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.

Comparing Cytology Brushes for Optimal Human Nasal Epithelial Cell Collection: Implications for Airway Disease Diagnosis and Research.

Primary nasal epithelial cells and culture models are used as important diagnostic, research and drug development tools for several airway diseases. Various instruments have been used for the collection of human nasal epithelial (HNE) cells but no global consensus yet exists regarding the optimal tool. This study compares the efficiency of two cytology brushes (Olympus (2 mm diameter) and Endoscan (8 mm diameter)) in collecting HNE cells. The study involved two phases, with phase one comparing the yield, morphology and cilia beat frequency (CBF) of cells collected from paediatric participants using each of the two brushes. Phase two compared nasal brushing under general anaesthetic and in the awake state, across a wide age range, via the retrospective audit of the use of the Endoscan brush in 145 participants. Results indicated no significant difference in CBF measurements between the two brushes, suggesting that the choice of brush does not compromise diagnostic accuracy. However, the Endoscan brush collected significantly more total and live cells than the Olympus brush, making it a more efficient option. Importantly, the Endoscan brush is more cost-effective, with a notable price difference between the two brushes.

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.

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.

Treatment of Cystic Fibrosis: From Gene- to Cell-Based Therapies.

Prognosis of patients with cystic fibrosis (CF) varies extensively despite recent advances in targeted therapies that improve CF transmembrane conductance regulator (CFTR) function. Despite being a multi-organ disease, extensive lung tissue destruction remains the major cause of morbidity and mortality. Progress towards a curative treatment strategy that implements a CFTR gene addition-technology to the patients' lungs has been slow and not yet developed beyond clinical trials. Improved delivery vectors are needed to overcome the body's defense system and ensure an efficient and consistent clinical response before gene therapy is suitable for clinical care. Cell-based therapy-which relies on functional modification of allogenic or autologous cells ex vivo, prior to transplantation into the patient-is now a therapeutic reality for various diseases. For CF, pioneering research has demonstrated proof-of-principle for allogenic transplantation of cultured human airway stem cells into mouse airways. However, applying a cell-based therapy to the human airways has distinct challenges. We review CF gene therapies using viral and non-viral delivery strategies and discuss current advances towards autologous cell-based therapies. Progress towards identification, correction, and expansion of a suitable regenerative cell, as well as refinement of pre-cell transplant lung conditioning protocols is discussed.