People with cystic fibrosis (CF) suffer from a multi-organ disorder caused by loss-of-function variants in the gene encoding the epithelial anion channel cystic fibrosis transmembrane conductance regulator (CFTR). Tremendous progress has been made in both basic and clinical sciences over the past three decades since the identification of the CFTR gene. Over 90% of people with CF now have access to therapies targeting dysfunctional CFTR. This success was made possible by numerous studies in the field that incrementally paved the way for the development of small molecules known as CFTR modulators. The advent of CFTR modulators transformed this life-threatening illness into a treatable disease by directly binding to the CFTR protein and correcting defects induced by pathogenic variants. In this chapter, we trace the trajectory of structural and functional studies that brought CF therapies from bench to bedside, with an emphasis on mechanistic understanding of CFTR modulators.

Dysfunction of the epithelial anion channel cystic fibrosis transmembrane conductance regulator (CFTR) causes a wide spectrum of disease, including cystic fibrosis (CF) and CFTR-related diseases (CFTR-RDs). Here, we investigate genotype-phenotype-CFTR function relationships using human nasal epithelial (hNE) cells from a small cohort of non-CF subjects and individuals with CF and CFTR-RDs and genotypes associated with either residual or minimal CFTR function using electrophysiological techniques. Collected hNE cells were either studied directly with the whole-cell patch-clamp technique or grown as primary cultures at an air-liquid interface after conditional reprogramming. The properties of cAMP-activated whole-cell Cl- currents in freshly isolated hNE cells identified them as CFTR-mediated. Their magnitude varied between hNE cells from individuals within the same genotype and decreased in the rank order: non-CF > CFTR residual function > CFTR minimal function. CFTR-mediated whole-cell Cl- currents in hNE cells isolated from fully differentiated primary cultures were identical to those in freshly isolated hNE cells in both magnitude and behaviour, demonstrating that conditional reprogramming culture is without effect on CFTR expression and function. For the cohort of subjects studied, CFTR-mediated whole-cell Cl- currents in hNE cells correlated well with CFTR-mediated transepithelial Cl- currents measured in vitro with the Ussing chamber technique, but not with those determined in vivo with the nasal potential difference assay. Nevertheless, they did correlate with the sweat Cl- concentration of study subjects. Thus, this study highlights the complexity of genotype-phenotype-CFTR function relationships, but emphasises the value of conditionally reprogrammed hNE cells in CFTR research and therapeutic testing. KEY POINTS: The genetic disease cystic fibrosis is caused by pathogenic variants in the cystic fibrosis transmembrane conductance regulator (CFTR), an ion channel, which controls anion flow across epithelia lining ducts and tubes in the body. This study investigated CFTR function in nasal epithelial cells from people with cystic fibrosis and CFTR variants with a range of disease severity. CFTR function varied widely in nasal epithelial cells depending on the identity of CFTR variants, but was unaffected by conditional reprogramming culture, a cell culture technique used to grow large numbers of patient-derived cells. Assessment of CFTR function in vitro in nasal epithelial cells and epithelia, and in vivo in the nasal epithelium and sweat gland highlights the complexity of genotype-phenotype-CFTR function relationships.

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator ( CFTR ) gene. Defective CFTR leads to accumulation of dehydrated viscous mucus within the small intestine, luminal acidification and altered intestinal motility, resulting in blockage. These changes promote gut microbial dysbiosis, adversely influencing the normal proliferation and differentiation of intestinal epithelial cells. Using Illumina 16S rRNA gene sequencing and immunohistochemistry, we assessed changes in mucosa-attached microbiome and epithelial cell profile in the small intestine of CF mice and a CF patient compared to wild-type mice and non-CF humans. We found increased abundance of pro-inflammatory Escherichia and depletion of beneficial secondary bile-acid producing bacteria in the ileal mucosa-attached microbiome of CFTR-null mice. The ileal mucosa in a CF patient was dominated by a non- aeruginosa Pseudomonas species and lacked numerous beneficial anti-inflammatory and short-chain fatty acid-producing bacteria. In the ileum of both CF mice and a CF patient, the number of absorptive enterocytes, Paneth and glucagon-like peptide 1 and 2 secreting L-type enteroendocrine cells were decreased, whereas stem and goblet cell numbers were increased. These changes in mucosa-attached microbiome and epithelial cell profile suggest that microbiota-host interactions may contribute to intestinal CF disease development with implications for therapy.

The gasotransmitter carbon monoxide (CO) regulates fluid and electrolyte movements across epithelial tissues. However, its action on anion channels is incompletely understood. Here, we investigate the direct action of CO on the cystic fibrosis transmembrane conductance regulator (CFTR) by applying CO-releasing molecules (CO-RMs) to the intracellular side of excised inside-out membrane patches from cells heterologously expressing wild-type human CFTR. Addition of increasing concentrations of tricarbonyldichlororuthenium(II) dimer (CORM-2) (1-300 μM) inhibited CFTR channel activity, whereas the control RuCl3 (100 μM) was without effect. CORM-2 predominantly inhibited CFTR by decreasing the frequency of channel openings and, hence, open probability (Po). But, it also reduced current flow through open channels with very fast kinetics, particularly at elevated concentrations. By contrast, the chemically distinct CO-releasing molecule CORM-3 inhibited CFTR by decreasing Po without altering current flow through open channels. Neither depolarizing the membrane voltage nor raising the ATP concentration on the intracellular side of the membrane affected CFTR inhibition by CORM-2. Interestingly, CFTR inhibition by CORM-2, but not by CFTRinh-172, was prevented by prior enhancement of channel activity by the clinically approved CFTR potentiator ivacaftor. Similarly, when added after CORM-2, ivacaftor completely relieved CFTR inhibition. In conclusion, CORM-2 has complex effects on wild-type human CFTR consistent with allosteric inhibition and open-channel blockade. Inhibition of CFTR by CO-releasing molecules suggests that CO regulates CFTR activity and that the gasotransmitter has tissue-specific effects on epithelial ion transport. The action of ivacaftor on CFTR Cl- channels inhibited by CO potentially expands the drug's clinical utility.

Cystic Fibrosis (CF) is a common lethal genetic disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel. Misfolding and degradation of CFTR are the hallmarks of the predominant mutation, F508del, located in the first nucleotide binding domain (NBD1). While the mutation is known to affect the thermal stability of NBD1 and assembly of CFTR domains, the molecular events that lead to misfolding of F508del-CFTR remain elusive. Here, we demonstrate that NBD1 of CFTR can adopt an alternative conformation that departs from the canonical NBD fold previously observed for CFTR and other ATP-binding cassette (ABC) transporter proteins. Crystallography studies reveal that this conformation involves a topological reorganization of the β-subdomain of NBD1. This alternative state is adopted by wild-type CFTR in cells and enhances channel activity. Single-molecule fluorescence resonance energy transfer microscopy shows that the equilibrium between the conformations is regulated by ATP binding. Under destabilizing conditions, however, this conformational flexibility enables unfolding of the β-subdomain. Our data indicate that in wild-type CFTR switching to this topologically-swapped conformation of NBD1 regulates channel function, but, in the presence of the F508del mutation, it allows domain misfolding and subsequent protein degradation. Our work provides a framework to design conformation-specific therapeutics to prevent noxious transitions.

With the clinical approval of Trikafta (elexacaftor-tezacaftor-ivacaftor) (https://www.fda.gov/news-events/press-announcements/fda-approves-new-breakthrough-therapy-cystic-fibrosis), most people with cystic fibrosis (CF) have a drug therapy targeting the root cause of their disease, mutations in the epithelial anion channel cystic fibrosis transmembrane conductance regulator (CFTR). However, the fight against CF is certainly not over. Now, attention is focused on the remaining 10% of CF mutations, the hardest group to tackle with drugs. A large fraction of these mutations are nonsense mutations, which introduce premature termination codons (PTCs) into the CFTR mRNA, leading to severe defects in protein production. Building on previous studies (e.g. Sharma et al. 2018), in this issue of The Journal of Physiology, Yeh & Hwang (2020) demonstrate that not all PTC mutations are created equal, showing that PTC location and identity influences the therapeutic strategy to rescue CFTR. Nonsense mutations cause many genetic diseases, including CF, Duchenne muscular dystrophy and β-thalassemia. The PTCs they introduce activate nonsense-mediated decay (NMD) to degrade the faulty mRNA, preventing the production of abnormal proteins. Any PTC-containing mRNA, which escapes NMD detection produces a truncated protein susceptible to degradation by the quality control machinery, which regulates protein production. Using the antibiotic G418, Howard et al. (1996) first demonstrated that PTCs can be suppressed by small molecules, which alter the fidelity of translation termination, leading to the production of full-length protein. Although there is great interest in the therapeutic potential of PTC suppression, the efficacy of these reagents remains limited. Yeh & Hwang (2020) investigated three PTC mutations in the CFTR gene, which cause severe CF disease: E60X, G542X and W1282X (https://cftr2.org/). Although E60X is rare, G542X and W1282X are the second and fifth most common CF mutations with worldwide frequencies of ∼5% and ∼1%, respectively. E60X affects a residue in the N-terminal lasso motif, G542X a residue in the first nucleotide-binding domain (NBD1) in the middle of the protein and W1282X a residue in NBD2 near the C-terminus. Based on their location, these mutations occur in regions that contribute to the regulation of CFTR channel gating. However, only W1282X would be expected to produce a truncated polypeptide with an intact CFTR pore. The authors expressed the PTC mutations in heterologous cells and studied them with sensitive biochemical and functional assays and pharmacological tools that enhance CFTR expression and function. Consistent with previous results (e.g. Haggie et al. 2017), the truncated polypeptide of W1282X-CFTR had a very severe gating defect, but partial function was restored to it using CFTR correctors and potentiators, which transport mutant CFTR to the plasma membrane and enhance its activity. The authors identified two populations of W1282X-CFTR Cl− channels in cell-free membrane patches. Most channels were ATP-insensitive, representing truncated proteins lacking a large part of NBD2. However, a small fraction was ATP-sensitive, representing full-length CFTR protein with an intact NBD2, produced by endogenous PTC suppression. G542X generated tiny CFTR-mediated whole-cell Cl− currents, but no CFTR protein was detected biochemically. Because single-channel studies revealed that G542X-CFTR had wild-type CFTR-like conductance properties, these tiny currents represent full-length CFTR protein with the PTC endogenously suppressed. Neither G418 nor the CFTR corrector lumacaftor increased current magnitude, but the CFTR potentiator GLPG1837, which shares a common mechanism of action with ivacaftor, restored wild-type levels of channel gating to G542X-CFTR. Two E60X-CFTR proteins were detected, which represent N-terminally truncated proteins produced by translation reinitiation using downstream methionine residues (Carroll et al. 1995). However, Yeh & Hwang (2020) concluded that these proteins were non-functional based on the tiny size of E60X-CFTR-mediated whole-cell Cl− currents. Interestingly, the single-channel behaviour of E60X-CFTR in cells treated with G418, but not lumacaftor, resembled that of wild-type CFTR and GLPG1837 strongly enhanced channel gating to supraphysiological levels. Intrigued by the single-channel behaviour of E60X-CFTR after G418 treatment, Yeh & Hwang (2020) investigated the identity of the amino acid residue inserted during PTC suppression. Previous studies (e.g. Xue et al. 2017) had identified the specific amino acid residues inserted during the suppression of PTC mutations in CFTR and investigated their consequences for CFTR protein expression and transepithelial ion transport. For E60X, the inserted residues are tyrosine (Y) and glutamine (Q). The single-channel behaviour of E60Y-CFTR resembled lumacaftor-treated E60X-CFTR and was characterized by defective channel gating. By contrast, E60Q-CFTR possessed the same single-channel conductance and gating pattern as wild-type CFTR and E60X-CFTR treated with G418. Yeh & Hwang (2020) interpreted these data to suggest that distinct near-cognate tRNAs are employed by endogenous and drug-mediated PTC suppression, leading to the insertion of different amino acids. Depending on the identity of the inserted amino acid, the resulting full-length protein might have none, some or wild-type CFTR function. The authors' results with G542X-CFTR highlighted this point, where neither endogenous nor drug-mediated PTC suppression resulted in single-channel behaviour comparable to those of the amino acids inserted at this PTC. The elegant mechanistic study of Yeh & Hwang (2020) is important for several reasons. First, it confirms that the location of the PTC mutation determines its consequences for CFTR expression and function, and highlights that these can be multiple just like many other CF mutations. Second, it demonstrates that the amino acid inserted during PTC suppression can have deleterious effects on CFTR. Finally, it emphasizes the need for different therapeutic strategies for distinct PTC mutations. These include combinations of CFTR correctors and potentiators together with small molecules that suppress PTCs and inhibit NMD (e.g. Laselva et al. 2019). Future studies should investigate these therapeutic strategies using patient-derived cells that endogenously express CFTR. D.N.S. is the recipient of a Vertex Innovation Award. I.S.-G. is the recipient of two Vertex Innovation Awards and a member of the scientific boards of Eloxx, PTC Therapeutics and Vertex Pharmaceuticals. The authors have approved the final version of the manuscript and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed. A.H. and I.S.-G. are supported by Vaincre La Mucoviscidose, Cystic Fibrosis Foundation, Agence Nationale pour la Recherche and Programme Hospitalier de Recherche Clinique and D.N.S. by Cystic Fibrosis Foundation Therapeutics and Cystic Fibrosis Trust.

Cross-species comparative studies have highlighted differences between human and mouse cystic fibrosis transmembrane conductance regulator (CFTR), the epithelial Cl- channel defective in cystic fibrosis (CF). Here, we compare the impact of the most common CF mutation F508del on the function of human and mouse CFTR heterologously expressed in mammalian cells and their response to CFTR modulators using the iodide efflux and patch-clamp techniques. Once delivered to the plasma membrane, human F508del-CFTR exhibited a severe gating defect characterized by infrequent channel openings and was thermally unstable, deactivating within minutes at 37°C. By contrast, the F508del mutation was without effect on the gating pattern of mouse CFTR, and channel activity demonstrated thermostability at 37°C. Strikingly, at all concentrations tested, the clinically approved CFTR potentiator ivacaftor was without effect on the mouse F508del-CFTR Cl- channel. Moreover, eight CFTR potentiators, including ivacaftor, failed to generate CFTR-mediated iodide efflux from CHO cells expressing mouse F508del-CFTR. However, they all produced CFTR-mediated iodide efflux with human F508del-CFTR-expressing CHO cells, while fifteen CFTR correctors rescued the plasma membrane expression of both human and mouse F508del-CFTR. Interestingly, the CFTR potentiator genistein enhanced CFTR-mediated iodide efflux from CHO cells expressing either human or mouse F508del-CFTR, whereas it only potentiated human F508del-CFTR Cl- channels in cell-free membrane patches, suggesting that its action on mouse F508del-CFTR is indirect. Thus, the F508del mutation has distinct effects on human and mouse CFTR Cl- channels.

BACKGROUND AND PURPOSE Rescue of F508del-cystic fibrosis (CF) transmembrane conductance regulator (CFTR), the most common CF mutation, requires small molecules that overcome protein processing, stability and channel gating defects. Here, we investigate F508del-CFTR rescue by CFFT-004, a small molecule designed to independently correct protein processing and channel gating defects. EXPERIMENTAL APPROACH Using CFTR-expressing recombinant cells and CF patient-derived bronchial epithelial cells, we studied CFTR expression by Western blotting and channel gating and stability with the patch-clamp and Ussing chamber techniques. KEY RESULTS Chronic treatment with CFFT-004 improved modestly F508del-CFTR processing, but not its plasma membrane stability. By contrast, CFFT-004 rescued F508del-CFTR channel gating better than C18, an analogue of the clinically used CFTR corrector lumacaftor. Subsequent acute addition of CFFT-004, but not C18, potentiated F508del-CFTR channel gating. However, CFFT-004 was without effect on A561E-CFTR, a CF mutation with a comparable mechanism of CFTR dysfunction as F508del-CFTR. To investigate the mechanism of action of CFFT-004, we used F508del-CFTR revertant mutations. Potentiation by CFFT-004 was unaffected by revertant mutations, but correction was abolished by the revertant mutation G550E. These data suggest that correction, but not potentiation, by CFFT-004 might involve nucleotide-binding domain 1 of CFTR. CONCLUSIONS AND IMPLICATIONS CFFT-004 is a dual-acting small molecule with independent corrector and potentiator activities that partially rescues F508del-CFTR in recombinant cells and native airway epithelia. The limited efficacy and potency of CFFT-004 suggests that combinations of small molecules targeting different defects in F508del-CFTR might be a more effective therapeutic strategy than a single agent.

Ivacaftor is the first drug to target directly defects in the cystic fibrosis transmembrane conductance regulator (CFTR), which causes cystic fibrosis (CF). To understand better how ivacaftor potentiates CFTR channel gating, here we investigated the effects of temperature on its action. As a control, we studied the benzimidazolone UCCF-853, which potentiates CFTR by a different mechanism. Using the patch-clamp technique and cells expressing recombinant CFTR, we studied the single-channel behavior of wild-type and F508del-CFTR, the most common CF mutation. Raising the temperature of the intracellular solution from 23 to 37°C increased the frequency but reduced the duration of wild-type and F508del-CFTR channel openings. Although the open probability ( Po) of wild-type CFTR increased progressively as temperature was elevated, the relationship between Poand temperature for F508del-CFTR was bell-shaped with a maximum Poat ~30°C. For wild-type CFTR and to a greatly reduced extent F508del-CFTR, the temperature dependence of channel gating was asymmetric with the opening rate demonstrating greater temperature sensitivity than the closing rate. At all temperatures tested, ivacaftor and UCCF-853 potentiated wild-type and F508del-CFTR. Strikingly, ivacaftor but not UCCF-853 abolished the asymmetric temperature dependence of CFTR channel gating. At all temperatures tested, Povalues of wild-type CFTR in the presence of ivacaftor were approximately double those of F508del-CFTR, which were equivalent to or greater than those of wild-type CFTR at 37°C in the absence of the drug. We conclude that the principal effect of ivacaftor is to promote channel opening to abolish the temperature dependence of CFTR channel gating.

Ortho-Phenylene bis-ureas serve as anionophores in cells expressing halide-sensitive yellow fluorescent protein, as well as in synthetic vesicles. Activities can reach high levels, and are strongly dependent on the deliverability of the transporters.

Treatment of individuals with cystic fibrosis (CF) has been transformed by small molecule therapies that target select pathogenic variants in the CF transmembrane conductance regulator (CFTR). To expand treatment eligibility, we stably expressed 43 rare missense CFTR variants associated with moderate CF from a single site in the genome of human CF bronchial epithelial (CFBE41o-) cells. The magnitude of drug response was highly correlated with residual CFTR function for the potentiator ivacaftor, the corrector lumacaftor, and ivacaftor-lumacaftor combination therapy. Response of a second set of 16 variants expressed stably in Fischer rat thyroid (FRT) cells showed nearly identical correlations. Subsets of variants were identified that demonstrated statistically significantly higher responses to specific treatments. Furthermore, nearly all variants studied in CFBE cells (40 of 43) and FRT cells (13 of 16) demonstrated greater response to ivacaftor-lumacaftor combination therapy than either modulator alone. Together, these variants represent 87% of individuals in the CFTR2 database with at least 1 missense variant. Thus, our results indicate that most individuals with CF carrying missense variants are (a) likely to respond modestly to currently available modulator therapy, while a small fraction will have pronounced responses, and (b) likely to derive the greatest benefit from combination therapy.

Synonymous single nucleotide polymorphisms (sSNPs) are considered neutral for protein function, as by definition they exchange only codons, not amino acids. We identified an sSNP that modifies the local translation speed of the cystic fibrosis transmembrane conductance regulator (CFTR), leading to detrimental changes to protein stability and function. This sSNP introduces a codon pairing to a low-abundance tRNA that is particularly rare in human bronchial epithelia, but not in other human tissues, suggesting tissue-specific effects of this sSNP. Up-regulation of the tRNA cognate to the mutated codon counteracts the effects of the sSNP and rescues protein conformation and function. Our results highlight the wide-ranging impact of sSNPs, which invert the programmed local speed of mRNA translation and provide direct evidence for the central role of cellular tRNA levels in mediating the actions of sSNPs in a tissue-specific manner.

Key points The cystic fibrosis transmembrane conductance regulator (CFTR), which is defective in the genetic disease cystic fibrosis (CF), forms a gated pathway for chloride movement regulated by intracellular ATP. To understand better CFTR function, we investigated the regulation of channel openings by intracellular pH. We found that short‐lived channel closures during channel openings represent subtle changes in the structure of CFTR that are regulated by intracellular pH, in part, at ATP‐binding site 1 formed by the nucleotide‐binding domains. Our results provide a framework for future studies to understand better the regulation of channel openings, the dysfunction of CFTR in CF and the action of drugs that repair CFTR gating defects. AbstractCystic fibrosis transmembrane conductance regulator (CFTR) is an ATP‐gated Cl− channel defective in the genetic disease cystic fibrosis (CF). The gating behaviour of CFTR is characterized by bursts of channel openings interrupted by brief, flickery closures, separated by long closures between bursts. Entry to and exit from an open burst is controlled by the interaction of ATP with two ATP‐binding sites, sites 1 and 2, in CFTR. To understand better the kinetic basis of CFTR intraburst gating, we investigated the single‐channel activity of human CFTR at different intracellular pH (pHi) values. When compared with the control (pHi 7.3), acidifying pHi to 6.3 or alkalinizing pHi to 8.3 and 8.8 caused small reductions in the open‐time constant (τo) of wild‐type CFTR. By contrast, the fast closed‐time constant (τcf), which describes the short‐lived closures that interrupt open bursts, was greatly increased at pHi 5.8 and 6.3. To analyse intraburst kinetics, we used linear three‐state gating schemes. All data were satisfactorily modelled by the C1 ↔ O ↔ C2 kinetic scheme. Changing the intracellular ATP concentration was without effect on τo, τcf and their responses to pHi changes. However, mutations that disrupt the interaction of ATP with ATP‐binding site 1, including K464A, D572N and the CF‐associated mutation G1349D all abolished the prolongation of τcf at pHi 6.3. Taken together, our data suggest that the regulation of CFTR intraburst gating is distinct from the ATP‐dependent mechanism that controls channel opening and closing. However, our data also suggest that ATP‐binding site 1 modulates intraburst gating.

Cystic fibrosis (CF) is caused by mutations that disrupt the plasma membrane expression, stability, and function of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. Two small molecules, the CFTR corrector lumacaftor and the potentiator ivacaftor, are now used clinically to treat CF, although some studies suggest that they have counteracting effects on CFTR stability. Here, we investigated the impact of these compounds on the instability of F508del-CFTR, the most common CF mutation. To study individual CFTR Cl- channels, we performed single-channel recording, whereas to assess entire CFTR populations, we used purified CFTR proteins and macroscopic CFTR Cl- currents. At 37 degrees C, low temperature-rescued F508del-CFTR more rapidly lost function in cell-free membrane patches and showed altered channel gating and current flow through open channels. Compared with purified wild-type CFTR, the full-length F508del-CFTR was about 10 degrees C less thermostable. Lumacaftor partially stabilized purified full-length F508del-CFTR and slightly delayed deactivation of individual F508del-CFTR Cl- channels. By contrast, ivacaftor further destabilized full-length F508del-CFTR and accelerated channel deactivation. Chronic (prolonged) co-incubation of F508del-CFTR-expressing cells with lumacaftor and ivacaftor deactivated macroscopic F508del-CFTR Cl- currents. However, at the single-channel level, chronic co-incubation greatly increased F508del-CFTR channel activity and temporal stability in most, but not all, cell-free membrane patches. We conclude that chronic lumacaftor and ivacaftor co-treatment restores stability in a small subpopulation of F508del-CFTR Cl- channels but that the majority remain destabilized. A fuller understanding of these effects and the characterization of the small F508del-CFTR subpopulation might be crucial for CF therapy development.

The use of recombinant adeno-associated viruses (rAAVs) is highly prevalent in musculoskeletal gene therapies due to their versatility, high transduction efficiency, natural tropism and vector genome persistence for years. As the largest organ in the body, treatment of skeletal muscle for widespread and sufficient therapeutic gene expression is highly challenging. In addition to disease-specific hurdles, vector genome loss, off-target gene transfer and immune responses to treatment can diminish the overall benefit of rAAV therapies. A variety of approaches have been developed to overcome these challenges and improve musculoskeletal targeting of rAAVs. This review focuses on recent advancements and remaining obstacles in creating optimal rAAV-based therapies for musculoskeletal application.

Key points Malfunction of the cystic fibrosis transmembrane conductance regulator (CFTR), a gated pathway for chloride movement, causes the common life‐shortening genetic disease cystic fibrosis (CF). Towards the development of a sheep model of CF, we have investigated the function of sheep CFTR. We found that sheep CFTR was noticeably more active than human CFTR, while the most common CF mutation, F508del, had reduced impact on sheep CFTR function. Our results demonstrate that subtle changes in protein structure have marked effects on CFTR function and the consequences of the CF mutation F508del. AbstractCross‐species comparative studies are a powerful approach to understanding the epithelial Cl− channel cystic fibrosis transmembrane conductance regulator (CFTR), which is defective in the genetic disease cystic fibrosis (CF). Here, we investigate the single‐channel behaviour of ovine CFTR and the impact of the most common CF mutation, F508del‐CFTR, using excised inside‐out membrane patches from transiently transfected CHO cells. Like human CFTR, ovine CFTR formed a weakly inwardly rectifying Cl− channel regulated by PKA‐dependent phosphorylation, inhibited by the open‐channel blocker glibenclamide. However, for three reasons, ovine CFTR was noticeably more active than human CFTR. First, single‐channel conductance was increased. Second, open probability was augmented because the frequency and duration of channel openings were increased. Third, with enhanced affinity and efficacy, ATP more strongly stimulated ovine CFTR channel gating. Consistent with these data, the CFTR modulator phloxine B failed to potentiate ovine CFTR Cl− currents. Similar to its impact on human CFTR, the F508del mutation caused a temperature‐sensitive folding defect, which disrupted ovine CFTR protein processing and reduced membrane stability. However, the F508del mutation had reduced impact on ovine CFTR channel gating in contrast to its marked effects on human CFTR. We conclude that ovine CFTR forms a regulated Cl− channel with enhanced conductance and ATP‐dependent channel gating. This phylogenetic analysis of CFTR structure and function demonstrates that subtle changes in structure have pronounced effects on channel function and the consequences of the CF mutation F508del.