Hierarchical assembly of tryptophan zipper peptides into stress-relaxing bioactive hydrogels.

Soft materials in nature are formed through reversible supramolecular assembly of biological polymers into dynamic hierarchical networks. Rational design has led to self-assembling peptides with structural similarities to natural materials. However, recreating the dynamic functional properties inherent to natural systems remains challenging. Here we report the discovery of a short peptide based on the tryptophan zipper (trpzip) motif, that shows multiscale hierarchical ordering that leads to emergent dynamic properties. Trpzip hydrogels are antimicrobial and self-healing, with tunable viscoelasticity and unique yield-stress properties that allow immediate harvest of embedded cells through a flick of the wrist. This characteristic makes Trpzip hydrogels amenable to syringe extrusion, which we demonstrate with examples of cell delivery and bioprinting. Trpzip hydrogels display innate bioactivity, allowing propagation of human intestinal organoids with apical-basal polarization. Considering these extensive attributes, we anticipate the Trpzip motif will prove a versatile building block for supramolecular assembly of soft materials for biotechnology and medicine.

Q1291H-CFTR molecular dynamics simulations and ex vivo theratyping in nasal epithelial models and clinical response to elexacaftor/tezacaftor/ivacaftor in a Q1291H/F508del patient.

Background: Cystic fibrosis (CF) is caused by a wide spectrum of mutations in the CF transmembrane conductance regulator (CFTR) gene, with some leading to non-classical clinical presentations. We present an integrated in vivo, in silico and in vitro investigation of an individual with CF carrying the rare Q1291H-CFTR allele and the common F508del allele. At age 56 years, the participant had obstructive lung disease and bronchiectasis, qualifying for Elexacaftor/Tezacaftor/Ivacaftor (ETI) CFTR modulator treatment due to their F508del allele. Q1291H CFTR incurs a splicing defect, producing both a normally spliced but mutant mRNA isoform and a misspliced isoform with a premature termination codon, causing nonsense mediated decay. The effectiveness of ETI in restoring Q1291H-CFTR is largely unknown. Methods: We collected clinical endpoint measurements, including forced expiratory volume in 1 s percent predicted (FEV1pp) and body mass index (BMI), and examined medical history. In silico simulations of the Q1291H-CFTR were compared to Q1291R, G551D, and wild-type (WT)-CFTR. We quantified relative Q1291H CFTR mRNA isoform abundance in patient-derived nasal epithelial cells. Differentiated pseudostratified airway epithelial cell models at air liquid interface were created and ETI treatment impact on CFTR was assessed by electrophysiology assays and Western blot. Results: The participant ceased ETI treatment after 3 months due to adverse events and no improvement in FEV1pp or BMI. In silico simulations of Q1291H-CFTR identified impairment of ATP binding similar to known gating mutants Q1291R and G551D-CFTR. Q1291H and F508del mRNA transcripts composed 32.91% and 67.09% of total mRNA respectively, indicating 50.94% of Q1291H mRNA was misspliced and degraded. Mature Q1291H-CFTR protein expression was reduced (3.18% ± 0.60% of WT/WT) and remained unchanged with ETI. Baseline CFTR activity was minimal (3.45 ± 0.25 µA/cm2) and not enhanced with ETI (5.73 ± 0.48 µA/cm2), aligning with the individual's clinical evaluation as a non-responder to ETI. Conclusion: The combination of in silico simulations and in vitro theratyping in patient-derived cell models can effectively assess CFTR modulator efficacy for individuals with non-classical CF manifestations or rare CFTR mutations, guiding personalized treatment strategies and optimizing clinical outcomes.

Molecular and Functional Characteristics of Airway Epithelium under Chronic Hypoxia.

Localized and chronic hypoxia of airway mucosa is a common feature of progressive respiratory diseases, including cystic fibrosis (CF). However, the impact of prolonged hypoxia on airway stem cell function and differentiated epithelium is not well elucidated. Acute hypoxia alters the transcription and translation of many genes, including the CF transmembrane conductance regulator (CFTR). CFTR-targeted therapies (modulators) have not been investigated in vitro under chronic hypoxic conditions found in CF airways in vivo. Nasal epithelial cells (hNECs) derived from eight CF and three non-CF participants were expanded and differentiated at the air-liquid interface (26-30 days) at ambient and 2% oxygen tension (hypoxia). Morphology, global proteomics (LC-MS/MS) and function (barrier integrity, cilia motility and ion transport) of basal stem cells and differentiated cultures were assessed. hNECs expanded at chronic hypoxia, demonstrating epithelial cobblestone morphology and a similar proliferation rate to hNECs expanded at normoxia. Hypoxia-inducible proteins and pathways in stem cells and differentiated cultures were identified. Despite the stem cells' plasticity and adaptation to chronic hypoxia, the differentiated epithelium was significantly thinner with reduced barrier integrity. Stem cell lineage commitment shifted to a more secretory epithelial phenotype. Motile cilia abundance, length, beat frequency and coordination were significantly negatively modulated. Chronic hypoxia reduces the activity of epithelial sodium and CFTR ion channels. CFTR modulator drug response was diminished. Our findings shed light on the molecular pathophysiology of hypoxia and its implications in CF. Targeting hypoxia can be a strategy to augment mucosal function and may provide a means to enhance the efficacy of CFTR modulators.

Assessment of Pre-Clinical Liver Models Based on Their Ability to Predict the Liver-Tropism of Adeno-Associated Virus Vectors.

The liver is a prime target for in vivo gene therapies using recombinant adeno-associated viral vectors. Multiple clinical trials have been undertaken for this target in the past 15 years; however, we are still to see market approval of the first liver-targeted adeno-associated virus (AAV)-based gene therapy. Inefficient expression of the therapeutic transgene, vector-induced liver toxicity and capsid, and/or transgene-mediated immune responses reported at high vector doses are the main challenges to date. One of the contributing factors to the insufficient clinical outcomes, despite highly encouraging preclinical data, is the lack of robust, biologically and clinically predictive preclinical models. To this end, this study reports findings of a functional evaluation of 6 AAV vectors in 12 preclinical models of the human liver, with the aim to uncover which combination of models is the most relevant for the identification of AAV capsid variant for safe and efficient transgene delivery to primary human hepatocytes. The results, generated by studies in models ranging from immortalized cells, iPSC-derived and primary hepatocytes, and primary human hepatic organoids to in vivo models, increased our understanding of the strengths and weaknesses of each system. This should allow the development of novel gene therapies targeting the human liver.

S945L-CFTR molecular dynamics, functional characterization and tezacaftor/ivacaftor efficacy in vivo and in vitro in matched pediatric patient-derived cell models.

Cystic Fibrosis (CF) results from over 400 different disease-causing mutations in the CF Transmembrane Conductance Regulator (CFTR) gene. These CFTR mutations lead to numerous defects in CFTR protein function. A novel class of targeted therapies (CFTR modulators) have been developed that can restore defects in CFTR folding and gating. This study aimed to characterize the functional and structural defects of S945L-CFTR and interrogate the efficacy of modulators with two modes of action: gating potentiator [ivacaftor (IVA)] and folding corrector [tezacaftor (TEZ)]. The response to these modulators in vitro in airway differentiated cell models created from a participant with S945L/G542X-CFTR was correlated with in vivo clinical outcomes of that participant at least 12 months pre and post modulator therapy. In this participants' airway cell models, CFTR-mediated chloride transport was assessed via ion transport electrophysiology. Monotherapy with IVA or TEZ increased CFTR activity, albeit not reaching statistical significance. Combination therapy with TEZ/IVA significantly (p = 0.02) increased CFTR activity 1.62-fold above baseline. Assessment of CFTR expression and maturation via western blot validated the presence of mature, fully glycosylated CFTR, which increased 4.1-fold in TEZ/IVA-treated cells. The in vitro S945L-CFTR response to modulator correlated with an improvement in in vivo lung function (ppFEV1) from 77.19 in the 12 months pre TEZ/IVA to 80.79 in the 12 months post TEZ/IVA. The slope of decline in ppFEV1 significantly (p = 0.02) changed in the 24 months post TEZ/IVA, becoming positive. Furthermore, there was a significant improvement in clinical parameters and a fall in sweat chloride from 68 to 28 mmol/L. The mechanism of dysfunction of S945L-CFTR was elucidated by in silico molecular dynamics (MD) simulations. S945L-CFTR caused misfolding of transmembrane helix 8 and disruption of the R domain, a CFTR domain critical to channel gating. This study showed in vitro and in silico that S945L causes both folding and gating defects in CFTR and demonstrated in vitro and in vivo that TEZ/IVA is an efficacious modulator combination to address these defects. As such, we support the utility of patient-derived cell models and MD simulations in predicting and understanding the effect of modulators on CFTR function on an individualized basis.

Quantifying Intracellular Viral Pathogen: Specimen Preparation, Visualization and Quantification of Multiple Immunofluorescent Signals in Fixed Human Airway Epithelium Cultured at Air-Liquid Interface.

Infection control and aggressive antibiotic therapy play an important role in the management of airway infections in individuals with cystic fibrosis (CF). The responses of airway epithelial cells to pathogens are likely to contribute to the pathobiology of CF lung disease. Primary airway epithelial cells obtained from individuals with CF, cultured and differentiated at air-liquid interface (ALI), effectively mimic the structure and function of the in vivo airway epithelium. With the recent respiratory viral pandemics, ALI cultures were extensively used to model respiratory infections in vitro to facilitate physiologically relevant respiratory research. Immunofluorescence staining and imaging were used as an effective tool to provide a fundamental understanding of host-pathogen interactions and for exploring the therapeutic potential of novel or repurposed drugs. Therefore, we described an optimized quantitative fluorescence microscopy assay for the wholemount staining and imaging of epithelial cell markers to identify distinct cell populations and pathogen-specific targets in ALI cultures of human airway epithelial cells grown on permeable support insert membranes. We present a detailed methodology using a graphical user interface (GUI) package to quantify the detected signals on a tiled whole membrane. Our method provided an imaging strategy of the entire membrane, overcoming the common issue of undersampling and enabling unbiased quantitative analysis.

Molecular Dynamics and Theratyping in Airway and Gut Organoids Reveal R352Q-CFTR Conductance Defect.

A significant challenge to making targeted cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapies accessible to all individuals with cystic fibrosis (CF) are many mutations in the CFTR gene that can cause CF, most of which remain uncharacterized. Here, we characterized the structural and functional defects of the rare CFTR mutation R352Q, with a potential role contributing to intrapore chloride ion permeation, in patient-derived cell models of the airway and gut. CFTR function in differentiated nasal epithelial cultures and matched intestinal organoids was assessed using an ion transport assay and forskolin-induced swelling assay, respectively. CFTR potentiators (VX-770, GLPG1837, and VX-445) and correctors (VX-809, VX-445, with or without VX-661) were tested. Data from R352Q-CFTR were compared with data of 20 participants with mutations with known impact on CFTR function. R352Q-CFTR has residual CFTR function that was restored to functional CFTR activity by CFTR potentiators but not the corrector. Molecular dynamics simulations of R352Q-CFTR were carried out, which indicated the presence of a chloride conductance defect, with little evidence supporting a gating defect. The combination approach of in vitro patient-derived cell models and in silico molecular dynamics simulations to characterize rare CFTR mutations can improve the specificity and sensitivity of modulator response predictions and aid in their translational use for CF precision medicine.

Air-Liquid-Interface Differentiated Human Nose Epithelium: A Robust Primary Tissue Culture Model of SARS-CoV-2 Infection.

The global urgency to uncover medical countermeasures to combat the COVID-19 pandemic caused by the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) has revealed an unmet need for robust tissue culture models that faithfully recapitulate key features of human tissues and disease. Infection of the nose is considered the dominant initial site for SARS-CoV-2 infection and models that replicate this entry portal offer the greatest potential for examining and demonstrating the effectiveness of countermeasures designed to prevent or manage this highly communicable disease. Here, we test an air-liquid-interface (ALI) differentiated human nasal epithelium (HNE) culture system as a model of authentic SARS-CoV-2 infection. Progenitor cells (basal cells) were isolated from nasal turbinate brushings, expanded under conditionally reprogrammed cell (CRC) culture conditions and differentiated at ALI. Differentiated cells were inoculated with different SARS-CoV-2 clinical isolates. Infectious virus release into apical washes was determined by TCID50, while infected cells were visualized by immunofluorescence and confocal microscopy. We demonstrate robust, reproducible SARS-CoV-2 infection of ALI-HNE established from different donors. Viral entry and release occurred from the apical surface, and infection was primarily observed in ciliated cells. In contrast to the ancestral clinical isolate, the Delta variant caused considerable cell damage. Successful establishment of ALI-HNE is donor dependent. ALI-HNE recapitulate key features of human SARS-CoV-2 infection of the nose and can serve as a pre-clinical model without the need for invasive collection of human respiratory tissue samples.

Molecular dynamics and functional characterization of I37R-CFTR lasso mutation provide insights into channel gating activity.

Characterization of I37R, a mutation located in the lasso motif of the CFTR chloride channel, was conducted by theratyping several CFTR modulators from both potentiator and corrector classes. Intestinal current measurements in rectal biopsies, forskolin-induced swelling (FIS) in intestinal organoids, and short circuit current measurements in organoid-derived monolayers from an individual with I37R/F508del CFTR genotype demonstrated that the I37R-CFTR results in a residual function defect amenable to treatment with potentiators and type III, but not type I, correctors. Molecular dynamics of I37R using an extended model of the phosphorylated, ATP-bound human CFTR identified an altered lasso motif conformation which results in an unfavorable strengthening of the interactions between the lasso motif, the regulatory (R) domain, and the transmembrane domain 2 (TMD2). Structural and functional characterization of the I37R-CFTR mutation increases understanding of CFTR channel regulation and provides a potential pathway to expand drug access to CF patients with ultra-rare genotypes.

Collection, Expansion, and Differentiation of Primary Human Nasal Epithelial Cell Models for Quantification of Cilia Beat Frequency.

Measurements of cilia function (beat frequency, pattern) have been established as diagnostic tools for respiratory diseases such as primary ciliary dyskinesia. However, the wider application of these techniques is limited by the extreme susceptibility of ciliary function to changes in environmental factors e.g., temperature, humidity, and pH. In the airway of patients with Cystic Fibrosis (CF), mucus accumulation impedes cilia beating. Cilia function has been investigated in primary airway cell models as an indicator of CF Transmembrane conductance Regulator (CFTR) channel activity. However, considerable patient-to-patient variability in cilia beating frequency has been found in response to CFTR-modulating drugs, even for patients with the same CFTR mutations. Furthermore, the impact of dysfunctional CFTR-regulated chloride secretion on ciliary function is poorly understood. There is currently no comprehensive protocol demonstrating sample preparation of in vitro airway models, image acquisition, and analysis of Cilia Beat Frequency (CBF). Standardized culture conditions and image acquisition performed in an environmentally controlled condition would enable consistent, reproducible quantification of CBF between individuals and in response to CFTR-modulating drugs. This protocol describes the quantification of CBF in three different airway epithelial cell model systems: 1) native epithelial sheets, 2) air-liquid interface models imaged on permeable support inserts, and 3) extracellular matrix-embedded three-dimensional organoids. The latter two replicate in vivo lung physiology, with beating cilia and production of mucus. The ciliary function is captured using a high-speed video camera in an environment-controlled chamber. Custom-built scripts are used for the analysis of CBF. Translation of CBF measurements to the clinic is envisioned to be an important clinical tool for predicting response to CFTR-modulating drugs on a per-patient basis.

Significant functional differences in differentiated Conditionally Reprogrammed (CRC)- and Feeder-free Dual SMAD inhibited-expanded human nasal epithelial cells.

BACKGROUND: Patient-derived airway cells differentiated at Air Liquid Interface (ALI) are valuable models for Cystic fibrosis (CF) precision therapy. Different culture expansion methods have been established to extend expansion capacity of airway basal cells, while retaining functional airway epithelium physiology. Considerable variation in response to CFTR modulators is observed in cultures even within the same CFTR genotype and despite the use of similar ALI culture techniques. We aimed to address culture expansion method impact on differentiation. METHODS: Nasal epithelial brushings from 14 individuals (CF=9; non-CF=5) were collected, then equally divided and expanded under conditional reprogramming culture (CRC) and feeder-serum-free "dual-SMAD inhibition" (SMADi) methods. Expanded cells from each culture were differentiated with proprietary PneumaCult™-ALI media. Morphology (Immunofluorescence), global proteomics (LC-MS/MS) and function (barrier integrity, cilia motility, and ion transport) were compared in CRCALI and SMADiALI under basal and CFTR corrector treated (VX-809) conditions. RESULTS: No significant difference in the structural morphology or baseline global proteomics profile were observed. Barrier integrity and cilia motility were significantly different, despite no difference in cell junction morphology or cilia abundance. Epithelial Sodium Channels and Calcium-activated Chloride Channel activity did not differ but CFTR mediated chloride currents were significantly reduced in SMADiALI compare to their CRCALI counterparts. CONCLUSION: Alteration of cellular physiological function in vitro were more prominent than structural and differentiation potential in airway ALI. Since initial expansion culture conditions significantly influence CFTR activity, this could lead to false conclusions if data from different labs are compared against each other without specific reference ranges.

Novel Antioxidant Therapy with the Immediate Precursor to Glutathione, γ-Glutamylcysteine (GGC), Ameliorates LPS-Induced Cellular Stress in In Vitro 3D-Differentiated Airway Model from Primary Cystic Fibrosis Human Bronchial Cells.

Systemic glutathione deficiency, inflammation, and oxidative stress are hallmarks of cystic fibrosis (CF), an inherited disease that causes persistent lung infections and severe damage to the respiratory system and many of the body organs. Improvements to current antioxidant therapeutic strategies are needed. The dietary supplement, γ-glutamylcysteine (GGC), which is the immediate precursor to glutathione, rapidly boosts cellular glutathione levels following a single dose in healthy individuals. Efficacy of GGC against oxidative stress induced by Pseudomonas aeruginosa, which is a common and chronic pathogen infecting lungs of CF patients, remains unassessed. Primary mucocilliary differentiated airway (bronchial and/or nasal) epithelial cells were created from four individuals with CF. Airway oxidative stress and inflammation was induced by P. aeruginosa lipopolysaccharide (LPS). Parameters including global proteomics alterations, cell redox state (glutathione, oxidative stress), pro-inflammatory mediators (IL-8, IDO-1), and cellular health (membrane integrity, stress granule formation, cell metabolic viability) were assayed under six experimental conditions: (1) Mock, (2) LPS-challenged (3) therapeutic, (4) prophylactic (5) therapeutic and prophylactic and (6) GGC alone. Proteomic analysis identified perturbation of several pathways related to cellular respiration and stress responses upon LPS challenge. Most of these were resolved when cells were treated with GGC. While GGC did not resolve LPS-induced IL-8 and IDO-1 activity, it effectively attenuated LPS-induced oxidative stress and stress granule formation, while significantly increasing total intracellular glutathione levels, metabolic viability and improving epithelial cell barrier integrity. Both therapeutic and prophylactic treatments were successful. Together, these findings indicate that GGC has therapeutic potential for treatment and prevention of oxidative stress-related damage to airways in cystic fibrosis.

Human Primary Epithelial Cell Models: Promising Tools in the Era of Cystic Fibrosis Personalized Medicine.

Cystic fibrosis (CF) is an inherited disorder where individual disease etiology and response to therapeutic intervention is impacted by CF transmembrane regulator (CFTR) mutations and other genetic modifiers. CFTR regulates multiple mechanisms in a diverse range of epithelial tissues. In this Review, we consolidate the latest updates in the development of primary epithelial cellular model systems relevant for CF. We discuss conventional two-dimensional (2-D) airway epithelial cell cultures, the backbone of in vitro cellular models to date, as well as improved expansion protocols to overcome finite supply of the cellular source. We highlight a range of strategies for establishment of three dimensional (3-D) airway and intestinal organoid models and evaluate the limitations and potential improvements in each system, focusing on their application in CF. The in vitro CFTR functional assays in patient-derived organoids allow for preclinical pharmacotherapy screening to identify responsive patients. It is likely that organoids will be an invaluable preclinical tool to unravel disease mechanisms, design novel treatments, and enable clinicians to provide personalized management for patients with CF.

Proteomic Analysis of Extracellular HMGB1 Identifies Binding Partners and Exposes Its Potential Role in Airway Epithelial Cell Homeostasis.

The release of damage-associated molecular patterns (DAMPs) by airway epithelial cells is believed to play a crucial role in the initiation and development of chronic airway conditions such as asthma and chronic obstructive pulmonary disease (COPD). Intriguingly, the classic DAMP high-mobility group box-1 (HMGB1) is detected in the culture supernatant of airway epithelial cells under basal conditions, indicating a role for HMGB1 in the regulation of epithelial cellular and immune homeostasis. To gain contextual insight into the potential role of HMGB1 in airway epithelial cell homeostasis, we used the orthogonal and complementary methods of high-resolution clear native electrophoresis, immunoprecipitation, and pull-downs coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) to profile HMGB1 and its binding partners in the culture supernatant of unstimulated airway epithelial cells. We found that HMGB1 presents exclusively as a protein complex under basal conditions. Moreover, protein network analysis performed on 185 binding proteins revealed 14 that directly associate with HMGB1: amyloid precursor protein, F-actin-capping protein subunit alpha-1 (CAPZA1), glyceraldehyde-3 phosphate dehydrogenase (GAPDH), ubiquitin, several members of the heat shock protein family (HSPA8, HSP90B1, HSP90AA1), XRCC5 and XRCC6, high mobility group A1 (HMGA1), histone 3 (H3F3B), the FACT (facilitates chromatin transcription) complex constituents SUPT1H and SSRP1, and heterogeneous ribonucleoprotein K (HNRNPK). These studies provide a new understanding of the extracellular functions of HMGB1 in cellular and immune homeostasis at the airway mucosal surface and could have implications for therapeutic targeting.

The SPARC protein: an overview of its role in lung cancer and pulmonary fibrosis and its potential role in chronic airways disease.

The SPARC (secreted protein acidic and rich in cysteine) protein is matricellular molecule regulating interactions between cells and their surrounding extracellular matrix (ECM). This protein thus governs fundamental cellular functions such as cell adhesion, proliferation and differentiation. SPARC also regulates the expression and activity of numerous growth factors and matrix metalloproteinases essential for ECM degradation and turnover. Studies in SPARC-null mice have revealed a critical role for SPARC in tissue development, injury and repair and in the regulation of the immune response. In the lung, SPARC drives pathological responses in non-small cell lung cancer and idiopathic pulmonary fibrosis by promoting microvascular remodelling and excessive deposition of ECM proteins. Remarkably, although chronic airway conditions such as asthma and chronic obstructive pulmonary disease (COPD) involve significant remodelling in both the airway and vascular compartments, the role of SPARC in these conditions has thus far been overlooked. In this review, we discuss the role of SPARC in lung cancer and pulmonary fibrosis, as well as potential mechanisms by which it may contribute to the disease process in asthma and COPD.