded protein response (UPR) genes. This pathway regulates a group of genes that control proinflammatory and metabolic responses in different immune cells; however, the metabolic state of immune cells and the role of this pathway in CF remain elusive. Our results indicate that only innate immune cells from CF patients present increased levels of ER stress, mainly affecting neutrophils, monocytes, and macrophages. An overactive IRE1α-XBP1 pathway reprograms CF M1 macrophages toward an increased metabolic state, with increased glycolytic rates and mitochondrial function, associated with exaggerated production of TNF and IL-6. This hyper-metabolic state, seen in CF macrophages, is reversed by inhibiting the RNase domain of IRE1α, thereby decreasing the increased glycolic rates, mitochondrial function and inflammation. Altogether, our results indicate that innate immune cells from CF patients are primarily affected by ER stress. Moreover, the IRE1α-XBP1 pathway of the UPR is responsible for the hyper-metabolic state seen in CF macrophages, which is associated with the exaggerated inflammatory response. Modulating ER stress, metabolism and inflammation, by targeting IRE1α, may improve the metabolic fitness of macrophages, and other immune cells in CF and other immune-related disorders.
PMID: 31428093 [PubMed – in process]
The CXCL12/CXCR4 Signaling Axis Retains Neutrophils at Inflammatory Sites in Zebrafish.
Front Immunol. 2019;10:1784
Authors: Isles HM, Herman KD, Robertson AL, Loynes CA, Prince LR, Elks PM, Renshaw SA
The inappropriate retention of neutrophils at inflammatory sites is a major driver of the excessive tissue damage characteristic of respiratory inflammatory diseases including COPD, ARDS, and cystic fibrosis. The molecular programmes which orchestrate neutrophil recruitment to inflammatory sites through chemotactic guidance have been well-studied. However, how neutrophil sensitivity to these cues is modulated during inflammation resolution is not understood. The identification of neutrophil reverse migration as a mechanism of inflammation resolution and the ability to modulate this therapeutically has identified a new target to treat inflammatory disease. Here we investigate the role of the CXCL12/CXCR4 signaling axis in modulating neutrophil retention at inflammatory sites. We used an in vivo tissue injury model to study neutrophilic inflammation using transgenic zebrafish larvae. Expression of cxcl12a and cxcr4b during the tissue damage response was assessed using in situ hybridization and analysis of RNA sequencing data. CRISPR/Cas9 was used to knockdown cxcl12a and cxcr4b in zebrafish larvae. The CXCR4 antagonist AMD3100 was used to block the Cxcl12/Cxcr4 signaling axis pharmacologically. We identified that cxcr4b and cxcl12a are expressed at the wound site in zebrafish larvae during the inflammatory response. Following tail-fin transection, removal of neutrophils from inflammatory sites is significantly increased in cxcr4b and cxcl12a CRISPR knockdown larvae. Pharmacological inhibition of the Cxcl12/Cxcr4 signaling axis accelerated resolution of the neutrophil component of inflammation, an effect caused by an increase in neutrophil reverse migration. The findings of this study suggest that CXCR4/CXCL12 signaling may play an important role in neutrophil retention at inflammatory sites, identifying a potential new target for the therapeutic removal of neutrophils from the lung in chronic inflammatory disease.
PMID: 31417560 [PubMed – in process]
Advances In Gene Therapy For Cystic Fibrosis Lung Disease.
Hum Mol Genet. 2019 Jul 23;:
Authors: Yan Z, McCray PB, Engelhardt JF
Cystic fibrosis (CF) is a multi-organ recessive genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Gene therapy efforts have focused on treating the lung, since it manifests the most significant life-threatening disease. Over two decades have past since the first CF lung gene therapy trials and significant advances in the therapeutic implementation of pharmacologic CFTR modulators have renewed the field’s focus on developing gene therapies for the 10% of CF patients these modulators cannot help. This review summarizes recent progress made in developing vectors for airway transduction and CF animal models required for understanding the relevant cellular targets in the lung and testing the efficacy of gene therapy approaches. We also highlight future opportunities in emerging gene editing strategies that may offer advantages for treating diseases like CF where the gene target is highly regulated at the cellular level. The outcomes of CF lung gene therapy trials will likely inform productive paths towards gene therapy for other complex genetic disorders, while also advancing treatments for all CF patients.
PMID: 31332440 [PubMed – as supplied by publisher]
Discovering the chloride pathway in the CFTR channel.
Cell Mol Life Sci. 2019 Jul 20;:
Authors: Farkas B, Tordai H, Padányi R, Tordai A, Gera J, Paragi G, Hegedűs T
Cystic fibrosis (CF), a lethal monogenic disease, is caused by pathogenic variants of the CFTR chloride channel. The majority of CF mutations affect protein folding and stability leading overall to diminished apical anion conductance of epithelial cells. The recently published cryo-EM structures of full-length human and zebrafish CFTR provide a good model to gain insight into structure-function relationships of CFTR variants. Although, some of the structures were determined in the phosphorylated and ATP-bound active state, none of the static structures showed an open pathway for chloride permeation. Therefore, we performed molecular dynamics simulations to generate a conformational ensemble of the protein and used channel detecting algorithms to identify conformations with an opened channel. Our simulations indicate a main intracellular entry at TM4/6, a secondary pore at TM10/12, and a bottleneck region involving numerous amino acids from TM1, TM6, and TM12 in accordance with experiments. Since chloride ions entered the pathway in our equilibrium simulations, but did not traverse the bottleneck region, we performed metadynamics simulations, which revealed two possible exits. One of the chloride ions exits includes hydrophobic lipid tails that may explain the lipid-dependency of CFTR function. In summary, our in silico study provides a detailed description of a potential chloride channel pathway based on a recent cryo-EM structure and may help to understand the gating of the CFTR chloride channel, thus contributing to novel strategies to rescue dysfunctional mutants.
PMID: 31327045 [PubMed – as supplied by publisher]
Lumacaftor-ivacaftor in the treatment of cystic fibrosis: design, development and place in therapy.
Drug Des Devel Ther. 2019;13:2405-2412
Authors: Connett GJ
Lumacaftor-ivacaftor is a combination of two small molecule therapies targeting the basic defect in cystic fibrosis (CF) at a cellular level. It is a precision medicine and its effects are specific to individuals with two copies of the p.Phe508del gene mutation. The drug combination works by restoring functioning CF transmembrane conductance regulator (CFTR) protein in cell surface membranes and was the first CFTR modulator licensed for the homozygous p.Phe508del genotype. The drug is a combination of a CFTR corrector and potentiator. Lumacaftor, the corrector, works by increasing the trafficking of CFTR proteins to the outer cell membrane. Ivacaftor, the potentiator, works by enabling the opening of what would otherwise be a dysfunctional chloride channel. In vivo lumacaftor-ivacaftor improves Phe508del-CFTR activity in airways, sweat ducts and intestine to approximately 10-20% of normal CFTR function with greater reductions in sweat chloride levels in children versus adults. Its use results in a modest improvement in lung function and a decreased rate of subsequent decline. Perhaps more importantly, those treated report increased levels of well-being and their rate of respiratory exacerbations is significantly improved. This review traces the development and use of this combination of CFTR modulators, the first licensed drug for treating the homozygous p.Phe508del CF genotype at the intracellular level by correcting the protein defect.
PMID: 31409974 [PubMed – in process]
Targeted Activation of Cystic Fibrosis Transmembrane Conductance Regulator.
Mol Ther. 2019 Jul 15;:
Authors: Villamizar O, Waters SA, Scott T, Saayman S, Grepo N, Urak R, Davis A, Jaffe A, Morris KV
Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. The majority of CFTR mutations result in impaired chloride channel function as only a fraction of the mutated CFTR reaches the plasma membrane. The development of a therapeutic approach that facilitates increased cell-surface expression of CFTR could prove clinically relevant. Here, we evaluate and contrast two molecular approaches to activate CFTR expression. We find that an RNA-guided nuclease null Cas9 (dCas9) fused with a tripartite activator, VP64-p65-Rta can activate endogenous CFTR in cultured human nasal epithelial cells from CF patients. We also find that targeting BGas, a long non-coding RNA involved in transcriptionally modulating CFTR expression with a gapmer, induced both strong knockdown of BGas and concordant activation of CFTR. Notably, the gapmer can be delivered to target cells when generated as electrostatic particles with recombinant HIV-Tat cell penetrating peptide (CPP), when packaged into exosomes, or when loaded into lipid nanoparticles (LNPs). Treatment of patient-derived human nasal epithelial cells containing F508del with gapmer-CPP, gapmer-exosomes, or LNPs resulted in increased expression and function of CFTR. Collectively, these observations suggest that CRISPR/dCas-VPR (CRISPR) and BGas-gapmer approaches can target and specifically activate CFTR.
PMID: 31383454 [PubMed – as supplied by publisher]
Nanotechnology based therapeutics for lung disease.
Thorax. 2019 Jul 08;:
Authors: Doroudian M, MacLoughlin R, Poynton F, Prina-Mello A, Donnelly SC
Nanomedicine is a multidisciplinary research field with an integration of traditional sciences such as chemistry, physics, biology and materials science. The application of nanomedicine for lung diseases as a relatively new area of interdisciplinary science has grown rapidly over the last 10 years. Promising research outcomes suggest that nanomedicine will revolutionise the practice of medicine, through the development of new approaches in therapeutic agent delivery, vaccine development and nanotechnology-based medical detections. Nano-based approaches in the diagnosis and treatment of lung diseases will, in the not too distant future, change the way we practise medicine. This review will focus on the current trends and developments in the clinical translation of nanomedicine for lung diseases, such as in the areas of lung cancer, cystic fibrosis, asthma, bacterial infections and COPD.
PMID: 31285360 [PubMed – as supplied by publisher]
CFTR-PTEN-dependent mitochondrial metabolic dysfunction promotes Pseudomonas aeruginosa airway infection.
Sci Transl Med. 2019 Jul 03;11(499):
Authors: Riquelme SA, Lozano C, Moustafa AM, Liimatta K, Tomlinson KL, Britto C, Khanal S, Gill SK, Narechania A, Azcona-Gutiérrez JM, DiMango E, Saénz Y, Planet P, Prince A
Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a tumor suppressor best known for regulating cell proliferation and metabolism. PTEN forms a complex with the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) at the plasma membrane, and this complex is known to be functionally impaired in CF. Here, we demonstrated that the combined effect of PTEN and CFTR dysfunction stimulates mitochondrial activity, resulting in excessive release of succinate and reactive oxygen species. This environment promoted the colonization of the airway by Pseudomonas aeruginosa, bacteria that preferentially metabolize succinate, and stimulated an anti-inflammatory host response dominated by immune-responsive gene 1 (IRG1) and itaconate. The recruitment of myeloid cells induced by these strains was inefficient in clearing the infection and increased numbers of phagocytes accumulated under CFTR-PTEN axis dysfunction. This central metabolic defect in mitochondrial function due to impaired PTEN activity contributes to P. aeruginosa infection in CF.
PMID: 31270271 [PubMed – in process]
Enabling Synthesis of ABBV-2222, A CFTR Corrector for the Treatment of Cystic Fibrosis.
Org Lett. 2019 Jul 01;:
Authors: Greszler SN, Shelat B, Voight EA
An enabling preclinical synthetic route to cystic fibrosis candidate ABBV-2222 is described. Two stereoselective steps provide access to an aminochroman intermediate with excellent control, and a late-stage demethylation/difluoromethylation sequence provides efficient access to the target molecule.
PMID: 31259557 [PubMed – as supplied by publisher]
The Emerging Role of Neutrophil Extracellular Traps in Respiratory Disease.
Chest. 2019 Jun 29;:
Authors: Twaddell SH, Baines KJ, Grainge C, Gibson PG
Neutrophil extracellular traps (NETs) are extrusions of intracellular DNA and attached granular material which enable bacterial killing. NETs are increasingly recognised for their role in the pathogenesis of respiratory disease. NETs are composed of a complex mix of intracellular derived material which neutrophils organise within the cytoplasm then expel in a non-directed manner in the vicinity of invading organisms. Combined, these trap and destroy multiple genera of microbes including bacteria, fungi, viruses and protozoans, limiting infection especially where phagocytosis is not possible. Initially NET formation was thought to be a terminal event for neutrophils, however it is now apparent that some neutrophils survive this process becoming anuclear and may drive ongoing tissue damage. NETs are now known to be directly cytotoxic to lung epithelium and endothelium and their excessive production is seen in pneumonia and acute lung injury as well as several chronic diseases, including COPD, asthma and cystic fibrosis. NETs also appear to play a role in both tumour defence and dissemination depending on the local microenvironment and the specific tumour subtype. It is becoming increasingly apparent that NET formation can be a positive or negative influence on multiple respiratory pathologies and that simply globally reducing or increasing NET formation is unlikely to be a therapeutic success. Rather, as our understanding grows, it is likely that targeted NET up or downregulation along with destruction or protection of already formed NETs may be become an additional point of intervention for respiratory physicians.
PMID: 31265835 [PubMed – as supplied by publisher]
Evaluation of eluforsen, a novel RNA oligonucleotide for restoration of CFTR function in in vitro and murine models of p.Phe508del cystic fibrosis.
PLoS One. 2019;14(6):e0219182
Authors: Beumer W, Swildens J, Leal T, Noel S, Anthonijsz H, van der Horst G, Kuiperij-Boersma H, Potman M, van Putten C, Biasutto P, Platenburg G, de Jonge H, Henig N, Ritsema T
Cystic fibrosis (CF) is caused by mutations in the gene encoding the epithelial chloride channel CF transmembrane conductance regulator (CFTR) protein. The most common mutation is a deletion of three nucleotides leading to the loss of phenylalanine at position 508 (p.Phe508del) in the protein. This study evaluates eluforsen, a novel, single-stranded, 33-nucleotide antisense oligonucleotide designed to restore CFTR function, in in vitro and in vivo models of p.Phe508del CF. The aims of the study were to demonstrate cellular uptake of eluforsen, and its efficacy in functional restoration of p.Phe508del-CFTR both in vitro and in vivo. In vitro, the effect of eluforsen was investigated in human CF pancreatic adenocarcinoma cells and human bronchial epithelial cells. Two mouse models were used to evaluate eluforsen in vivo. In vitro, eluforsen improved chloride efflux in CF pancreatic adenocarcinoma cell cultures and increased short-circuit current in primary human bronchial epithelial cells, both indicating restoration of CFTR function. In vivo, eluforsen was taken up by airway epithelium following oro-tracheal administration in mice, resulting in systemic exposure of eluforsen. In female F508del-CFTR mice, eluforsen significantly increased CFTR-mediated saliva secretion (used as a measure of CFTR function, equivalent to the sweat test in humans). Similarly, intranasal administration of eluforsen significantly improved nasal potential difference (NPD), and therefore CFTR conductance, in two CF mouse models. These findings indicate that eluforsen improved CFTR function in cell and animal models of p.Phe508del-CFTR-mediated CF and supported further development of eluforsen in human clinical trials, where eluforsen has also been shown to improve CFTR activity as measured by NPD.
PMID: 31251792 [PubMed – in process]
Emerging gene therapies for cystic fibrosis.
Expert Rev Respir Med. 2019 Jun 19;:
Authors: Miah KM, Hyde SC, Gill DR
Introduction: Cystic fibrosis (CF) remains a life-threatening genetic disease, with few clinically effective treatment options. Gene therapy and gene editing strategies offer the potential for a one-time CF cure, irrespective of the CFTR mutation class. Areas covered: We review emerging gene therapies and gene delivery strategies for the treatment of CF particularly viral and non-viral approaches with potential to treat CF. Expert opinion: It was initially anticipated that the challenge of developing a gene therapy for CF lung disease would be met relatively easily. Following early proof-of-concept clinical studies, CF gene therapy has entered a new era with innovative vector designs, approaches to subvert the humoral immune system and increase gene delivery and gene correction efficiencies. Developments include integrating adenoviral vectors, rapamycin loaded nanoparticles and lung-tropic lentiviral vectors. The characterisation of novel cell types in the lung epithelium, including pulmonary ionocytes, may also encourage cell type-specific targeting for CF correction. We anticipate preclinical studies to further validate these strategies, which should pave the way for clinical trials. We also expect gene editing efficiencies to improve to clinically translatable levels, given advancements in viral and non-viral vectors. Overall, gene delivery technologies look more convincing in producing an effective CF gene therapy.
PMID: 31215818 [PubMed – as supplied by publisher]
Effectivenesss of ivacaftor in severe cystic fibrosis patients and non-G551D gating mutations.
Pediatr Pulmonol. 2019 Jun 25;:
Authors: Salvatore D, Carnovale V, Iacotucci P, Braggion C, Castellani C, Cimino G, Colangelo C, Francalanci M, Leonetti G, Lucidi V, Manca A, Vitullo P, Ferrara N
BACKGROUND: Ivacaftor is a significant innovation in the treatment of cystic fibrosis (CF) with gating mutations. A substantial percentage of patients with CF have severe lung involvement, but these patients are usually excluded from phase III clinical trials. Thus, the effectiveness of ivacaftor in this population has not been fully determined.
METHODS: Data were collected from Italian CF centers with patients enrolled in an ivacaftor compassionate use programme (percent predicted [pp] forced expiratory volume in 1 second [FEV1 ] < 40%, or on lung transplant waiting list, or with a fast worsening trend of lung function). Data were collected for 1 year before and 1 year after ivacaftor commencement. RESULTS: Thirteen patients received ivacaftor for a median of 320 days. Mean (SD) ppFEV1 increased from 35.1% (14.3%) before treatment to 46.6% (18.8%) after 12 months of treatment (absolute increase 11.5%, relative increase 32.8%). Mean distance of the 6-minute walking test improved significantly, from 535.1 m before to 611.6 m after 12 months of treatment (P = .002). The number of pulmonary exacerbations decreased significantly, from 57 during the year before ivacaftor to 28 in the year following ivacaftor (P = .0048). Five of the 13 patients (38.5%) had no exacerbations during the 12 months after starting ivacaftor. Median weight increased significantly, from 52.7 kg to 55.6 kg (P = .0031). Mean (SD) sweat chloride concentration decreased significantly, from 99.5 (22.8) mmol/L to 39.3 (15.8) mmol/L (P < .0001). No safety concerns were registered. CONCLUSIONS: Ivacaftor was safe and effective in patients with CF with severe lung disease and non-G551D gating mutations. PMID: 31237430 [PubMed - as supplied by publisher] PubMed:31237430
Cystic fibrosis drug ivacaftor stimulates CFTR channels at picomolar concentrations.
Elife. 2019 Jun 17;8:
Authors: Csanády L, Töröcsik B
The devastating inherited disease cystic fibrosis (CF) is caused by mutations of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) anion channel. The recent approval of the CFTR potentiator drug ivacaftor (Vx-770) for the treatment of CF patients has marked the advent of causative CF therapy. Currently, thousands of patients are being treated with the drug, and its molecular mechanism of action is under intensive investigation. Here we determine the solubility profile and true stimulatory potency of Vx-770 towards wild-type (WT) and mutant human CFTR channels in cell-free patches of membrane. We find that its aqueous solubility is ~200-fold lower (~60 nanomolar), whereas the potency of its stimulatory effect is >100-fold higher, than reported, and is unexpectedly fully reversible. Strong, but greatly delayed, channel activation by picomolar Vx-770 identifies multiple sequential slow steps in the activation pathway. These findings provide solid guidelines for the design of in vitro studies using Vx-770.
PMID: 31205003 [PubMed – as supplied by publisher]
Airway regeneration using iPS cell-derived airway epithelial cells with Cl- channel function.
Channels (Austin). 2019 Dec;13(1):227-234
Authors: Yoshie S, Omori K, Hazama A
induced pluripotent stem (iPS) cells can be differentiated into various cell types, including airway epithelial cells, since they have the capacity for self-renewal and pluripotency. Thus, airway epithelial cells generated from iPS cells are expected to be potent candidates for use in airway regeneration and the treatment of airway diseases such as cystic fibrosis (CF). Recently, it was reported that iPS cells can be differentiated into airway epithelial cells according to the airway developmental process. These studies demonstrate that airway epithelial cells generated from iPS cells are equivalent to their in vivo counterparts. However, it has not been evaluated in detail whether these cells exhibit physiological functions and are fully mature. Airway epithelial cells adequately control water volume on the airway surface via the function of Cl- channels. Reasonable environments on the airway surface cause ciliary movement with a constant rhythm and maintain airway clearance. Therefore, the generation of functional airway epithelial cells/tissues with Cl- channel function from iPS cells will be indispensable for cell/tissue replacement therapy, the development of a reliable airway disease model, and the treatment of airway disease. This review highlights the generation of functional airway epithelial cells from iPS cells and discusses the remaining challenges to the generation of functional airway epithelial cells for airway regeneration and the treatment of airway disease.
PMID: 31198082 [PubMed – in process]
Augmentation of CFTR function in human bronchial epithelial cells via SLC6A14-dependent amino acid uptake: Implications for treatment of Cystic Fibrosis.
Am J Respir Cell Mol Biol. 2019 Jun 12;:
Authors: Ahmadi S, Wu YS, Li M, Ip W, Lloyd-Kuzik A, Di Paola M, Du K, Xia S, Lew A, Bozoky Z, Forman-Kay J, Bear CE, Gonska T
RATIONALE: SLC6A14 mediated L-arginine transport has been shown to augment the residual anion channel activity of the major mutant, F508del-CFTR, in the murine gastrointestinal tract. It is not yet known if this transporter augments residual and pharmacological corrected F508del-CFTR in primary airway epithelia.
OBJECTIVE: To determine the role of L-arginine uptake via SLC6A14 in modifying F508del-CFTR channel activity in airway cells from CF patients.
METHODS: Human bronchial epithelial cells from lung explants of non-CF (HBE) and CF patients (CF-HBE) were used for H3-flux, airway surface liquid (ASL) and Ussing chamber studies. We used α-methyltryptophan (α-MT) as specific inhibitor for SLC6A14. CFBE41o-, a commonly used CF airway cell line, was employed for studying the mechanism of the functional interaction between SLC6A14 and F508del-CFTR.
MAIN RESULTS: SLC6A14 is functionally expressed in CF-HBE cells. L-arginine uptake via SLC6A14 augmented F508del-CFTR function at baseline and after treatment with lumacaftor. SLC6A14 mediated L-arginine uptake also increased the ASL in CF-HBE cells. Using CFBE41o cells we showed that the positive SLC6A14 effect was mainly dependent on the nitric oxide synthase activity, nitrogen oxides including nitric oxide (NO) and phosphorylation by protein kinase G. These finding were confirmed in CF-HBE as iNOS inhibition abrogated the functional interaction between SLC6A14 and pharmacological corrected F508del-CFTR.
CONCLUSION: SLC6A14-mediated L-arginine transport augments residual F508del-CFTR channel function via a non-canonical, NO pathway. This effect is enhanced with increasing pharmacological rescue of F508del-CFTR to the membrane. The current study demonstrates how endogenous pathways can be utilized for the development of companion therapy in CF.
PMID: 31189070 [PubMed – as supplied by publisher]
Novel AAV-mediated gene delivery system corrects CFTR function in pigs.
Am J Respir Cell Mol Biol. 2019 Jun 11;:
Authors: Cooney AL, Thornell IM, Singh BK, Shah VS, Stoltz DA, McCray PB, Zabner J, Sinn PL
Cystic fibrosis (CF) is an autosomal recessive disease caused by a mutant cystic fibrosis transmembrane conductance regulator (CFTR) gene and characterized by chronic bacterial lung infections and inflammation. Complementation with functional CFTR normalizes anion transport across the airway surface. Adeno-associated virus (AAV) is a useful vector for gene therapy because of its low immunogenicity and ability to persist for months to years. However, episomal expression may decrease following cell division and ultimately require readministration. To overcome this, we designed an integrating AAV-based CFTR expressing vector termed PB/AAV carrying CFTR flanked by the terminal repeats (TRs) of the piggyBac transposon. With co-delivery of piggyBac transposase, PB/AAV can integrate into the host genome. Because of the AAV packaging constraints, careful consideration was required to ensure that the vector would package and express its CFTR cDNA cargo. In this short-term study, PB/AAV-CFTR was aerosolized to the airways of CF pigs in the absence of the transposase. Two weeks later, transepithelial Cl- current was restored in freshly excised tracheal and bronchus tissue. Additionally, we observed an increase in tracheal airway surface liquid (ASL) pH and bacterial killing as compared to untreated CF pigs. ASL from primary airway cells cultured from treated CF pigs exhibited increased pH correlating with decreased viscosity. Together, these results show that complementing CFTR in CF pigs with PB/AAV rescues the anion transport defect in a large animal CF model. Delivery of this integrating viral vector system to airway progenitor cells could lead to persistent, life-long expression in vivo.
PMID: 31184507 [PubMed – as supplied by publisher]
Antisense oligonucleotide eluforsen is safe and improves respiratory symptoms in F508DEL cystic fibrosis.
J Cyst Fibros. 2019 Jun 07;:
Authors: Drevinek P, Pressler T, Cipolli M, De Boeck K, Schwarz C, Bouisset F, Boff M, Henig N, Paquette-Lamontagne N, Montgomery S, Perquin J, Tomkinson N, den Hollander W, Elborn JS
BACKGROUND: Eluforsen is an antisense oligonucleotide designed to bind to the mRNA region around the F508-encoding deletion and restore the cystic fibrosis transmembrane conductance regulator (CFTR) protein function in the airway epithelium. We assessed the safety and tolerability, pharmacokinetics and exploratory measures of efficacy of inhaled eluforsen in cystic fibrosis (CF) patients homozygous for the F508del-CFTR mutation.
METHODS: This randomised, double-blind, placebo-controlled, dose escalation 1b study recruited adult CF subjects with a FEV1 > 70% predicted in four single ascending dose cohorts and four multiple ascending dose cohorts. Primary objectives were safety and tolerability. Secondary endpoints included pharmacokinetics, percent predicted forced expiratory volume in 1 s (ppFEV1), and Cystic Fibrosis Questionnaire-Revised (CFQ-R) Respiratory Symptom Score (RSS).
RESULTS: Single and multiple doses of inhaled eluforsen up to 50 mg were safe and well tolerated. A maximum tolerated dose was not established. Systemic exposure was low in all cohorts and lung function remained stable throughout the study. Three of four eluforsen-treated groups in the MAD study demonstrated an improvement in CFQ-R RSS at end of treatment with adjusted mean change from baseline values ranging from 6.4 to 12.7 points. In comparison, there was a mean decrease of 6.5 points in the placebo group from baseline to end of treatment.
CONCLUSIONS: Inhaled eluforsen up to 50 mg dosed 3 times per week for 4 weeks was safe and well tolerated, showed low systemic exposure, and demonstrated improvement in CFQ-R RSS, a relevant measure of clinical benefit in CF patients.
PMID: 31182369 [PubMed – as supplied by publisher]
Network Biology Identifies Novel Regulators of CFTR Trafficking and Membrane Stability.
Front Pharmacol. 2019;10:619
Authors: Loureiro CA, Santos JD, Matos AM, Jordan P, Matos P, Farinha CM, Pinto FR
In cystic fibrosis, the most common disease-causing mutation is F508del, which causes not only intracellular retention and degradation of CFTR, but also defective channel gating and decreased membrane stability of the small amount that reaches the plasma membrane (PM). Thus, pharmacological correction of mutant CFTR requires targeting of multiple cellular defects in order to achieve clinical benefit. Although small-molecule compounds have been identified and commercialized that can correct its folding or gating, an efficient retention of F508del CFTR at the PM has not yet been explored pharmacologically despite being recognized as a crucial factor for improving functional rescue of chloride transport. In ongoing efforts to determine the CFTR interactome at the PM, we used three complementary approaches: targeting proteins binding to tyrosine-phosphorylated CFTR, protein complexes involved in cAMP-mediated CFTR stabilization at the PM, and proteins selectively interacting at the PM with rescued F508del-CFTR but not wt-CFTR. Using co-immunoprecipitation or peptide-pull down strategies, we identified around 400 candidate proteins through sequencing of complex protein mixtures using the nano-LC Triple TOF MS technique. Key candidate proteins were validated for their robust interaction with CFTR-containing protein complexes and for their ability to modulate the amount of CFTR expressed at the cell surface of bronchial epithelial cells. Here, we describe how we explored the abovementioned experimental datasets to build a protein interaction network with the aim of identifying novel pharmacological targets to rescue CFTR function in cystic fibrosis (CF) patients. We identified and validated novel candidate proteins that were essential components of the network but not detected in previous proteomic analyses.
PMID: 31231217 [PubMed]
Focus on TRP channels in cystic fibrosis.
Cell Calcium. 2019 May 30;81:29-37
Authors: Grebert C, Becq F, Vandebrouck C
The Transient Receptor Potential (TRP) protein superfamily is a group of cation channels expressed in various cell types and involved in respiratory diseases such as cystic fibrosis (CF), the genetic disease caused by CF Transmembrane conductance Regulator (CFTR) mutations. In human airway epithelial cells, there is growing evidence for a functional link between CFTR and TRP channels. TRP channels contribute to transmitting extracellular signals into the cells and, in an indirect manner, to CFTR activity via a Ca2+ rise signaling. Indeed, mutated CFTR-epithelial cells are characterized by an increased Ca2+ influx and, on the opposite, by a decreased of magnesium influx, both being mediated by TRP channels. This increasing cellular Ca2+ triggers the activation of calcium-activated chloride channels (CaCC) or CFTR itself, via adenylyl cyclase, PKA and tyrosine kinases activation, but also leads to an exaltation of the inflammatory response. Another shortcoming in mutated CFTR-epithelial cells is a [Mg2+]i decrease, associated with impaired TRPM7 functioning. This deregulation has to be taken into consideration in CF physiopathology, as Mg2+ is required for ATP hydrolysis and CFTR activity. The modulation of druggable TRP channels could supplement CF therapy either an anti-inflammatory drug or for CFTR potentiation, according to the balance between exacerbation and respite phases. The present paper focus on TRPA1, TRPC6, TRPM7, TRPV2, TRPV4, TRPV6 and ORAI 1, the proteins identified, for now, as dysfunctional channels, in CF cells.
PMID: 31176886 [PubMed – as supplied by publisher]
Antibiofilm efficacy of nitric oxide-releasing alginates against cystic fibrosis bacterial pathogens.
ACS Infect Dis. 2019 May 28;:
Authors: Ahonen MJR, Dorrier JM, Schoenfisch MH
Colonization of the lungs by biofilm-forming pathogens is a major cause of mortality in cystic fibrosis (CF). In CF patients, these pathogens are difficult to treat due to the additional protection provided by both the biofilm exopolysaccharide matrix and thick, viscous mucus. The antibiofilm efficacy of nitric oxide (NO)-releasing alginates were evaluated against Pseudomonas aeruginosa, Burkholderia cepacia, Staphylococcus aureus, and methicillin-resistant S. aureus biofilms in both aerobic and anaerobic environments. Varying the amine precursor grafted onto alginate oligosaccharides imparted tunable NO storage (~0.1-0.3 µmol/mg) and release kinetics (~4-40 min half-lives) in the artificial sputum media used for biofilm testing. The NO-releasing alginates were highly antibacterial against the four CF-relevant pathogens, achieving a 5-log reduction in biofilm viability after 24-h treatment, with biocidal efficacy dependent on NO-release kinetics. Aerobic biofilms required greater starting NO doses to achieve killing relative to the anaerobic biofilms. Relative to tobramycin (MBEC24h ≥2000 µg/mL) and vancomycin (MBEC24h ≥1000 µg/mL), the NO-releasing alginates proved to be more effective (NO dose ≤520 µg/mL) regardless of growth conditions.
PMID: 31136714 [PubMed – as supplied by publisher]
A Systematic Review of the Clinical Efficacy and Safety of CFTR Modulators in Cystic Fibrosis.
Sci Rep. 2019 May 10;9(1):7234
Authors: Habib AR, Kajbafzadeh M, Desai S, Yang CL, Skolnik K, Quon BS
Several placebo-controlled trials have been recently published evaluating novel therapies targeting the defective CFTR protein. This systematic review examines the clinical efficacy and safety of CFTR modulators in individuals with cystic fibrosis (CF) with specific genetic mutations. Online sources were searched for placebo-controlled, parallel-design clinical trials investigating CFTR modulators from January 1, 2005 to March 31, 2018. The primary outcome of interest was FEV1% predicted (ppFEV1). Fourteen RCTs met our eligibility criteria. The largest improvement in ppFEV1 favouring treatment was observed for ivacaftor (IVA) in G551D individuals (≥6 years old). Both tezacaftor-ivacaftor (TEZ-IVA) and lumacaftor-ivacaftor (LUM-IVA) also improved ppFEV1 in F508del homozygous individuals but there was increased reporting of respiratory adverse events with LUM-IVA compared to placebo. IVA also significantly improved ppFEV1 in a sub-group of individuals ≥18 years old with an R117H mutation. No significant improvements in ppFEV1 were observed for IVA, LUM, or TEZ in F508del homozygous individuals, LUM or LUM-IVA in F508del heterozygous individuals, or ataluren in individuals with a nonsense mutation. Significant improvements in ppFEV1 and other clinical outcomes were observed for IVA in G551D individuals, TEV-IVA and LUM-IVA in F508del homozygous individuals, and IVA in adults with a R117H mutation.
PMID: 31076617 [PubMed – in process]
Identification of GLPG/ABBV-2737, a Novel Class of Corrector, Which Exerts Functional Synergy With Other CFTR Modulators.
Front Pharmacol. 2019;10:514
Authors: de Wilde G, Gees M, Musch S, Verdonck K, Jans M, Wesse AS, Singh AK, Hwang TC, Christophe T, Pizzonero M, Van der Plas S, Desroy N, Cowart M, Stouten P, Nelles L, Conrath K
The deletion of phenylalanine at position 508 (F508del) in cystic fibrosis transmembrane conductance regulator (CFTR) causes a severe defect in folding and trafficking of the chloride channel resulting in its absence at the plasma membrane of epithelial cells leading to cystic fibrosis. Progress in the understanding of the disease increased over the past decades and led to the awareness that combinations of mechanistically different CFTR modulators are required to obtain meaningful clinical benefit. Today, there remains an unmet need for identification and development of more effective CFTR modulator combinations to improve existing therapies for patients carrying the F508del mutation. Here, we describe the identification of a novel F508del corrector using functional assays. We provide experimental evidence that the clinical candidate GLPG/ABBV-2737 represents a novel class of corrector exerting activity both on its own and in combination with VX809 or GLPG/ABBV-2222.
PMID: 31143125 [PubMed]
Structural analogues of roscovitine rescue the intracellular traffic and the function of ER-retained ABCB4 variants in cell models.
Sci Rep. 2019 Apr 30;9(1):6653
Authors: Vauthier V, Ben Saad A, Elie J, Oumata N, Durand-Schneider AM, Bruneau A, Delaunay JL, Housset C, Aït-Slimane T, Meijer L, Falguières T
Adenosine triphosphate binding cassette transporter, subfamily B member 4 (ABCB4) is the transporter of phosphatidylcholine at the canalicular membrane of hepatocytes. ABCB4 deficiency, due to genetic variations, is responsible for progressive familial intrahepatic cholestasis type 3 (PFIC3) and other rare biliary diseases. Roscovitine is a molecule in clinical trial that was shown to correct the F508del variant of cystic fibrosis transmembrane conductance regulator (CFTR), another ABC transporter. In the present study, we hypothesized that roscovitine could act as a corrector of ABCB4 traffic-defective variants. Using HEK and HepG2 cells, we showed that roscovitine corrected the traffic and localisation at the plasma membrane of ABCB4-I541F, a prototypical intracellularly retained variant. However, roscovitine caused cytotoxicity, which urged us to synthesize non-toxic structural analogues. Roscovitine analogues were able to correct the intracellular traffic of ABCB4-I541F in HepG2 cells. Importantly, the phospholipid secretion activity of this variant was substantially rescued by three analogues (MRT2-235, MRT2-237 and MRT2-243) in HEK cells. We showed that these analogues also triggered the rescue of intracellular traffic and function of two other intracellularly retained ABCB4 variants, i.e. I490T and L556R. Our results indicate that structural analogues of roscovitine can rescue genetic variations altering the intracellular traffic of ABCB4 and should be considered as therapeutic means for severe biliary diseases caused by this class of variations.
PMID: 31040306 [PubMed – in process]
Restoration of F508-del Function by Transcomplementation: The Partners Meet in the Endoplasmic Reticulum.
Cell Physiol Biochem. 2019;52(6):1267-1279
Authors: Bergbower EAS, Sabirzhanova I, Boinot C, Guggino WB, Cebotaru L
BACKGROUND/AIMS: Because of the small size of adeno-associated virus, AAV, the cystic fibrosis conductance regulator, CFTR, cDNA is too large to fit within AAV and must be truncated. We report here on two truncated versions of CFTR, which, when inserted into AAV1 and used to infect airway cells, rescue F508-del CFTR via transcomplementation. The purpose of this study is to shed light on where in the cell transcomplementation occurs and how it results in close association between the endogenous F508-del and truncated CFTR.
METHODS: We treated CF airway cells (CFBE41o-) with AAV2/1 (AAV2 inverted terminal repeats/AAV1 capsid) containing truncated forms of CFTR, ∆264 and ∆27-264 CFTR, who can restore the function of F508-del by transcomplementation. We addressed the aims of the study using a combination of confocal microscopy and short circuit currents measurements. For the latter, CF bronchial epithelial cells (CFBE) were grown on permeable supports.
RESULTS: We show that both F508del and the truncation mutants colocalize in the ER and that both the rescued F508-del and the transcomplementing mutants reach the plasma membrane together. There was significant fluorescence resonance energy transfer (FRET) between F508-del and the transcomplementing mutants within the endoplasmic reticulum (ER), suggesting that transcomplementation occurs through a bimolecular interaction. We found that transcomplementation could increase the Isc in CFBE41o- cells stably expressing additional wt-CFTR or F508-del and in parental CFBE41o- cells expressing endogenous levels of F508-del.
CONCLUSION: We conclude that the functional rescue of F508-del by transcomplementation occurs via a bimolecular interaction that most likely begins in the ER and continues at the plasma membrane. These results come at an opportune time for developing a gene therapy for CF and offer new treatment options for a wide range of CF patients.
PMID: 31026390 [PubMed – in process]
Exploring the effect of chirality on the therapeutic potential of N-alkyl-deoxyiminosugars: anti-inflammatory response to Pseudomonas aeruginosa infections for application in CF lung disease.
Eur J Med Chem. 2019 Apr 25;175:63-71
Authors: De Fenza M, D’Alonzo D, Esposito A, Munari S, Loberto N, Santangelo A, Lampronti I, Tamanini A, Rossi A, Ranucci S, De Fino I, Bragonzi A, Aureli M, Bassi R, Tironi M, Lippi G, Gambari R, Cabrini G, Palumbo G, Dechecchi MC, Guaragna A
In the frame of a research program aimed to explore the relationship between chirality of iminosugars and their therapeutic potential, herein we report the synthesis of N-akyl l-deoxyiminosugars and the evaluation of the anti-inflammatory properties of selected candidates for the treatment of Pseudomonas aeruginosa infections in Cystic Fibrosis (CF) lung disease. Target glycomimetics were prepared by the shortest and most convenient approach reported to date, relying on the use of the well-known PS-TPP/I2 reagent system to prepare reactive alkoxyalkyl iodides, acting as key intermediates. Iminosugars ent-1-3 demonstrated to efficiently reduce the inflammatory response induced by P. aeruginosa in CuFi cells, either alone or in synergistic combination with their d-enantiomers, by selectively inhibiting NLGase. Surprisingly, the evaluation in murine models of lung disease showed that the amount of ent-1 required to reduce the recruitment of neutrophils was 40-fold lower than that of the corresponding d-enantiomer. The remarkably low dosage of the l-iminosugar, combined with its inability to act as inhibitor for most glycosidases, is expected to limit the onset of undesired effects, which are typically associated with the administration of its d-counterpart. Biological results herein obtained place ent-1 and congeners among the earliest examples of l-iminosugars acting as anti-inflammatory agents for therapeutic applications in Cystic Fibrosis.
PMID: 31075609 [PubMed – as supplied by publisher]
Impact of CFTR-modulating drugs on GH-IGF-1 axis impairment in adult patients with cystic fibrosis.
J Endocrinol Invest. 2019 Apr 20;:
Authors: Pascucci C, De Biase RV, Savi D, Quattrucci S, Gnessi L, Lubrano C, Lenzi A
INTRODUCTION: A new class of drugs in the treatment of cystic fibrosis (CF) includes two agents: lumacaftor, which corrects CFTR channel protein, and ivacaftor, which increases CFTR channel activity. In our previous study we recruited 50 stable adults with CF and 16 of them showed growth hormone deficit (GHD): 7 patients severe and 9 patients partial GHD.
MATERIAL AND METHODS: We decided to re-evaluate ten patients with the GHRH + arginine test of whom only five were treated with lumacaftor/ivacaftor.
RESULTS: All CF patients in therapy with lumacaftor/ivacaftor showed a marked improvement in GHD. Two patients moved from a severe GHD to a normal response to the GH/IGF-1 axis test, and three patients who had partial GHD moved to normal response.
CONCLUSION: The pituitary gland may be damaged by CF disease and could benefit of the action of correcting drugs.
PMID: 31006073 [PubMed – as supplied by publisher]
AMPs as Anti-biofilm Agents for Human Therapy and Prophylaxis.
Adv Exp Med Biol. 2019;1117:257-279
Authors: Shahrour H, Ferrer-Espada R, Dandache I, Bárcena-Varela S, Sánchez-Gómez S, Chokr A, Martínez-de-Tejada G
Microbial cells show a strong natural tendency to adhere to surfaces and to colonize them by forming complex communities called biofilms. In this growth mode, biofilm-forming cells encase themselves inside a dense matrix which efficiently protects them against antimicrobial agents and effectors of the immune system. Moreover, at the physiological level, biofilms contain a very heterogeneous cell population including metabolically inactive organisms and persisters, which are highly tolerant to antibiotics. The majority of human infectious diseases are caused by biofilm-forming microorganisms which are responsible for pathologies such as cystic fibrosis, infective endocarditis, pneumonia, wound infections, dental caries, infections of indwelling devices, etc. AMPs are well suited to combat biofilms because of their potent bactericidal activity of broad spectrum (including resting cells and persisters) and their ability to first penetrate and then to disorganize these structures. In addition, AMPs frequently synergize with antimicrobial compounds and were recently reported to repress the molecular pathways leading to biofilm formation. Finally, there is a very active research to develop AMP-containing coatings that can prevent biofilm formation by killing microbial cells on contact or by locally releasing their active principle. In this chapter we will describe these strategies and discuss the perspectives of the use of AMPs as anti-biofilm agents for human therapy and prophylaxis.
PMID: 30980362 [PubMed – in process]
Microbial Metabolites in Cystic Fibrosis: a Target for Future Therapy?
Am J Respir Cell Mol Biol. 2019 Apr 09;:
Authors: Widder S, Knapp S
PMID: 30965015 [PubMed – as supplied by publisher]
Antibacterial Properties and Efficacy of a Novel SPLUNC1-Derived Antimicrobial Peptide, α4-Short, in a Murine Model of Respiratory Infection.
MBio. 2019 Apr 09;10(2):
Authors: Jiang S, Deslouches B, Chen C, Di ME, Di YP
Multidrug resistance (MDR) by bacterial pathogens constitutes a global health crisis, and resistance to treatment displayed by biofilm-associated infections (e.g., cystic fibrosis, surgical sites, and medical implants) only exacerbates a problem that is already difficult to overcome. Antimicrobial peptides (AMPs) are a promising class of therapeutics that may be useful in the battle against antibiotic resistance, although certain limitations have hindered their clinical development. The goal of this study was to examine the therapeutic potential of novel AMPs derived from the multifunctional respiratory host defense protein SPLUNC1. Using standard growth inhibition and antibiofilm assays, we demonstrated that a novel structurally optimized AMP, α4-short, was highly effective against the most common group of MDR bacteria while showing broad-spectrum bactericidal and antibiofilm activities. With negligible hemolysis and toxicity to white blood cells, the new peptide also demonstrated in vivo efficacy when delivered directly into the airway in a murine model of Pseudomonas aeruginosa-induced respiratory infection. The data warrant further exploration of SPLUNC1-derived AMPs with optimized structures to assess the potential application to difficult-to-cure biofilm-associated infections.IMPORTANCE The rise of superbugs underscores the urgent need for novel antimicrobial agents. Antimicrobial peptides (AMPs) have the ability to kill superbugs regardless of resistance to traditional antibiotics. However, AMPs often display a lack of efficacy in vivo. Sequence optimization and engineering are promising but may result in increased host toxicity. We report here the optimization of a novel AMP (α4-short) derived from the multifunctional respiratory protein SPLUNC1. The AMP α4-short demonstrated broad-spectrum activity against superbugs as well as in vivo efficacy in the P. aeruginosa pneumonia model. Further exploration for clinical development is warranted.
PMID: 30967458 [PubMed – in process]
Functional effects of the microbiota in chronic respiratory disease.
Lancet Respir Med. 2019 Apr 08;:
Authors: Budden KF, Shukla SD, Rehman SF, Bowerman KL, Keely S, Hugenholtz P, Armstrong-James DPH, Adcock IM, Chotirmall SH, Chung KF, Hansbro PM
The composition of the lung microbiome is increasingly well characterised, with changes in microbial diversity or abundance observed in association with several chronic respiratory diseases such as asthma, cystic fibrosis, bronchiectasis, and chronic obstructive pulmonary disease. However, the precise effects of the microbiome on pulmonary health and the functional mechanisms by which it regulates host immunity are only now beginning to be elucidated. Bacteria, viruses, and fungi from both the upper and lower respiratory tract produce structural ligands and metabolites that interact with the host and alter the development and progression of chronic respiratory diseases. Here, we review recent advances in our understanding of the composition of the lung microbiome, including the virome and mycobiome, the mechanisms by which these microbes interact with host immunity, and their functional effects on the pathogenesis, exacerbations, and comorbidities of chronic respiratory diseases. We also describe the present understanding of how respiratory microbiota can influence the efficacy of common therapies for chronic respiratory disease, and the potential of manipulation of the microbiome as a therapeutic strategy. Finally, we highlight some of the limitations in the field and propose how these could be addressed in future research.
PMID: 30975495 [PubMed – as supplied by publisher]
Cytomegalovirus – an unrecognised potential contributor to cystic fibrosis disease progression?
Eur Respir J. 2019 Apr 07;:
Authors: Parkins MD, Ramos KJ, Goss CH, Somayaji R
Cytomegalovirus (CMV) is a common human beta-herpes virus most notable for causing visceral disease in profoundly immune-suppressed populations, and congenital infections. However, an increasing body of work has demonstrated that CMV seropositivity is associated with a number of chronic medical conditions including heart disease and dementia – potentially related to the effects of chronic inflammation. We hypothesided that the outcomes of individuals with cystic fibrosis (CF), a chronic inflammatory disease, could similarly be associated with CMV-status. We performed a single-centre retrospective study of all 71 individuals with CF referred for lung transplantation from our CF centre between 1991-2017 and assessed how CMV serostatus associated with patient pre-transplant outcomes. We observed CMV IgG positivity was associated with disproportionate progression to end-stage lung disease as defined by death/or transplantation in our cohort (27.2 versus 35.1 years, difference 7.95 (95% CI 3.61-12.29 years), p<0.001) which remained significant following adjustment for confounders (difference 6.96 (95% CI 2.51-11.41 years). CMV may represent a potentially important modifier of CF lung disease, warranting further study. PMID: 30956206 [PubMed - as supplied by publisher] PubMed:30956206
Clinical papers of the year 2018 – Cystic fibrosis.
Paediatr Respir Rev. 2019 Apr 05;:
Authors: Balfour-Lynn IM
This paper reviews the most important clinical papers in cystic fibrosis published in 2018, having searched all the literature on Pubmed. Focus is on CFTR modulator therapy, randomised controlled trials, and infection/microbiology issues.
PMID: 31053358 [PubMed – as supplied by publisher]
Bacteriophage PEV20 and Ciprofloxacin Combination Treatment Enhances Removal of Pseudomonas aeruginosa Biofilm Isolated from Cystic Fibrosis and Wound Patients.
AAPS J. 2019 Apr 04;21(3):49
Authors: Chang RYK, Das T, Manos J, Kutter E, Morales S, Chan HK
Antibiotic resistance in Pseudomonas aeruginosa biofilms necessitates the need for novel antimicrobial therapy with anti-biofilm properties. Bacteriophages (phages) are recognized as an ideal biopharmaceutical for combating antibiotic-resistant bacteria especially when used in combination with antibiotics. However, previous studies primarily focused on using phages against of P. aeruginosa biofilms of laboratory strains. In the present study, biofilms of six P. aeruginosa isolated from cystic fibrosis and wound patients, and one laboratory strain was treated singly and with combinations of anti-Pseudomonas phage PEV20 and ciprofloxacin. Of these strains, three were highly susceptible to the phage, while one was partially resistant and one was completely resistant. Combination treatment with PEV20 and ciprofloxacin enhanced biofilm eradication compared with single treatment. Phage and ciprofloxacin synergy was found to depend on phage-resistance profile of the target bacteria. Furthermore, phage and ciprofloxacin combination formulation protected the lung epithelial and fibroblast cells from P. aeruginosa and promoted cell growth. The results demonstrated that thorough screening of phage-resistance is crucial for designing phage-antibiotic formulation. The addition of highly effective phage could reduce the ciprofloxacin concentration required to combat P. aeruginosa infections associated with biofilm in cystic fibrosis and wound patients.
PMID: 30949776 [PubMed – in process]
Cystic Fibrosis Transmembrane Conductance Regulator: A Possible New Target for Photodynamic Therapy Enhances Wound Healing.
Adv Wound Care (New Rochelle). 2019 Oct 01;8(10):476-486
Authors: Chiu WT, Tran TV, Pan SC, Huang HK, Chen YC, Wong TW
Objective: Cell migration is an essential process in skin wound healing. Photodynamic therapy (PDT) enhances wound healing by photoactivating a photosensitizer with a specific wavelength of light. Cystic fibrosis transmembrane conductance regulator (CFTR) is an ion channel expressed in multiple layers of keratinocytes. Recent studies showed that the activation of CFTR-related downstream signaling affects skin wound healing. We examined whether indocyanine green (ICG)-mediated PDT-enhanced cell migration is related to CFTR activation. Approach: The spatial and temporal expression levels of CFTR and proteins involved in focal adhesion, including focal adhesion kinase (FAK) and paxillin, were evaluated during cell migration in vitro and in vivo for wound healing. Results: ICG-PDT-conditioned medium collected from cells exposed to 5 J/cm2 near-infrared light in the presence of 100 μg/mL ICG activated CFTR and enhanced HaCaT cell migration. The expression of phosphorylated FAK Tyr861 and phosphorylated paxillin in focal adhesions was spatially and temporally regulated in parallel by ICG-PDT-conditioned medium. Curcumin, a nonspecific activator of CFTR, further increased PDT-enhanced cell migration, whereas inhibition of CFTR and FAK delayed cell migration. The involvement of CFTR in ICG-PDT-enhanced skin wound healing was confirmed in a mouse back skin wound model. Innovation: CFTR is a potential new therapeutic target in ICG-PDT to enhance wound healing. Conclusion: ICG-PDT-enhanced cell migration may be related to activation of the CFTR and FAK pathway. Conditioned medium collected from ICG-PDT may be useful for treating patients with chronic skin ulcer by regulating CFTR expression in keratinocytes.
PMID: 31456905 [PubMed]
Roles, Actions, and Therapeutic Potential of Specialized Pro-resolving Lipid Mediators for the Treatment of Inflammation in Cystic Fibrosis.
Front Pharmacol. 2019;10:252
Authors: Recchiuti A, Mattoscio D, Isopi E
Non-resolving inflammation is the main mechanism of morbidity and mortality among patients suffering from cystic fibrosis (CF), the most common life-threatening human genetic disease. Resolution of inflammation is an active process timely controlled by endogenous specialized pro-resolving lipid mediators (SPMs) produced locally in inflammatory loci to restrain this innate response, prevent further damages to the host, and permit return to homeostasis. Lipoxins, resolvins, protectins, and maresins are SPM derived from polyunsaturated fatty acids that limit excessive leukocyte infiltration and pro-inflammatory signals, stimulate innate microbial killing, and enhance resolution. Their unique chemical structures, receptors, and bioactions are being elucidated. Accruing data indicate that SPMs carry protective functions against unrelenting inflammation and infections in preclinical models and human CF systems. Here, we reviewed their roles and actions in controlling resolution of inflammation, evidence for their impairment in CF, and proofs of principle for their exploitation as innovative, non-immunosuppressive drugs to address inflammation and infections in CF.
PMID: 31001110 [PubMed]
Safety and efficacy of treatment with lumacaftor in combination with ivacaftor in younger patients with cystic fibrosis.
Expert Rev Respir Med. 2019 Mar 30;:
Authors: Cheng PC, Alexiou S, Rubenstein RC
INTRODUCTION: Cystic fibrosis (CF) is the most common autosomal recessive disorder affecting approximately 70,000 people worldwide. The lack of functional cystic fibrosis transmembrane conductance regulator (CFTR) causes dysregulation of epithelial fluid transport in the lungs, gastrointestinal tract, and sweat glands. Areas covered: The most common disease-causing CFTR mutation, F508del, is present in nearly 75% of those affected and results in a defective protein. Therapies to improve the function of this mutant protein have the promise to reduce morbidity and mortality in the majority of patients with CF. The combination of lumacaftor, which corrects the aberrant intracellular trafficking of F508del, and ivacaftor, which potentiates CFTR function, is known as OrkambiTM, and is the first drug approved for the treatment of cystic fibrosis (CF) in patients who are homozygous for F508del. OrkambiTM is currently approved for use in children aged 2 and older based on recent data from open-label Phase 3 clinical safety studies. In older patients, treatment with lumacaftor/ivacaftor is associated with a modest, statistically significant improvement in lung function and reduced pulmonary exacerbations in placebo-controlled trials; these findings are also observed in Phase IV observational studies. While severe side effects are rare, chest tightness, elevation of transaminases, and cataracts have been reported and recommendations for monitoring have been established. Expert opinion: OrkambiTM somewhat improves clinical outcomes for people with CF who are homozygous for the F508del mutation, and does so with a reasonable safety profile. This therapy represents a major advance in the therapy for CF, but further advances are needed, perhaps with addition of a third agent to this combination small molecule therapy, in order to expand both the targeted population and beneficial effects.
PMID: 30929526 [PubMed – as supplied by publisher]
Vitamin D inhibits pro-inflammatory cytokines in the airways of cystic fibrosis patients infected by Pseudomonas aeruginosa- pilot study.
Ital J Pediatr. 2019 Mar 29;45(1):41
Authors: Olszowiec-Chlebna M, Koniarek-Maniecka A, Brzozowska A, Błauż A, Rychlik B, Stelmach I
BACKGROUND: Vitamin D plays an important role in inflammatory responses after antigen exposure. Interleukin-23 (Il-23) promotes Il-17-dependent inflammation during Pseudomonas aeruginosa (P. aeruginosa) pulmonary infection. We aimed to compare the ability of calcitriol and cholecalciferol to modulate the inflammatory response of the CF airways infected with P. aeruginosa.
METHODS: This was a randomized, placebo-controlled, double-blind, cross-over trial. Twenty-three patients with CF (aged 6-19), chronically infected by P. aeruginosa were randomly assigned to: calcitriol group receiving 1,25(OH)2D 0,5 mcg daily or cholecalciferol group receiving cholecalciferol 1000 IU daily for three months. The levels of Il-23 and Il-17A in the exhaled breath concentrate (EBC) were measured. Calcium-phosphorus balance was also evaluated (serum concentration of calcium, phosphorus, 25OHD, parathormone (PTH) and calcium/creatinine ratio in urine). Data were analyzed using means of Stata/Special Edition, release 14.2. A level of P < 0.05 was considered statistically significant. RESULTS: The level of Il-17A in EBC significantly decreased in calcitriol group from 0,475 pg/mL (± SD 0,515 pg/mL) to 0,384 pg/mL (± SD 0,429 pg/mL) (p = 0,008); there was no change in cholecalciferol group (p = 0,074). The level of Il-23 in EBC did not significantly change in calcitriol group (p = 0,086); there was significant decrease in cholecalciferol group from 8,90 pg/mL (± SD 4,07 pg/mL) to 7,33 pg/mL (± SD 3,88 pg/mL) (p = 0,001). In calcitriol group serum phosphorus and PTH significantly decreased (p = 0,021 and p = 0,019 respectively), the concentration of calcium significantly increased (p = 0,001); there were no changes in cholecalciferol group. CONCLUSIONS: Both analogs of vitamin D revealed their anti-inflammatory effect and reduced the level of Il-17A and Il-23 in the airway of CF patients with chronic P. aeruginosa infection. We observed improvement in calcium-phosphorus metabolism after supplementation with calcitriol, without adverse effects. It is recommended to use vitamin D in CF patients. PMID: 30922377 [PubMed - in process] PubMed:30922377
Prevention of cystic fibrosis: The beginning of the end?
Sci Transl Med. 2019 Mar 27;11(485):
Authors: Ferkol TW
Prenatal and postnatal treatment with a CFTR modifier attenuates pathological changes in a ferret model of cystic fibrosis (Sun et al., this issue).
PMID: 30918110 [PubMed – in process]
In utero and postnatal VX-770 administration rescues multiorgan disease in a ferret model of cystic fibrosis.
Sci Transl Med. 2019 Mar 27;11(485):
Authors: Sun X, Yi Y, Yan Z, Rosen BH, Liang B, Winter MC, Evans TIA, Rotti PG, Yang Y, Gray JS, Park SY, Zhou W, Zhang Y, Moll SR, Woody L, Tran DM, Jiang L, Vonk AM, Beekman JM, Negulescu P, Van Goor F, Fiorino DF, Gibson-Corley KN, Engelhardt JF
Cystic fibrosis (CF) is a multiorgan disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). In patients with CF, abnormalities initiate in several organs before birth. However, the long-term impact of these in utero pathologies on disease pathophysiology is unclear. To address this issue, we generated ferrets harboring a VX-770 (ivacaftor)-responsive CFTR G551D mutation. In utero VX-770 administration provided partial protection from developmental pathologies in the pancreas, intestine, and male reproductive tract. Homozygous CFTR G551D/G551D animals showed the greatest VX-770-mediated protection from these pathologies. Sustained postnatal VX-770 administration led to improved pancreatic exocrine function, glucose tolerance, growth and survival, and to reduced mucus accumulation and bacterial infections in the lung. VX-770 withdrawal at any age reestablished disease, with the most rapid onset of morbidity occurring when withdrawal was initiated during the first 2 weeks after birth. The results suggest that CFTR is important for establishing organ function early in life. Moreover, this ferret model provides proof of concept for in utero pharmacologic correction of genetic disease and offers opportunities for understanding CF pathogenesis and improving treatment.
PMID: 30918114 [PubMed – in process]
Plasma membrane-localized TMEM16 proteins are indispensable for expression of CFTR.
J Mol Med (Berl). 2019 Mar 26;:
Authors: Benedetto R, Ousingsawat J, Cabrita I, Pinto M, Lérias JR, Wanitchakool P, Schreiber R, Kunzelmann K
The cystic fibrosis transmembrane conductance regulator (CFTR) is the secretory chloride channel in epithelial tissues that has a central role in cystic fibrosis (CF) lung and gastrointestinal disease. A recent publication demonstrates a close association between CFTR and TMEM16A, the calcium-activated chloride channel. Thus, no CFTR chloride currents could be detected in airways and large intestine from mice lacking epithelial expression of TMEM16A. Here, we demonstrate that another plasma membrane-localized TMEM16 paralogue, TMEM16F, can compensate for the lack of TMEM16A. Using TMEM16 knockout mice, human lymphocytes, and a number of human cell lines with endogenous protein expression or heterologous expression, we demonstrate that CFTR can only function in the presence of either TMEM16A or TMEM16F. Double knockout of intestinal epithelial TMEM16A/F expression did not produce offsprings, suggesting a lethal phenotype in utero. Plasma membrane-localized TMEM16A or TMEM16F is required for exocytosis and expression of CFTR in the plasma membrane. TMEM16A/F proteins may therefore have an impact on disease severity in CF. KEY MESSAGES: • Cystic fibrosis is caused by the defective Cl- channel cystic fibrosis transmembrane conductance regulator (CFTR). • A close relationship exists between CFTR and the calcium-activated chloride channels TMEM16A/TMEM16F. • In conditional airway and intestinal knockout mice, lymphocytes from Scott disease patients and in overexpressing cells, CFTR is not functional in the absence of TMEM16A and TMEM16F. • TMEM16A and TMEM16F support membrane exocytosis and are essential for plasma membrane insertion of CFTR.
PMID: 30915480 [PubMed – as supplied by publisher]
Brevenal, a Marine Natural Product, is Anti-Inflammatory and an Immunomodulator of Macrophage and Lung Epithelial Cells.
Mar Drugs. 2019 Mar 20;17(3):
Authors: Keeler DM, Grandal MK, McCall JR
Chronic respiratory diseases, including chronic obstructive pulmonary disease (COPD), cystic fibrosis, and asthma, are some of the leading causes of illness and fatalities worldwide. The search for novel treatments led to the exploration of marine natural products as drug candidates to combat the debilitating effects of mucus accumulation and chronic inflammation. Previous research showed that an alga-derived compound, brevenal, could attenuate the effects of inflammatory agents, but the mechanisms by which it exerted its effects remained unclear. We investigated the effects of brevenal on lipopolysaccharide (LPS) induced cytokine/chemokine production from murine macrophages and human lung epithelial cells. It was found that brevenal reduces proinflammatory mediator secretion while preserving anti-inflammatory secretion from these cells. Furthermore, we found that brevenal does not alter cell surface Toll-like receptor 4 (TLR4) expression, thereby maintaining the cells’ ability to respond to bacterial infection. However, brevenal does alter macrophage activation states, as demonstrated by reduced expression of both M1 and M2 phenotype markers, indicating this putative anti-inflammatory drug shifts innate immune cells to a less active state. Such a mechanism of action would be ideal for reducing inflammation in the lung, especially with patients suffering from chronic respiratory diseases, where inflammation can be lethal.
PMID: 30897777 [PubMed – in process]
Cell-Selective Regulation of CFTR Gene Expression: Relevance to Gene Editing Therapeutics.
Genes (Basel). 2019 Mar 19;10(3):
Authors: Swahn H, Harris A
The cystic fibrosis transmembrane conductance regulator (CFTR) gene is an attractive target for gene editing approaches, which may yield novel therapeutic approaches for genetic diseases such as cystic fibrosis (CF). However, for gene editing to be effective, aspects of the three-dimensional (3D) structure and cis-regulatory elements governing the dynamic expression of CFTR need to be considered. In this review, we focus on the higher order chromatin organization required for normal CFTR locus function, together with the complex mechanisms controlling expression of the gene in different cell types impaired by CF pathology. Across all cells, the CFTR locus is organized into an invariant topologically associated domain (TAD) established by the architectural proteins CCCTC-binding factor (CTCF) and cohesin complex. Additional insulator elements within the TAD also recruit these factors. Although the CFTR promoter is required for basal levels of expression, cis-regulatory elements (CREs) in intergenic and intronic regions are crucial for cell-specific and temporal coordination of CFTR transcription. These CREs are recruited to the promoter through chromatin looping mechanisms and enhance cell-type-specific expression. These features of the CFTR locus should be considered when designing gene-editing approaches, since failure to recognize their importance may disrupt gene expression and reduce the efficacy of therapies.
PMID: 30893953 [PubMed]
Choline Supplementation in Cystic Fibrosis-The Metabolic and Clinical Impact.
Nutrients. 2019 Mar 18;11(3):
Authors: Bernhard W, Lange R, Graepler-Mainka U, Engel C, Machann J, Hund V, Shunova A, Hector A, Riethmüller J
BACKGROUND: Choline is essential for the synthesis of liver phosphatidylcholine (PC), parenchymal maintenance, bile formation, and lipoprotein assembly to secrete triglycerides. In choline deficiency, the liver accretes choline/PC at the expense of lung tissue, thereby impairing pulmonary PC homoeostasis. In cystic fibrosis (CF), exocrine pancreas insufficiency results in impaired cleavage of bile PC and subsequent fecal choline loss. In these patients, the plasma choline concentration is low and correlates with lung function. We therefore investigated the effect of choline supplementation on plasma choline/PC concentration and metabolism, lung function, and liver fat.
METHODS: 10 adult male CF patients were recruited (11/2014⁻1/2016), and orally supplemented with 3 × 1 g choline chloride for 84 (84⁻91) days. Pre-/post-supplementation, patients were spiked with 3.6 mg/kg [methyl-D₉]choline chloride to assess choline/PC metabolism. Mass spectrometry, spirometry, and hepatic nuclear resonance spectrometry served for analysis.
RESULTS: Supplementation increased plasma choline from 4.8 (4.1⁻6.2) µmol/L to 10.5 (8.5⁻15.5) µmol/L at d84 (p < 0.01). Whereas plasma PC concentration remained unchanged, D₉-labeled PC was decreased (12.2 [10.5⁻18.3] µmol/L vs. 17.7 [15.5⁻22.4] µmol/L, p < 0.01), indicating D₉-tracer dilution due to higher choline pools. Supplementation increased Forced Expiratory Volume in 1 second percent of predicted (ppFEV1) from 70.0 (50.9⁻74.8)% to 78.3 (60.1⁻83.9)% (p < 0.05), and decreased liver fat from 1.58 (0.37⁻8.82)% to 0.84 (0.56⁻1.17)% (p < 0.01). Plasma choline returned to baseline concentration within 60 h. CONCLUSIONS: Choline supplementation normalized plasma choline concentration and increased choline-containing PC precursor pools in adult CF patients. Improved lung function and decreased liver fat suggest that in CF correcting choline deficiency is clinically important. Choline supplementation of CF patients should be further investigated in randomized, placebo-controlled trials. PMID: 30889905 [PubMed - in process] PubMed:30889905
Autophagy suppresses the pathogenic immune response to dietary antigens in cystic fibrosis.
Cell Death Dis. 2019 Mar 15;10(4):258
Authors: Villella VR, Esposito S, Ferrari E, Monzani R, Tosco A, Rossin F, Castaldo A, Silano M, Marseglia GL, Romani L, Barlev NA, Piacentini M, Raia V, Kroemer G, Maiuri L
Under physiological conditions, a finely tuned system of cellular adaptation allows the intestinal mucosa to maintain the gut barrier function while avoiding excessive immune responses to non-self-antigens from dietary origin or from commensal microbes. This homeostatic function is compromised in cystic fibrosis (CF) due to loss-of-function mutations in the CF transmembrane conductance regulator (CFTR). Recently, we reported that mice bearing defective CFTR are abnormally susceptible to a celiac disease-like enteropathy, in thus far that oral challenge with the gluten derivative gliadin elicits an inflammatory response. However, the mechanisms through which CFTR malfunction drives such an exaggerated response to dietary protein remains elusive. Here we demonstrate that the proteostasis regulator/transglutaminase 2 (TGM2) inhibitor cysteamine restores reduced Beclin 1 (BECN1) protein levels in mice bearing cysteamine-rescuable F508del-CFTR mutant, either in homozygosis or in compound heterozygosis with a null allele, but not in knock-out CFTR mice. When cysteamine restored BECN1 expression, autophagy was increased and gliadin-induced inflammation was reduced. The beneficial effects of cysteamine on F508del-CFTR mice were lost when these mice were backcrossed into a Becn1 haploinsufficient/autophagy-deficient background. Conversely, the transfection-enforced expression of BECN1 in human intestinal epithelial Caco-2 cells mitigated the pro-inflammatory cellular stress response elicited by the gliadin-derived P31-43 peptide. In conclusion, our data provide the proof-of-concept that autophagy stimulation may mitigate the intestinal malfunction of CF patients.
PMID: 30874543 [PubMed – in process]
Cystic fibrosis transmembrane conductance regulator modulators: Present and future in cystic fibrosis treatment. A review.
Arch Argent Pediatr. 2019 Apr 01;117(2):e131-e136
Authors: De la Hoz D, Villamil Osorio M, Restrepo-Gualteros SM
Cystic fibrosis transmembrane conductance regulator (CFTR) modulators are the present and future of drug management for patients with cystic fibrosis. The objective of this article is to review this therapeutic option. Scientific articles were reviewed by searching the MedLine database, which is available through the Cystic Fibrosis Foundation’s official website, from 2009 to 2018, in English. Twelve articles about the current status of research in CFTR modulators were selected without restrictions regarding the type of study. To date, the United States Food and Drug Administration has approved three modulators: ivacaftor, lumacaftor + ivacaftor, and tezacaftor + ivacaftor, while other 11 drugs are being studied in different investigation phases. CFTR modulator therapy is a developing reality aimed at the highest goal of personalized medicine and promises to improve the quality of life of cystic fibrosis patients.
PMID: 30869491 [PubMed – in process]
Lentiviral Vectors for the Treatment and Prevention of Cystic Fibrosis Lung Disease.
Genes (Basel). 2019 Mar 14;10(3):
Authors: Marquez Loza LI, Yuen EC, McCray PB
Despite the continued development of cystic fibrosis transmembrane conductance regulator (CFTR) modulator drugs for the treatment of cystic fibrosis (CF), the need for mutation agnostic treatments remains. In a sub-group of CF individuals with mutations that may not respond to modulators, such as those with nonsense mutations, CFTR gene transfer to airway epithelia offers the potential for an effective treatment. Lentiviral vectors are well-suited for this purpose because they transduce nondividing cells, and provide long-term transgene expression. Studies in primary cultures of human CF airway epithelia and CF animal models demonstrate the long-term correction of CF phenotypes and low immunogenicity using lentiviral vectors. Further development of CF gene therapy requires the investigation of optimal CFTR expression in the airways. Lentiviral vectors with improved safety features have minimized insertional mutagenesis safety concerns raised in early clinical trials for severe combined immunodeficiency using γ-retroviral vectors. Recent clinical trials using improved lentiviral vectors support the feasibility and safety of lentiviral gene therapy for monogenetic diseases. While work remains to be done before CF gene therapy reaches the bedside, recent advances in lentiviral vector development reviewed here are encouraging and suggest it could be tested in clinical studies in the near future.
PMID: 30875857 [PubMed]
A Practical Review of Proteasome Pharmacology.
Pharmacol Rev. 2019 Apr;71(2):170-197
Authors: Thibaudeau TA, Smith DM
The ubiquitin proteasome system (UPS) degrades individual proteins in a highly regulated fashion and is responsible for the degradation of misfolded, damaged, or unneeded cellular proteins. During the past 20 years, investigators have established a critical role for the UPS in essentially every cellular process, including cell cycle progression, transcriptional regulation, genome integrity, apoptosis, immune responses, and neuronal plasticity. At the center of the UPS is the proteasome, a large and complex molecular machine containing a multicatalytic protease complex. When the efficiency of this proteostasis system is perturbed, misfolded and damaged protein aggregates can accumulate to toxic levels and cause neuronal dysfunction, which may underlie many neurodegenerative diseases. In addition, many cancers rely on robust proteasome activity for degrading tumor suppressors and cell cycle checkpoint inhibitors necessary for rapid cell division. Thus, proteasome inhibitors have proven clinically useful to treat some types of cancer, especially multiple myeloma. Numerous cellular processes rely on finely tuned proteasome function, making it a crucial target for future therapeutic intervention in many diseases, including neurodegenerative diseases, cystic fibrosis, atherosclerosis, autoimmune diseases, diabetes, and cancer. In this review, we discuss the structure and function of the proteasome, the mechanisms of action of different proteasome inhibitors, various techniques to evaluate proteasome function in vitro and in vivo, proteasome inhibitors in preclinical and clinical development, and the feasibility for pharmacological activation of the proteasome to potentially treat neurodegenerative disease.
PMID: 30867233 [PubMed – in process]
Clinical validation of an evidence-based method to adjust Pancreatic Enzyme Replacement Therapy through a prospective interventional study in paediatric patients with Cystic Fibrosis.
PLoS One. 2019;14(3):e0213216
Authors: Calvo-Lerma J, Hulst J, Boon M, Colombo C, Masip E, Ruperto M, Fornés-Ferrer V, van der Wiel E, Claes I, Garriga M, Roca M, Crespo-Escobar P, Bulfamante A, Woodcock S, Martínez-Barona S, Andrés A, de Boeck K, Ribes-Koninckx C, MyCyFAPP project
BACKGROUND: A method to adjust Pancreatic Enzyme Replacement Therapy in Cystic Fibrosis is not currently available.
OBJECTIVES: To assess the in vivo efficacy of a method to adjust the dose of enzymatic supplement in CF extrapolated from previous in vitro digestion studies (theoretical optimal dose, TOD). Secondly, to assess how individual patient characteristics influence the expected coefficient of fat absorption (CFA) and thus to identify an individual correction factor to improve TOD.
METHODS: A prospective interventional study in 43 paediatric patients with CF from 5 European centres. They followed a 24h fixed diet with the theoretical optimal dose for each meal. Faecal collection was carried out between colorimetric markers in order to include all the faeces corresponding to the fixed diet. Beta regression models were applied to assess the associations of individual patient characteristics with the CFA.
RESULTS: Median CFA was 90% (84, 94% 1st, 3rd Q.) with no significant differences among centres. Intestinal transit time was positively associated with CFA (p = 0.007), but no statistical associations were found with and age, gender, phenotype or BMI. Regression model showed no improvement of the in vitro predicted theoretical optimal dose when taking individual patient characteristics into account.
CONCLUSION: Strict adherence to the theoretical optimal dose of enzymatic supplement for a prescribed meal, led to median CFA levels at the clinical target of 90% with a low variability between patients. The proposed method can be considered as a first approach for an evidence-based method in PERT dosing based on food characteristics. Results have to be confirmed in free dietary settings.
PMID: 30861039 [PubMed – in process]
Two CFTR mutations within codon 970 differently impact on the chloride channel functionality.
Hum Mutat. 2019 Mar 09;:
Authors: Amato F, Scudieri P, Musante I, Tomati V, Caci E, Comegna M, Maietta S, Manzoni F, di Lullo AM, De Wachter E, Vanderhelst E, Terlizzi V, Braggion C, Castaldo G, Galietta LJV
Pharmacological rescue of mutant CFTR in cystic fibrosis (CF) depends on the specific defect caused by different mutation classes. We asked whether a patient with the rare p.Gly970Asp (c.2909G>A) mutation could benefit from CFTR pharmacotherapy since a similar missense mutant p.Gly970Arg (c.2908G>C) was previously found to be sensitive to potentiators in vitro but not in vivo. By cDNA transfection, we found that both mutations are associated with defective CFTR function amenable to pharmacological treatment. However, analysis of mRNA from patients cells revealed that c.2908G>C impairs RNA splicing whereas c.2909G>A does not perturb splicing and leads to the expected p.Gly970Asp mutation. In agreement with these results, nasal epithelial cells from the p.Gly970Asp patient showed significant improvement of CFTR function upon pharmacological treatment. Our results underline the importance of controlling the effect of CF mutation at the mRNA level in order to determine if the pharmacotherapy of CFTR basic defect is appropriate. This article is protected by copyright. All rights reserved.
PMID: 30851139 [PubMed – as supplied by publisher]
Cystic Fibrosis Plasma Blunts the Immune Response to Bacterial Infection.
Am J Respir Cell Mol Biol. 2019 Mar 08;:
Authors: Zhang X, Pan A, Jia S, Ideozu JE, Woods K, Murkowski K, Hessner MJ, Simpson PM, Levy H
RATIONALE: Cystic fibrosis (CF) is caused by mutations of the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). It remains unclear whether the abnormal immune response in CF involves extrinsic signals released from the external or internal environment.
OBJECTIVES: To characterize the peripheral immune signatures in CF and its association with clinical phenotypes.
METHODS: Healthy peripheral blood mononuclear cells (PBMCs) were cultured with plasma from CF probands (CF) or healthy controls (HC) followed by nCounter gene and microRNA (miRNA) profiling. A discovery cohort of 12 CF and 12 HC and a validation cohort of 103 CF and 31 HC (our previous microarray data, GSE71799) were analyzed to characterize the composition of cultured immune cells and establish a miRNA‒mRNA network. Cell compositions and miRNA profiles were associated with clinical characteristics of the cohorts.
MEASUREMENTS AND MAIN RESULTS: Significantly differentially expressed genes and abundance of myeloid cells were downregulated in PMBCs after culture with CF plasma (p < 0.05). Top-ranked miRNAs that increased in response to CF plasma (adjusted p < 0.05) included miR-155 and miR-146a, which target many immune-related genes such as IL-8. Pseudomonas aeruginosa infection was negatively associated with abundance of monocytes and the presence of those regulatory miRNAs. CONCLUSIONS: Extrinsic signals in plasma from CF patients led to monocyte inactivation and miRNA upregulation in PBMC. An improved understanding of the immune effects of extrinsic factors in CF holds great promise for integrating immunomodulatory cell therapies into current treatment strategies in CF. PMID: 30848661 [PubMed - as supplied by publisher] PubMed:30848661
SMAD Signaling Restricts Mucous Cell Differentiation In Human Airway Epithelium.
Am J Respir Cell Mol Biol. 2019 Mar 08;:
Authors: Feldman MB, Wood M, Lapey A, Mou H
Mucin-secreting goblet cell metaplasia and hyperplasia (GCMH) is a common pathological phenotype in many human respiratory diseases including asthma, chronic obstructive pulmonary disease, cystic fibrosis, primary ciliary dyskinesia, and infections. A better understanding of how goblet cell quantities or proportions in the airway epithelium are regulated may provide novel therapeutic targets to mitigate GCMH in these devastating diseases. We identify canonical SMAD signaling as the principle pathway restricting goblet cell differentiation in human airway epithelium. Differentiated goblet cells express low levels of phosphorylated SMAD. Accordingly, inhibition of SMAD signaling markedly amplifies GCMH induced by mucous mediators. In contrast, SMAD signaling activation impedes goblet cell generation and accelerates the resolution of preexisting GCMH. SMAD signaling inhibition can override the suppressive effects imposed by a GABAergic receptor inhibitor, suggesting the GABAergic pathway likely operates through inhibition of SMAD signaling in regulating mucous differentiation. Collectively, our data demonstrate that SMAD signaling plays a determining role in mucous cell differentiation and thus raises the possibility that dysregulation of this pathway contributes to respiratory pathophysiology during airway inflammation and pulmonary diseases. Additionally, our study also highlights the potential for SMAD modulation as a therapeutic target in mitigating GCMH.
PMID: 30848657 [PubMed – as supplied by publisher]
The effects of cycled inhaled aztreonam on the cystic fibrosis (CF) lung microbiome.
J Cyst Fibros. 2019 Mar 08;:
Authors: Heirali AA, Acosta N, Storey DG, Workentine ML, Somayaji R, Laforest-Lapointe I, Leung W, Quon BS, Berthiaume Y, Rabin HR, Waddell BJ, Rossi L, Surette MG, Parkins MD
BACKGROUND: To improve clinical outcomes, cystic fibrosis (CF) patients with chronic Pseudomonas aeruginosa infections are prescribed inhaled anti-pseudomonal antibiotics. Although, a diverse microbial community exists within CF airways, little is known about how the CF microbiota influences patient outcomes. We hypothesized that organisms within the CF microbiota are affected by inhaled-antibiotics and baseline microbiome may be used to predict therapeutic response.
METHODS: Adults with chronic P. aeruginosa infection from four clinics were observed during a single 28-day on/off inhaled-aztreonam cycle. Patients performed serial sputum collection, CF-respiratory infection symptom scores (CRISS), and spirometry. Patients achieving a decrease of ≥2 CRISS by day 28 were categorized as subjective responders (SR). The airway microbiome was defined by Illumina MiSeq analysis of the 16S rRNA gene.
RESULTS: Thirty-seven patients (median 37.4 years and FEV1 44% predicted) were enrolled. No significant cohort-wide changes in the microbiome were observed between on/off AZLI cycles in either alpha- or beta-diversity metrics. However, at an individual level shifts were apparent. Twenty-one patients (57%) were SR and fourteen patients did not subjectively respond. While alpha-diversity metrics did not associate with response, patients who did not subjectively respond had a higher abundance of Staphylococcus and Streptococcus, and lower abundance of Haemophilus.
CONCLUSIONS: The CF microbiome is relatively resilient to AZLI perturbations. However, associated changes were observed at the individual patient level. The relative abundance of key “off-target” organisms associated with subjective improvements suggesting that the microbiome may be used as a tool to predict patient response – potentially improving outcomes.
PMID: 30857926 [PubMed – as supplied by publisher]
Pyruvate-depleting conditions induce biofilm dispersion and enhance the efficacy of antibiotics in killing biofilms in vitro and in vivo.
Sci Rep. 2019 Mar 06;9(1):3763
Authors: Goodwine J, Gil J, Doiron A, Valdes J, Solis M, Higa A, Davis S, Sauer K
The formation of biofilms is a developmental process initiated by planktonic cells transitioning to the surface, which comes full circle when cells disperse from the biofilm and transition to the planktonic mode of growth. Considering that pyruvate has been previously demonstrated to be required for the formation of P. aeruginosa biofilms, we asked whether pyruvate likewise contributes to the maintenance of the biofilm structure, with depletion of pyruvate resulting in dispersion. Here, we demonstrate that the enzymatic depletion of pyruvate coincided with the dispersion of established biofilms by S. aureus and laboratory and clinical P. aeruginosa isolates. The dispersion response was dependent on pyruvate fermentation pathway components but independent of proteins previously described to contribute to P. aeruginosa biofilm dispersion. Using porcine second-degree burn wounds infected with P. aeruginosa biofilm cells, we furthermore demonstrated that pyruvate depletion resulted in a reduction of biofilm biomass in vivo. Pyruvate-depleting conditions enhanced the efficacy of tobramycin killing of the resident wound biofilms by up to 5-logs. Our findings strongly suggest the management of pyruvate availability to be a promising strategy to combat biofilm-related infections by two principal pathogens associated with wound and cystic fibrosis lung infections.
PMID: 30842579 [PubMed – in process]
Nonsense Mediated RNA Decay Pathway Inhibition Restores Expression and Function of W1282X CFTR.
Am J Respir Cell Mol Biol. 2019 Mar 05;:
Authors: Keenan MM, Huang L, Jordan NJ, Wong E, Cheng Y, Valley HC, Mahiou J, Liang F, Bihler H, Mense M, Guo S, Monia BP
RATIONALE: The recessive genetic disease cystic fibrosis is caused by loss of function mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Approximately 10% of cystic fibrosis patients have at least one allele with a nonsense mutation in CFTR. Nonsense mutations generate premature termination codons that can subject mRNA transcripts to rapid degradation through the nonsense mediated mRNA decay (NMD) pathway. Currently, there are no approved therapies specifically targeting nonsense mutations in CFTR.
OBJECTIVES: Here, we identify antisense oligonucleotides (ASOs) targeting the nonsense mediated decay factor SMG1 to inhibit the NMD pathway and determine their effects on the W1282X CFTR mutation.
METHODS: First, we develop and validate two in vitro models of the W1282X CFTR mutation. Next, we treat these cells with antisense oligonucleotides to inhibit nonsense mediated decay and measure the effects of these treatments on W1282X expression and function.
MEASUREMENTS AND MAIN RESULTS: SMG1-ASO mediated NMD inhibition upregulates the RNA, protein and surface-localized protein expression of the truncated W1282X gene product. Additionally, these ASOs increase the CFTR chloride channel function in cells homozygous for the W1282X mutation.
CONCLUSIONS: Our approach suggests a new therapeutic strategy for patients harboring nonsense mutations and may be beneficial as a single agent in CF patients with the W1282X mutation.
PMID: 30836009 [PubMed – as supplied by publisher]
Small molecule-facilitated anion transporters in cells for a novel cystic fibrosis therapeutic approach.
Br J Pharmacol. 2019 Mar 01;:
Authors: Fiore M, Cossu C, Capurro V, Picco C, Ludovico A, Mielczarek M, Carreira-Barral I, Caci E, Baroni D, Quesada R, Moran O
BACKGROUND AND PURPOSE: Cystic fibrosis (CF) is a lethal autosomal recessive genetic disease that originates from the defective function of the CFTR protein, a cAMP-dependent anion channel involved in fluid transport across epithelium. Due to their capability to replace the ion transport independently from the genetic mutation that affects the CFTR, small synthetic transmembrane anion transporters, named anionophores, are candidates as new potential CF therapeutics.
EXPERIMENTAL APPROACH: With the aim of evaluating their impact on cell physiology, we have analysed the transport properties of five compounds, three prodigiosines and two tambjamines.
KEY RESULTS: All studied compounds are capable of transporting halides and bicarbonate across the cell membrane, with a higher transport capacity at acidic pH. Interestingly, the presence of these anionophores do not interfere with the activation of CFTR, and do not modify the action of the lumacaftor and ivacaftor, a CFTR-corrector and -potentiator, respectively.
CONCLUSION AND IMPLICATIONS: Their ability to transport chloride and bicarbonate when applied at low concentration take shape as a promising starting point for the development of novel CF-therapy drug candidates.
PMID: 30825185 [PubMed – as supplied by publisher]
Emerging Therapeutic Approaches for Cystic Fibrosis. From Gene Editing to Personalized Medicine.
Front Pharmacol. 2019;10:121
Authors: Pranke I, Golec A, Hinzpeter A, Edelman A, Sermet-Gaudelus I
An improved understanding of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) protein structure and the consequences of CFTR gene mutations have allowed the development of novel therapies targeting specific defects underlying CF. Some strategies are mutation specific and have already reached clinical development; some strategies include a read-through of the specific premature termination codons (read-through therapies, nonsense mediated decay pathway inhibitors for Class I mutations); correction of CFTR folding and trafficking to the apical plasma membrane (correctors for Class II mutations); and an increase in the function of CFTR channel (potentiators therapy for Class III mutations and any mutant with a residual function located at the membrane). Other therapies that are in preclinical development are not mutation specific and include gene therapy to edit the genome and stem cell therapy to repair the airway tissue. These strategies that are directed at the basic CF defects are now revolutionizing the treatment for patients and should positively impact their survival rates.
PMID: 30873022 [PubMed]
https://www.mdpi.com/2073-4425/10/3/180 [Read more…] about Transcriptome Profiling and Molecular Therapeutic Advances in Cystic Fibrosis: Recent Insights
Tetrafunctional Block Copolymers Promote Lung Gene Transfer in Newborn Piglets.
Mol Ther Nucleic Acids. 2019 Feb 26;16:186-193
Authors: Caballero I, Riou M, Hacquin O, Chevaleyre C, Barc C, Pezant J, Pinard A, Fassy J, Rezzonico R, Mari B, Heuzé-Vourc’h N, Pitard B, Vassaux G
Tetrafunctional block copolymers are molecules capable of complexing DNA. Although ineffective in vitro, studies in mice have shown that the tetrafunctional block copolymer 704 is a more efficient lung gene transfer agent than the cationic liposome GL67A, previously used in a phase II clinical trial in cystic fibrosis patients. In the present study, we compared the gene transfer capacity of the 704-DNA formulation and a cationic liposome-DNA formulation equivalent to GL67A in a larger-animal model, the newborn piglet. Our results indicate an efficacy of the 704-DNA formulation well above one order of magnitude higher than that of the cationic liposome-DNA formulation, with no elevated levels of interleukin-6 (IL-6), taken as a marker of inflammation. Transgene expression was heterogeneous within lung lobes, with expression levels that were below the detection threshold in some samples, while high in other samples. This heterogeneity is likely to be due to the bolus injection procedure as well as to the small volume of injection. The present study highlights the potential of tetrafunctional block copolymers as non-viral vectors for lung gene therapy.
PMID: 30897407 [PubMed – as supplied by publisher]
Japonicin-2LF could be potential drug candidate to control MRSA infection in cystic fibrosis patients…