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

Abstract

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.

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

“…hNECs cells were seeded (150,000 cells/insert) onto 6.5 mm 0.4 µm Transwell porous polyester membranes (Sigma CLS3470) pre-coated with collagen I. Cells were cultured as described previously ( 30 , 44 , 47 ). Briefly, cells were cultured with PneumaCult Ex Plus expansion media (STEMCELL Technologies 05040) until a confluent monolayer was reached (∼4 days).…”

“…Differentiated hNECs were lysed with TNI lysis buffer (0.5% Igepal CA-630, 50 mM Tris pH 7.5, 250 mM NaCl, 1 mM EDTA) ( 48 ) containing protease inhibitor cocktail (Roche 04693159001) for 30 min on ice. As previously described ( 30 ), lysates were sonicated using the Bioruptor Pico (Diagenode, Liège, Belgium) at 4°C for 20 cycles (30 s on, 30 s off). Lysates were then centrifuged at 14,000 rpm at 4°C for 20 min.…”

“…In parallel in silico Molecular Dynamics (MD) simulations provide a powerful tool to understand the structural basis of CFTR protein function and explore the conformational energy landscape caused by a CFTR mutation. The combination approach of using in vitro experiments and in silico MD simulations has been used previously to characterize rare CFTR mutations ( 30 – 33 ). Understanding the functional and structural changes caused by CFTR mutations is important for identifying which drug is most likely to benefit a patient.…”

Leukocyte-specific DNA methylation biomarkers and their implication for pathological epigenetic analysis

“…Isolated DNA was resuspended in RNAse-free water and quantified using Thermo Scientific Nano Drop Spectrophotometer. Intestinal organoids were cultured according to the established protocol [61] from crypts isolated from rectal biopsies as described previously [62]. The participants' provided written informed consent.…”

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

“…We describe a detailed stepwise protocol for the wholemount staining and imaging and the quantification of various epithelial cell markers and pathogens (such as the RNA virus SARS-CoV-2 antigen) in ALI cultures of CF and non-CF airway epithelial cells grown on membranes. The antibodies recommended in this protocol are well-established and were previously reported by our group [ 18 , 19 ] and by others ( Table S1 ). We extended this protocol with instructions for the quantification of the protein signal of interest.…”