The cystic fibrosis (CF) causing variant G542X harbours a premature translation stop signal in the cystic fibrosis transmembrane conductance regulator (CFTR) mRNA. This results in nonsense-mediated decay and loss of functional CFTR protein which leads to defective anion transport and the development of CF disease pathology. Currently available CF modulator therapies cannot be used to treat this variant. We used an adenine base editor (ABE8e Cas9) and guide RNA (sgRNA)/enhanced green fluorescent protein (EGFP) plasmids encapsulated in receptor targeted nanoparticles (RTN), delivered to Bmi-1 transduced basal human CF nasal epithelial cells harbouring the homozygous CFTR G542X variant, to convert the stop codon to G542R, a variant which is amenable to modulator therapy. ABE resulted in 17% of alleles edited to G542R and further selection of GFP fluorescent cells by FACS liberated a population with 52% G542R edited alleles with no editing of neighbouring adenines (A) and few off target edits using a gRNA homology-based approach. In cells differentiated at air-liquid-interface (ALI), 17% and 52% editing of CFTR G542X increased mRNA abundance. 52% editing alone or 17% and 52% editing of CFTR G542X plus treatment with CFTR modulators (VX-445/VX-661/VX-770; ETI/Trikafta/Kaftrio) increased epithelial CFTR protein expression, CFTR protein band C abundance, CFTR172 inhibitable anion transport, and changes in airway surface liquid height and pH in response to vasoactive intestinal peptide (VIP) stimulation. Epithelial scratch repair speed and directionality was also improved. These data provide proof-of-concept that ABE of G542X to G542R in human CF airway epithelial cells could provide a feasible therapy for this variant.

The respiratory tract possesses a highly regulated innate defense system that includes cilia-mediated mucociliary clearance (MCC). Efficient MCC relies on appropriate hydration of airway surfaces, which is controlled by a blend of transepithelial sodium and liquid absorption, as well as anion and liquid secretion. The latter is mediated primarily by the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel. Succinate is derived from parasites, microorganisms, and inflammatory cells, and its concentration increases in the airway surface liquid during infections, activating the G protein-coupled succinate receptor (SUCNR1), which acts as a succinate sensor. Because MCC is tightly regulated by second messengers, we tested the hypothesis that succinate signaling was linked to CFTR activity. We observed that SUCNR1 activation stimulated anion secretion, increased mucus transport, and induced tracheal constriction in mouse airways. In the CftrΔF508/ΔF508 mouse, increased mucus transport and tracheal constriction were not observed, whereas succinate-induced electrogenic anion secretion remained unaffected. Stimulation of normal human bronchial epithelial cells with succinate activated CFTR-dependent anion secretion and increased airway surface liquid height. Moreover, human bronchial epithelial cells derived from ΔF508-CF individuals that lacked succinate-induced anion secretion, unless incubated with elexacaftor-tezacaftor-ivacaftor, which restored succinate-induced anion secretion, confirmed the tight relationship between SUCNR1 signaling and CFTR function. We have identified a novel mechanism for regulating CFTR/MCC activation that is defective in cystic fibrosis airways. We propose that succinate acts as a danger molecule that alerts the airways to the presence of pathogens leading to a flushing out of the airways.

Rationale People with cystic fibrosis (pwCF) exhibit chronic and hyperactive neutrophilia which results in a progressive loss of lung function. CF neutrophils have elevated store operated Ca2+ entry (SOCE) relative to healthy non-CF neutrophils, which contributes to persistent neutrophilia. The vast majority of pwCF now take CFTR modulators such as elexacaftor/tezacaftor/ivacaftor (ETI), which effectively increase CFTR function in multiple organs including the lung. However, ETI’s impact on neutrophils is poorly understood. Orai1 is a plasma membrane Ca2+ channel that contributes to SOCE. We have developed a novel peptide (ELD607) that specifically inhibits Orai1, which we evaluated in CF neutrophils. Objectives To characterize Orai1/SOCE in neutrophils from pwCF taking ETI, and to evaluate the impact of SOCE inhibition by ELD607 on pwCF neutrophil Ca2+ signaling/function. Methods Peripheral blood neutrophils were isolated by negative selection. SOCE was characterized using fluorescent approaches. Protein expression was characterized by proteomics and confocal microscopy. Neutrophil degranulation was measured using a multiplex assay. Measurements and main results Proteomic analysis revealed major global differences between non-CF and pwCF neutrophils, despite use of ETI. Several proteins involved in SOCE, including Orai1, were significantly elevated in pwCF neutrophils. ELD607 dose-dependently inhibited SOCE, leading to reduced neutrophil degranulation. Ca2+ homeostasis was significantly elevated in pwCF compared to non-CF neutrophils. ELD607-sensitive SOCE inversely correlated with lung function (FEV1pp). Conclusions Our findings highlight SOCE as a novel biomarker of CF lung disease. ELD607 can be used to reduce SOCE and subsequent degranulation in CF neutrophils. We therefore hypothesize that ELD607 may be of benefit in the management of inflammation in pwCF.

Inhaled therapeutics have high potential for the treatment of chronic respiratory diseases of high unmet medical need, such as idiopathic pulmonary fibrosis (IPF). Preclinical and early clinical evidence show that cellular communication network factor 2 (CCN2), previously called connective tissue growth factor (CTGF), is a promising target for the treatment of IPF. In recent phase 3 clinical trials, however, systemic CCN2 inhibition failed to demonstrate a clinically meaningful benefit. Here, we present the preclinical profile of the inhaled anti-CCN2 Anticalin® protein PRS-220. Our study demonstrates that efficient pulmonary delivery directly translates into superior efficacy in relevant models of pulmonary fibrosis when compared to systemic CCN2 inhibition. Moreover, we present a holistic approach for the preclinical characterization of inhaled PRS-220 from state-of-the art in vitro and in vivo models to novel human ex vivo and in silico models, highlighting the advantage of inhaled drug delivery for treatment of respiratory disease.

E-cigarettes and heated tobacco products are marketed as safer combustible cigarette alternatives due to their perceived potential for reduced tobacco-related toxicant exposure; however, their relative safety remains controversial. In this study we utilized serum protease levels, established biomarkers of harm contributing to lung disease, to study the effects of alternate tobacco products. Twenty-one adults who smoke cigarettes completed three visits in a randomized crossover design, separated by a 48-h washout period. Participants used their usual brand of cigarette (UBC), e-cigarette (JUUL), and heated tobacco (IQOS). We quantified serum proteases (matrix metalloproteinase (MMP) 1, MMP9, and neutrophil elastase (NE) using graphene-based nanobiosensors. UBC delivered significantly greater peak nicotine concentrations compared to JUUL or IQOS. Every device increased peak serum protease levels. After adjustment for serum nicotine, JUUL use resulted in higher levels of NE and MMP1 compared to UBC. Hierarchical clustering revealed three distinct patterns of systemic protease production that agnostically grouped by device. We demonstrated that e-cigarettes, but not IQOS, exhibited increased risk of potentially pathogenic protease release compared to UBC. These data indicate the need for prospectively designed and fully powered studies of longer duration to better understand the relative risks of e-cigarettes, IQOS and cigarettes on protease activation.

Short palate lung and nasal epithelial clone 1 (SPLUNC1; gene name BPIFA1) is a secreted protein that is highly expressed in the nasopharyngeal and pulmonary systems. By data mining, we found that SPLUNC1 is also expressed in other organs, including the kidneys and the pituitary gland. SPLUNC1 is an asthma and cystic fibrosis gene modifier that also inversely correlates with the severity of bronchiectasis. Orai1 is a plasma membrane Ca2+ channel that is an essential regulator of the immune system. We previously found that SPLUNC1 binds to Orai1, causing it to be ubiquitinated, internalized and trafficked to the lysosome for degradation, thus reducing Ca2+ signaling. Here, we discuss how dysregulation of SPLUNC1–Orai1 interactions may contribute to hyperinflammation in multiple pulmonary diseases. We, and others, have also targeted Orai1 therapeutically, and we will also discuss how Orai1 inhibition may overcome SPLUNC1 deficiency and be beneficial for the treatment of chronic lung disease.

Background and Objectives: The rising popularity of new-generation electronic cigarettes (e-cig) like JUUL necessitates a better understanding of their impact on respiratory and other body systems, as the effects of JUUL’s components remain unclear. This study aimed to investigate the effects of JUUL components on ion channels and airway surface liquid (ASL) height in human bronchial epithelial cells (HBECs). Furthermore, the cytotoxic effects of these components were investigated in human embryonic kidney 293T (HEK293T) cells. Materials and Methods: The components tested included nicotine salt (NicSalt), benzoic acid (BA), sodium hydrogen tartrate (NaTar), propylene glycol/vegetable glycerin (PG/VG), freebase nicotine (FBNic) and nicotine salt+benzoic acid (NicSalt+BA). Each component was prepared at 100 µM, and HBECs were exposed for 24 h to measure ASL height, short-circuit current (Isc), and transepithelial electrical resistance (TEER). Results: Initial exposure (0 h) to these substances did not significantly alter ASL height. However, after 2 h, FBNic-treated HBECs exhibited a significant reduction in ASL height compared to NicSalt and other tested substances, with the most pronounced decrease observed at the 6th hour. This effect persisted over prolonged exposure, suggesting a cumulative impact on airway hydration and epithelial function. Additionally, adenosine administration did not induce a significant increase in ASL height. NicSalt, BA, and FBNic were found to disrupt ion balance in HBECs, affecting ion channels and ASL homeostasis while significantly decreasing TEER. In terms of cytotoxicity, NicSalt, and benzoic acid demonstrated minimal cytotoxicity at low concentrations, whereas FBNic showed significantly higher cytotoxicity at moderate levels. Conclusions: In conclusion, this study highlights that e-cigarette components can disrupt airway surface liquid homeostasis by affecting ion channel activity, compromise epithelial barrier integrity by reducing transepithelial electrical resistance, and emphasize the importance of their cytotoxic effects.

"Translational medicine" has been a buzzword for over two decades. The concept was intended to be lofty, to reflect a new "bench-to-bedside" approach to basic and clinical research that would bridge fields, close gaps, accelerate innovation, and shorten the time and effort it takes to bring novel technologies from basic discovery to clinical application. Has this approach been successful and lived up to its promise? Despite incredible scientific advances and innovations developed within academia, successful clinical translation into real-world solutions has been difficult. This has been particularly challenging within the pulmonary field, because there have been fewer U.S. Food and Drug Administration-approved drugs and higher failure rates for pulmonary therapies than with other common disease areas. The American Thoracic Society convened a working group with the goal of identifying major challenges related to the commercialization of technologies within the pulmonary space and opportunities to enhance this process. A survey was developed and administered to 164 participants within the pulmonary arena. This report provides a summary of these survey results. Importantly, this report identifies a number of poorly recognized challenges that exist in pulmonary academic settings, which likely contribute to diminished efficiency of commercialization efforts, ultimately hindering the rate of successful clinical translation. Because many innovations are initially developed in academic settings, this is a global public health issue that impacts the entire American Thoracic Society community. This report also summarizes key resources and opportunities and provides recommendations to enhance successful commercialization of pulmonary technologies.

The respiratory tract possesses a highly regulated innate defense system which includes efficient cilia-mediated mucus transport or mucociliary clearance (MCC). This essential process relies on appropriate hydration of airway surfaces which is controlled by a blend of transepithelial sodium and liquid absorption via the epithelial sodium channel (ENaC), and anion and liquid secretion, primarily regulated by the cystic fibrosis transmembrane conductance regulator (CFTR) channel. MCC is tightly regulated by second messenger signalling pathways. Succinate is derived from parasites, microorganisms and inflammatory cells, and its concentration increases in the airway surface liquid (ASL) during infections. Increases in ASL succinate activates the G-protein coupled succinate receptor (SUCNR1), which acts as a succinate sensor. Here, we tested the hypothesis that succinate signalling was linked to CFTR activity, ASL hydration and increased MCC. We observed that SUCNR1 activation stimulated anion secretion, increased mucus transport and induced bronchoconstriction in mouse airways. In parallel, stimulation of human bronchial epithelial cells (HBEC) with succinate activated anion secretion and increased ASL height. All functions activated by succinate/SUCNR1 were impeded when working with tissues and cells isolated from animal models or individuals affected cystic fibrosis (CF) or when CFTR was inhibited. Moreover, when HBECs derived from ΔF508 individuals were incubated with the triple drug combination of elexacaftor/tezacaftor/ivacaftor (ETI), succinate-induced anion secretion was restored, confirming the tight relationship between SUCNR1 signalling and CFTR function. Our results identify a novel activation pathway for CFTR that participates in the defence response of the airways, which is defective in CF. We propose that succinate acts as a danger molecule that alerts the airways to the presence of pathogens leading to a flushing out of the airways. ### Competing Interest Statement The authors have declared no competing interest.

Orai1 is a plasma membrane Ca2+ channel involved in store operated calcium entry (SOCE). SOCE can regulate cell growth, exocytosis, gene expression and inflammation. We previously found that short palate lung and nasal epithelial clone 1’s (SPLUNC1) sixth α-helix (α6) bound Orai1 to inhibit SOCE. SPLUNC1 was not proteolytically stable, so we developed ELD607, an 11 amino acid peptide based on SPLUNC1’s α6 region which was more stable and more potent than SPLUNC1/α6. Here, we studied ELD607’s mechanism of action. We overexpressed either Orai1- HA or Orai1-YFP in HEK293T cells to probe ELD607-Orai1 interactions by confocal microscopy. We also measured changes in Fluo-4 fluorescence in a multiplate reader as a marker of cytoplasmic Ca2+ levels. ELD607 internalized Orai1 independently of STIM1. Both 15 min and 3 h exposure to ELD607 similarly depleted Orai1 in the plasma membrane. However, 3 h exposure to ELD607 yielded greater inhibition of SOCE. ELD607 continued to colocalize with Orai1 after internalization and this process was dependent on the presence of the ubiquitin ligase NEDD4.2. Similarly, ELD607 increased the colocalization between Orai1 and ubiquitin. ELD607 also increased the colocalization between Orai1 and Rab5 and 7, but not Rab11, suggesting that Orai1 trafficked through early and late but not recycling endosomes. Finally, ELD607 caused Orai1, but not Orai2, Orai3, or STIM1 to traffic to lysosomes. We conclude that ELD607 rapidly binds to Orai1 and works in an identical fashion as full length SPLUNC1 by internalizing Orai1 and sending it to lysosomes, leading to a decrease in SOCE.