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

Abstract

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.

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

“…The simulation methodologies for this study are the same as those used previously ( Allan et al, 2022 ; Wong et al, 2022a ; Wong et al, 2022b ). In brief, an extended model of the CFTR protein bound to ATP under phosphorylating conditions (PDB ID: 6MSM) was embedded in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) model bilayer then solvated with a solution containing 150 mmol KCl.…”

“…Various patient derived cell models [reviewed in (10)] have been shown to replicate the genetic makeup of the individual they were created from. Importantly, CFTR functional testing in patient-derived cell models correlates with the in vivo clinical outcomes for that patient ( Dekkers et al, 2016 ; McGarry et al, 2017 ; Mutyam et al, 2017 ; McCarthy et al, 2018 ; Phuan et al, 2021 ; Terlizzi et al, 2021 ; Allan et al, 2022 ; Ciciriello et al, 2022 ). As such, they are useful tools to predict patient responsiveness to CFTR modulators and characterize rare mutations ( Awatade et al, 2018 ).…”

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

“…The reanalysis of cilia is often necessary following HNE cell culture and differentiation at the air–liquid interface to investigate the potential impact of secondary infections on CBF, which can modify ciliary function and lead to the potential misinterpretation of results in PCD assessment. Beyond PCD, HNE cell cultures have become increasingly important for diagnosis and studying the pathophysiology of, and developing drugs for, other airway diseases, such as allergic rhinitis, asthma and cystic fibrosis (CF) [ 9 , 11 , 12 , 13 , 14 , 15 , 16 ].…”

Molecular and Functional Characteristics of Airway Epithelium under Chronic Hypoxia

“…In vitro assessment of CFTR modulator efficacy in patient-derived cell models has accelerated drug regulatory processes and provided access to personalized treatment to individuals who would otherwise be ineligible for approved modulator therapy [ 20 , 21 , 22 ]. Several studies comparing in vitro and in vivo CFTR function reported a correlation between the two; however, the sample size of the studies was relatively small [ 20 , 23 , 24 , 25 ]. A recent study reported that patient-derived cell models could identify modulator-responsive patients but may not accurately predict the magnitude of clinical benefit [ 26 ].…”