Rare Trafficking CFTR Mutations Involve Distinct Cellular Retention Machineries and Require Different Rescuing Strategies

Most of the ~2100 CFTR variants so far reported are very rare and still uncharacterized regarding their cystic fibrosis (CF) disease liability. Since some may respond to currently approved modulators, characterizing their defect and response to these drugs is essential. Here we aimed characterizing the defect associated with four rare missense (likely Class II) CFTR variants and assess their rescue by corrector drugs. We produced CFBE cell lines stably expressing CFTR with W57G, R560S, H1079P and Q1100P, assessed their effect upon CFTR expression and maturation and their rescue by VX-661/VX-445 correctors. Results were validated by forskolin-induced swelling assay (FIS) using intestinal organoids from individuals bearing these variants. Finally, knock-down (KD) of genes previously shown to rescue F508del-CFTR was assessed on these mutants. Results show that all the variants preclude the production of mature CFTR, confirming them as Class II mutations. None of the variants responded to VX-661 but the combination rescued H1079P- and Q1100P-CFTR. The KD of factors that correct F508del-CFTR retention only marginally rescued R560S- and H1079P-CFTR. Overall, data evidence that Class II mutations induce distinct molecular defects that are neither rescued by the same corrector compounds nor recognized by the same cellular machinery, thus requiring personalized drug discovery initiatives.

Revisiting CFTR Interactions: Old Partners and New Players

Remarkable progress in CFTR research has led to the therapeutic development of modulators that rescue the basic defect in cystic fibrosis. There is continuous interest in studying CFTR molecular disease mechanisms as not all cystic fibrosis patients have a therapeutic option available. Addressing the basis of the problem by comprehensively understanding the critical molecular associations of CFTR interactions remains key. With the availability of CFTR modulators, there is interest in comprehending which interactions are critical to rescue CFTR and which are altered by modulators or CFTR mutations. Here, the current knowledge on interactions that govern CFTR folding, processing, and stability is summarized. Furthermore, we describe protein complexes and signal pathways that modulate the CFTR function. Primary epithelial cells display a spatial control of the CFTR interactions and have become a common system for preclinical and personalized medicine studies. Strikingly, the novel roles of CFTR in development and differentiation have been recently uncovered and it has been revealed that specific CFTR gene interactions also play an important role in transcriptional regulation. For a comprehensive understanding of the molecular environment of CFTR, it is important to consider CFTR mutation-dependent interactions as well as factors affecting the CFTR interactome on the cell type, tissue-specific, and transcriptional levels.

Molecular pathology of the R117H cystic fibrosis mutation is explained by loss of a hydrogen bond

The phosphorylation-activated anion channel CFTR is gated by an ATP hydrolysis cycle at its two cytosolic nucleotide binding domains, and is essential for epithelial salt-water transport. A large number of CFTR mutations cause cystic fibrosis. Since recent breakthrough in targeted pharmacotherapy, CFTR mutants with impaired gating are candidates for stimulation by potentiator drugs. Thus, understanding the molecular pathology of individual mutations has become important. The relatively common R117H mutation affects an extracellular loop, but nevertheless causes a strong gating defect. Here we identify a hydrogen bond between the side chain of arginine 117 and the backbone carbonyl group of glutamate 1124 in the cryo-electronmicroscopic structure of phosphorylated, ATP-bound CFTR. We address the functional relevance of that interaction for CFTR gating using macroscopic and microscopic inside-out patch-clamp recordings. Employing thermodynamic double-mutant cycles, we systematically track gating-state dependent changes in the strength of the R117-E1124 interaction. We find that the H-bond is formed only in the open state, but neither in the short-lived "flickery" nor in the long-lived 'interburst' closed state. Loss of this H-bond explains the strong gating phenotype of the R117H mutant, including robustly shortened burst durations and strongly reduced intraburst open probability. The findings may help targeted potentiator design.

Comprehensive Analysis of Combinatorial Pharmacological Treatments to Correct Nonsense Mutations in the CFTR Gene

Cystic fibrosis (CF) is caused by loss of function of the CFTR chloride channel. A substantial number of CF patients carry nonsense mutations in the CFTR gene. These patients cannot directly benefit from pharmacological correctors and potentiators that have been developed for other types of CFTR mutations. We evaluated the efficacy of combinations of drugs targeting at various levels the effects of nonsense mutations: SMG1i to protect CFTR mRNA from nonsense-mediated decay (NMD), G418 and ELX-02 for readthrough, VX-809 and VX-445 to promote protein maturation and function, PTI-428 to enhance CFTR protein synthesis. We found that the extent of rescue and sensitivity to the various agents is largely dependent on the type of mutation, with W1282X and R553X being the mutations most and least sensitive to pharmacological treatments, respectively. In particular, W1282X-CFTR was highly responsive to NMD suppression by SMG1i but also required treatment with VX-445 corrector to show function. In contrast, G542X-CFTR required treatment with readthrough agents and VX-809. Importantly, we never found cooperativity between the NMD inhibitor and readthrough compounds. Our results indicate that treatment of CF patients with nonsense mutations requires a precision medicine approach with the design of specific drug combinations for each mutation.

Molecular pathology of a cystic fibrosis mutation is explained by loss of a hydrogen bond

The phosphorylation-activated anion channel CFTR is gated by an ATP hydrolysis cycle at its two cytosolic nucleotide binding domains, and is essential for epithelial salt-water transport. A large number of CFTR mutations cause cystic fibrosis. Since recent breakthrough in targeted pharmacotherapy, CFTR mutants with impaired gating are candidates for stimulation by potentiator drugs. Thus, understanding the molecular pathology of individual mutations has become important. The relatively common R117H mutation affects an extracellular loop, but nevertheless causes a strong gating defect. Here we identify a hydrogen bond between the side chain of arginine 117 and the backbone carbonyl group of glutamate 1124 in the cryo-electronmicroscopic structure of phosphorylated, ATP-bound CFTR. We address the functional relevance of that interaction for CFTR gating using macroscopic and microscopic inside-out patch-clamp recordings. Employing thermodynamic double-mutant cycles, we systematically track gating-state dependent changes in the strength of the R117-E1124 interaction. We find that the H-bond is formed only in the open state, but neither in the short-lived "flickery" nor in the long-lived "interburst" closed state. Loss of this H-bond explains the entire gating phenotype of the R117H mutant, including robustly shortened burst durations and strongly reduced intraburst open probability. The findings may help targeted potentiator design.

Therapeutic Approaches for Patients with Cystic Fibrosis Not Eligible for Current CFTR Modulators

Cystic fibrosis is a severe autosomal recessive disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene encoding the CFTR protein, a chloride channel expressed in many epithelial cells. New drugs called CFTR modulators aim at restoring the CFTR protein function, and they will benefit many patients with cystic fibrosis in the near future. However, some patients bear rare mutations that are not yet eligible for CFTR modulators, although they might be amenable to these new disease-modifying drugs. Moreover, more than 10% of CFTR mutations do not produce any CFTR protein for CFTR modulators to act upon. The purpose of this review is to provide an overview of different approaches pursued to treat patients bearing mutations ineligible for CFTR modulators. One approach is to broaden the numbers of mutations eligible for CFTR modulators. This requires developing strategies to evaluate drugs in populations bearing very rare genotypes. Other approaches aiming at correcting the CFTR defect develop new mutation-specific or mutation-agnostic therapies for mutations that do not produce a CFTR protein: readthrough agents for nonsense mutations, nucleic acid-based therapies, RNA- or DNA-based, and cell-based therapies. Most of these approaches are in pre-clinical development or, for some of them, early clinical phases. Many hurdles and challenges will have to be solved before they can be safely translated to patients.

Elexacaftor is a CFTR potentiator and acts synergistically with ivacaftor during acute and chronic treatment

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), which lead to early death due to progressive lung disease. The development of small-molecule modulators that directly interact with CFTR to aid in protein folding (“correctors”) and/or increase channel function (“potentiators”) have proven to be highly effective in the therapeutic treatment of CF. Notably, incorporation of the next-generation CFTR corrector, elexacaftor, into a triple combination therapeutic (marketed as Trikafta) has shown tremendous clinical promise in treating CF caused by F508del-CFTR. Here, we report on a newly-described role of elexacaftor as a CFTR potentiator. We explore the acute and chronic actions, pharmacology, and efficacy of elexacaftor as a CFTR potentiator in restoring function to multiple classes of CFTR mutations. We demonstrate that the potentiating action of elexacaftor exhibits multiplicative synergy with the established CFTR potentiator ivacaftor in rescuing multiple CFTR class defects, indicating that a new combination therapeutic of ivacaftor and elexacaftor could have broad impact on CF therapies.

Functional correction of CFTR mutations in human airway epithelial cells using adenine base editors

Mutations in the CFTR gene that lead to premature stop codons or splicing defects cause cystic fibrosis (CF) and are not amenable to treatment by small-molecule modulators. Here, we investigate the use of adenine base editor (ABE) ribonucleoproteins (RNPs) that convert A•T to G•C base pairs as a therapeutic strategy for three CF-causing mutations. Using ABE RNPs, we corrected in human airway epithelial cells premature stop codon mutations (R553X and W1282X) and a splice-site mutation (3849 + 10 kb C > T). Following ABE delivery, DNA sequencing revealed correction of these pathogenic mutations at efficiencies that reached 38–82% with minimal bystander edits or indels. This range of editing was sufficient to attain functional correction of CFTR-dependent anion channel activity in primary epithelial cells from CF patients and in a CF patient-derived cell line. These results demonstrate the utility of base editor RNPs to repair CFTR mutations that are not currently treatable with approved therapeutics.