CFTR modulator therapy with elexacaftor/tezacaftor/ivacaftor (ETI) has been approved for people with CF and at least one F508del allele in Europe. In the US, the ETI label has been expanded to 177 rare CFTR mutations responsive in Fischer rat thyroid cells, including G85E, but not N1303K. However, knowledge on the effect of ETI on G85E or N1303K CFTR function remains limited. In vitro effects of ETI were measured in primary human nasal epithelial cultures (pHNECs) of a G85E homozygous patient and an N1303K homozygous patient. Effects of ETI therapy in vivo in these patients were assessed using clinical outcomes, including multiple breath washout and lung MRI, and the CFTR biomarkers sweat chloride concentration (SCC), nasal potential difference (NPD) and intestinal current measurement (ICM), before and after initiation of ETI. ETI increased CFTR-mediated chloride transport in G85E/G85E and N1303K/N1303K pHNECs. In the G85E/G85E and the N1303K/N1303K patient, we observed an improvement in lung function, SCC, and CFTR function in the respiratory and rectal epithelium after initiation of ETI. The approach of combining preclinical in vitro testing with subsequent in vivo verification can facilitate access to CFTR modulator therapy and enhance precision medicine for patients carrying rare CFTR mutations.

Pharmacological rescue of F508del-CFTR by the triple combination CFTR modulator therapy elexacaftor/tezacaftor/ivacaftor (ETI) leads to unprecedented clinical benefits in patients with cystic fibrosis (CF). However, previous studies in CF primary human airway epithelial cultures demonstrated that chronic treatment with the potentiator ivacaftor can render the F508del protein unstable, thus limiting restoration of CFTR chloride channel function. Even so, quantitative studies of this unwanted effect of ivacaftor on F508del channel function with dependency on cell culture conditions remain limited, and the impact of chronic ivacaftor exposure on restoration of mucociliary clearance that is impaired in patients with CF has not been studied. In patient-derived primary nasal epithelial cultures, we found that different culture conditions (UNC-ALI medium vs. PneumaCult medium) have profound effects on ETI-mediated restoration of F508del-CFTR function. Chronic treatment with ivacaftor as part of ETI triple therapy limited the rescue of F508del-CFTR chloride channel function when CF nasal epithelial cultures were grown in UNC-ALI medium but not in PneumaCult medium. In PneumaCult medium, both chronic and acute addition of ivacaftor as part of ETI treatment led to constitutive CFTR-mediated chloride secretion in the absence of exogenous cAMP-dependent stimulation. This constitutive CFTR-mediated chloride secretion was essential to improve viscoelastic properties of the mucus layer and to restore mucociliary transport on CF nasal epithelial cultures. Furthermore, nasal potential difference measurements in patients with CF showed that ETI restored constitutive F508del-CFTR activity in vivo. These results demonstrate that ivacaftor as a component of ETI therapy is essential to restore mucociliary clearance and suggest that this effect is facilitated by its constitutive activation of F508del channels following their folding correction in patients with CF.

Nanobodies have gained considerable attention as particularly promising biopharmaceuticals. However, nanobody-based modalities are currently limited to extracellular targets due to a lack of efficient delivery methods required to reach targets inside cells. In this study, we introduce cell-permeable nanobodies for targeting a disease-relevant intracellular protein, namely the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel with the most common cystic fibrosis (CF)-causing mutation F508del. We employ cell-penetrating peptides (CPPs) to deliver a CFTR-binding nanobody (NB1) that stabilizes misfolded F508del-CFTR and prevents its degradation to restore its function. Our data show that conjugation of a disulfide-linked CPP in combination with a cell-surface anchored CPP-additive enables intracellular delivery of NB1 into CF bronchial epithelial cells, which promotes maturation and trafficking of F508del-CFTR protein to the apical cell membrane. Furthermore, we demonstrate that the cell-permeable nanobody restores CFTR chloride channel function, which can be further enhanced by the clinically approved small molecule CFTR potentiator ivacaftor. This study highlights the use of cell-permeable nanobodies for modulation of protein function and illustrates their therapeutic potential as next-generation biopharmaceuticals for intracellular delivery and targeting. Abstract Figure:

SUMMARY: Defects in protein trafficking underlie many genetic diseases, including cystic fibrosis (CF), where the predominant F508del mutation destabilizes the cystic fibrosis transmembrane conductance regulator (CFTR) channel, leading to its degradation. To provide a protein-specific chaperone, we used lipid nanoparticles to deliver mRNA encoding T2a, a nanobody that thermally stabilizes CFTR via high-affinity binding to nucleotide-binding domain 1 (NBD1). When combined with clinically-approved correctors, T2a considerably improved F508del-CFTR maturation, plasma membrane expression, and channel activity. Single-channel recordings revealed that nanobody binding sustained channel activity by promoting both full open and sub-conductance gating states and protecting F508del-CFTR against thermal deactivation. Because T2a binding to NBD1 prevents the ATP-dependent NBD1-NBD2 association that drives canonical channel opening, the observation of channel activity in the presence of the nanobody suggested adoption of a novel open-channel CFTR structure. This inference was confirmed by cryo-EM analyses of CFTR in the presence of T2a, which revealed a novel open-channel conformation in which NBD1 binds to an alternative site on CFTR incompatible with NBD dimerization. Our findings establish a new paradigm to correct protein trafficking by stabilizing misfolded domains with targeted nanobodies and demonstrate a broadly applicable framework to treat CF and related protein misfolding diseases.

Background: Air-liquid interface (ALI) cultures of patient-derived primary nasal epithelial cells (pNEC) can support diagnostics and in vitro phenotyping of primary ciliary dyskinesia (PCD); however, low cell yield remains a limitation. We aimed to establish and optimize conditionally reprogrammed cell (CRC) technology for pNEC from individuals with PCD and use it as a diagnostic tool for inconclusive cases and for pharmacological testing. Methods: pNEC from 36 individuals (8 healthy controls (HC), 26 PCD, 2 PCD candidates) were expanded using CRC and differentiated at ALI. Ciliary beat frequency (CBF), mucociliary transport velocity (MCTV), and culture morphology were assessed. Further, we assessed the effect of the β-receptor agonist clenbuterol on cilia function in pNEC of HC, PCD (DNAH11, CCDC40) and PCD candidates harboring biallelic DNAH1 variants. Results: CRC expansion demonstrated a >90% success rate, yielding high cell counts (mean 9.65 × 10⁶). Differentiation at ALI resulted in pseudostratified ciliated pNEC cultures. CBF was comparable between native and cultured pNEC. MCTV was significantly reduced in PCD vs. HC cultures (p < 0.0001). Clenbuterol significantly increased CBF in HC and DNAH1, but not in CCDC40 or DNAH11 cultures. MCTV was significantly increased in HC by clenbuterol treatment. Conclusions: CRC technology provides a scalable platform for the expansion of pNEC that recapitulate in vivo ciliary function. By enabling genotype-specific pharmacological screening, CRC may serve as a tool for functional diagnostics and precision medicine in PCD.