Restoration of R117H CFTR folding and function in human airway cells through combination treatment with VX-809 and VX-770.

Cystic fibrosis (CF) is a lethal recessive genetic disease caused primarily by the F508del mutation in the CF transmembrane conductance regulator (CFTR). The potentiator VX-770 was the first CFTR modulator approved by the FDA for treatment of CF patients with the gating mutation G551D. Orkambi is a drug containing VX-770 and corrector VX809 and is approved for treatment of CF patients homozygous for F508del, which has folding and gating defects. At least 30% of CF patients are heterozygous for the F508del mutation with the other allele encoding for one of many different rare CFTR mutations. Treatment of heterozygous F508del patients with VX-809 and VX-770 has had limited success, so it is important to identify heterozygous patients that respond to CFTR modulator therapy. R117H is a more prevalent rare mutation found in over 2,000 CF patients. In this study we investigated the effectiveness of VX-809/VX-770 therapy on restoring CFTR function in human bronchial epithelial (HBE) cells from R117H/F508del CF patients. We found that VX-809 stimulated more CFTR activity in R117H/F508del HBEs than in F508del/F508del HBEs. R117H expressed exclusively in immortalized HBEs exhibited a folding defect, was retained in the ER, and degraded prematurely. VX-809 corrected the R117H folding defect and restored channel function. Because R117 is involved in ion conductance, VX-770 acted additively with VX-809 to restore CFTR function in chronically treated R117H/F508del cells. Although treatment of R117H patients with VX-770 has been approved, our studies indicate that Orkambi may be more beneficial for rescue of CFTR function in these patients.

Cystic fibrosis airway inflammation enables elexacaftor/tezacaftor/ivacaftor-mediated rescue of N1303K CFTR mutation.

Rescue of N1303K CFTR by highly effective modulator therapy (HEMT) is enabled by CF airway inflammation. These findings suggest that evaluation of HEMT for rare CFTR mutations must be performed under inflammatory conditions relevant to CF airways. https://bit.ly/3tTcoJE.

A Novel Co-Culture Model Reveals Enhanced CFTR Rescue in Primary Cystic Fibrosis Airway Epithelial Cultures with Persistent Pseudomonas aeruginosa Infection.

People with cystic fibrosis (pwCF) suffer from chronic and recurring bacterial lung infections that begin very early in life and contribute to progressive lung failure. CF is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, which encodes an ion channel important for maintaining the proper hydration of pulmonary surfaces. When CFTR function is ablated or impaired, airways develop thickened, adherent mucus that contributes to a vicious cycle of infection and inflammation. Therapeutics for pwCF, called CFTR modulators, target the CFTR defect directly, restoring airway surface hydration and mucociliary clearance. However, even with CFTR modulator therapy, bacterial infections persist. To develop a relevant model of diseased airway epithelium, we established a primary human airway epithelium culture system with persistent Pseudomonas aeruginosa infection. We used this model to examine the effects of CFTR modulators on CFTR maturation, CFTR function, and bacterial persistence. We found that the presence of P. aeruginosa increased CFTR mRNA, protein, and function. We also found that CFTR modulators caused a decrease in P. aeruginosa burden. These results demonstrate the importance of including live bacteria to accurately model the CF lung, and that understanding the effects of infection on CFTR rescue by CFTR modulators is critical to evaluating and optimizing drug therapies for all pwCF.

Established and novel human translational models to advance cystic fibrosis research, drug discovery, and optimize CFTR-targeting therapeutics.

To find a cure for cystic fibrosis, there has been tremendous progress in the development of treatments that target the basic defect in the protein channel, CFTR. However, 10% of cystic fibrosis patients have rare CFTR mutations that are still without an approved CFTR-targeting drug. To identify relevant therapies for these patients, culture models using nasal, bronchial, and rectal tissue from individual patients allow functional, biochemical, and cellular detection of drug-rescued CFTR. Additionally, novel systems such as induced pluripotent stem cell-derived models are utilized to characterize CFTR mutations and identify treatments. State-of-the-art translational models were instrumental for CFTR modulator development and may become important for gene-based drug discovery and other novel therapeutic strategies.

Small-molecule eRF3a degraders rescue CFTR nonsense mutations by promoting premature termination codon readthrough.

The vast majority of people with cystic fibrosis (CF) are now eligible for CF transmembrane regulator (CFTR) modulator therapy. The remaining individuals with CF harbor premature termination codons (PTCs) or rare CFTR variants with limited treatment options. Although the clinical modulator response can be reliably predicted using primary airway epithelial cells, primary cells carrying rare CFTR variants are scarce. To overcome this obstacle, cell lines can be created by overexpression of mouse Bmi-1 and human TERT (hTERT). Using this approach, we developed 2 non-CF and 6 CF airway epithelial cell lines, 3 of which were homozygous for the W1282X PTC variant. The Bmi-1/hTERT cell lines recapitulated primary cell morphology and ion transport function. The 2 F508del-CFTR cell lines responded robustly to CFTR modulators, which was mirrored in the parent primary cells and in the cell donors' clinical response. Cereblon E3 ligase modulators targeting eukaryotic release factor 3a (eRF3a) rescued W1282X-CFTR function to approximately 20% of WT levels and, when paired with G418, rescued G542X-CFTR function to approximately 50% of WT levels. Intriguingly, eRF3a degraders also diminished epithelial sodium channel (ENaC) function. These studies demonstrate that Bmi-1/hTERT cell lines faithfully mirrored primary cell responses to CFTR modulators and illustrate a therapeutic approach to rescue CFTR nonsense mutations.

A small-molecule inhibitor and degrader of the RNF5 ubiquitin ligase.

RNF5 E3 ubiquitin ligase has multiple biological roles and has been linked to the development of severe diseases such as cystic fibrosis, acute myeloid leukemia, and certain viral infections, emphasizing the importance of discovering small-molecule RNF5 modulators for research and drug development. The present study describes the synthesis of a new benzo[b]thiophene derivative, FX12, that acts as a selective small-molecule inhibitor and degrader of RNF5. We initially identified the previously reported STAT3 inhibitor, Stattic, as an inhibitor of dislocation of misfolded proteins from the endoplasmic reticulum (ER) lumen to the cytosol in ER-associated degradation. A concise structure-activity relationship campaign (SAR) around the Stattic chemotype led to the synthesis of FX12, which has diminished activity in inhibition of STAT3 activation and retains dislocation inhibitory activity. FX12 binds to RNF5 and inhibits its E3 activity in vitro as well as promoting proteasomal degradation of RNF5 in cells. RNF5 as a molecular target for FX12 was supported by the facts that FX12 requires RNF5 to inhibit dislocation and negatively regulates RNF5 function. Thus, this study developed a small-molecule inhibitor and degrader of the RNF5 ubiquitin ligase, providing a chemical biology tool for RNF5 research and therapeutic development.

Passive immunization with a polyclonal antiserum to the hemoglobin receptor of Haemophilus ducreyi confers protection against a homologous challenge in the experimental swine model of chancroid.

Haemophilus ducreyi, the etiologic agent of chancroid, has an obligate requirement for heme. Heme is acquired by H. ducreyi from its human host via TonB-dependent transporters expressed at its bacterial surface. Of 3 TonB-dependent transporters encoded in the genome of H. ducreyi, only the hemoglobin receptor, HgbA, is required to establish infection during the early stages of the experimental human model of chancroid. Active immunization with a native preparation of HgbA (nHgbA) confers complete protection in the experimental swine model of chancroid, using either Freund's or monophosphoryl lipid A as adjuvants. To determine if transfer of anti-nHgbA serum is sufficient to confer protection, a passive immunization experiment using pooled nHgbA antiserum was conducted in the experimental swine model of chancroid. Pigs receiving this pooled nHgbA antiserum were protected from a homologous, but not a heterologous, challenge. Passively transferred polyclonal antibodies elicited to nHgbA bound the surface of H. ducreyi and partially blocked hemoglobin binding by nHgbA, but were not bactericidal. Taken together, these data suggest that the humoral immune response to the HgbA vaccine is protective against an H. ducreyi infection, possibly by preventing acquisition of the essential nutrient heme.

DNAJB12 and Hsp70 triage arrested intermediates of N1303K-CFTR for endoplasmic reticulum-associated autophagy.

The transmembrane Hsp40 DNAJB12 and cytosolic Hsp70 cooperate on the endoplasmic reticulum's (ER) cytoplasmic face to facilitate the triage of nascent polytopic membrane proteins for folding versus degradation. N1303K is a common mutation that causes misfolding of the ion channel CFTR, but unlike F508del-CFTR, biogenic and functional defects in N1303K-CFTR are resistant to correction by folding modulators. N1303K is reported to arrest CFTR folding at a late stage after partial assembly of its N-terminal domains. N1303K-CFTR intermediates are clients of JB12-Hsp70 complexes, maintained in a detergent-soluble state, and have a relatively long 3-h half-life. ER-associated degradation (ERAD)-resistant pools of N1303K-CFTR are concentrated in ER tubules that associate with autophagy initiation sites containing WIPI1, FlP200, and LC3. Destabilization of N1303K-CFTR or depletion of JB12 prevents entry of N1303K-CFTR into the membranes of ER-connected phagophores and traffic to autolysosomes. In contrast, the stabilization of intermediates with the modulator VX-809 promotes the association of N1303K-CFTR with autophagy initiation machinery. N1303K-CFTR is excluded from the ER-exit sites, and its passage from the ER to autolysosomes does not require ER-phagy receptors. DNAJB12 operates in biosynthetically active ER microdomains to triage membrane protein intermediates in a conformation-specific manner for secretion versus degradation via ERAD or selective-ER-associated autophagy.

Airway Epithelial Inflammation In Vitro Augments the Rescue of Mutant CFTR by Current CFTR Modulator Therapies.

In cystic fibrosis (CF), defective biogenesis and activity of the cystic fibrosis transmembrane conductance regulator (CFTR) leads to airway dehydration and impaired mucociliary clearance, resulting in chronic airway infection and inflammation. The most common CFTR mutation, F508del, results in a processing defect in which the protein is retained in the endoplasmic reticulum and does not reach the apical surface. CFTR corrector compounds address this processing defect to promote mutant CFTR transfer to the apical membrane. When coupled with potentiators to increase CFTR channel activity, these drugs yield significant clinical benefits in CF patients carrying the F508del mutation. However, processing of CFTR and other proteins can be influenced by environmental factors such as inflammation, and the impact of airway inflammation on pharmacological activity of CFTR correctors is not established. The present study evaluated CFTR-rescuing therapies in inflamed CF airway epithelial cultures, utilizing models that mimic the inflammatory environment of CF airways. Primary bronchial epithelial cultures from F508del/F508del CF patients were inflamed by mucosal exposure to one of two inflammatory stimuli: 1) supernatant from mucopurulent material from CF airways with advanced lung disease, or 2) bronchoalveolar lavage fluid from pediatric CF patients. Cultures inflamed with either stimulus exhibited augmented F508del responses following therapy with correctors VX-809 or VX-661, and overcame the detrimental effects of chronic exposure to the CFTR potentiator VX-770. Remarkably, even the improved CFTR rescue responses resulting from a clinically effective triple therapy (VX-659/VX-661/VX-770) were enhanced by epithelial inflammation. Thus, the airway inflammatory milieu from late- and early-stage CF lung disease improves the efficacy of CFTR modulators, regardless of the combination therapy used. Our findings suggest that pre-clinical evaluation of CFTR corrector therapies should be performed under conditions mimicking the native inflammatory status of CF airways, and altering the inflammatory status of CF airways may change the efficacy of CFTR modulator therapies.

Immunization with the Haemophilus ducreyi hemoglobin receptor HgbA with adjuvant monophosphoryl lipid A protects swine from a homologous but not a heterologous challenge.

Haemophilus ducreyi, the etiological agent of chancroid, has a strict requirement for heme, which it acquires from its only natural host, humans. Previously, we showed that a vaccine preparation containing the native hemoglobin receptor HgbA purified from H. ducreyi class I strain 35000HP (nHgbAI) and administered with Freund's adjuvant provided complete protection against a homologous challenge. In the current study, we investigated whether nHgbAI dispensed with monophosphoryl lipid A (MPL), an adjuvant approved for use in humans, offered protection against a challenge with H. ducreyi strain 35000HP expressing either class I or class II HgbA (35000HPhgbAI and 35000HPhgbAII, respectively). Pigs immunized with the nHgbAI/MPL vaccine were protected against a challenge from homologous H. ducreyi strain 35000HPhgbAI but not heterologous strain 35000HPhgbAII, as evidenced by the isolation of only strain 35000HPhgbAII from nHgbAI-immunized pigs. Furthermore, histological analysis of the lesions showed striking differences between mock-immunized and nHgbAI-immunized animals challenged with strains 35000HPhgbAI but not those challenged with strain 35000HPhgbAII. Mock-immunized pigs were not protected from a challenge by either strain. The enzyme-linked immunosorbent assay (ELISA) activity of the nHgbAI/MPL antiserum was lower than the activity of antiserum from animals immunized with the nHgbAI/Freund's vaccine; however, anti-nHgbAI from both studies bound whole cells of 35000HPhgbAI better than 35000HPhgbAII and partially blocked hemoglobin binding to nHgbAI. In conclusion, despite eliciting lower antibody ELISA activity than the nHgbAI/Freund's, the nHgbAI/MPL vaccine provided protection against a challenge with homologous but not heterologous H. ducreyi, suggesting that a bivalent HgbA vaccine may be needed.

Modulation of endocytic trafficking and apical stability of CFTR in primary human airway epithelial cultures.

CFTR is a highly regulated apical chloride channel of epithelial cells that is mutated in cystic fibrosis (CF). In this study, we characterized the apical stability and intracellular trafficking of wild-type and mutant CFTR in its native environment, i.e., highly differentiated primary human airway epithelial (HAE) cultures. We labeled the apical pool of CFTR and subsequently visualized the protein in intracellular compartments. CFTR moved from the apical surface to endosomes and then efficiently recycled back to the surface. CFTR endocytosis occurred more slowly in polarized than in nonpolarized HAE cells or in a polarized epithelial cell line. The most common mutation in CF, DeltaF508 CFTR, was rescued from endoplasmic reticulum retention by low-temperature incubation but transited from the apical membrane to endocytic compartments more rapidly and recycled less efficiently than wild-type CFTR. Incubation with small-molecule correctors resulted in DeltaF508 CFTR at the apical membrane but did not restore apical stability. To stabilize the mutant protein at the apical membrane, we found that the dynamin inhibitor Dynasore and the cholesterol-extracting agent cyclodextrin dramatically reduced internalization of DeltaF508, whereas the proteasomal inhibitor MG-132 completely blocked endocytosis of DeltaF508. On examination of intrinsic properties of CFTR that may affect its apical stability, we found that N-linked oligosaccharides were not necessary for transport to the apical membrane but were required for efficient apical recycling and, therefore, influenced the turnover of surface CFTR. Thus apical stability of CFTR in its native environment is affected by properties of the protein and modulation of endocytic trafficking.

Accumulation and persistence of ivacaftor in airway epithelia with prolonged treatment.

BACKGROUND: Current dosing strategies of CFTR modulators are based on serum pharmacokinetics, but drug concentrations in target tissues such as airway epithelia are not known. Previous data suggest that CFTR modulators may accumulate in airway epithelia, and serum pharmacokinetics may not accurately predict effects of chronic treatment. METHODS: CF (F508del homozygous) primary human bronchial epithelial (HBE) cells grown at air-liquid interface were treated for 14 days with ivacaftor plus lumacaftor or ivacaftor plus tezacaftor, followed by a 14-day washout period. At various intervals during treatment and washout phases, drug concentrations were measured via mass spectrometry, electrophysiological function was assessed in Ussing chambers, and mature CFTR protein was quantified by Western blotting. RESULTS: During treatment, ivacaftor accumulated in CF-HBEs to a much greater extent than either lumacaftor or tezacaftor and remained persistently elevated even after 14 days of washout. CFTR activity peaked at 7 days of treatment but diminished with further ivacaftor accumulation, though remained above baseline even after washout. CONCLUSIONS: Intracellular accrual and persistence of CFTR modulators during and after chronic treatment suggest complex pharmacokinetic and pharmacodynamic properties within airway epithelia that are not predicted by serum pharmacokinetics. Direct measurement of drugs in target tissues may be needed to optimize dosing strategies, and the persistence of CFTR modulators after treatment cessation has implications for personalized medicine approaches.

Bioactive Thymosin Alpha-1 Does Not Influence F508del-CFTR Maturation and Activity.

Deletion of phenylalanine 508 (F508del) in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel is the most frequent mutation causing cystic fibrosis (CF). F508del-CFTR is misfolded and prematurely degraded. Recently thymosin a-1 (Tα-1) was proposed as a single molecule-based therapy for CF, improving both F508del-CFTR maturation and function by restoring defective autophagy. However, three independent laboratories failed to reproduce these results. Lack of reproducibility has been ascribed by the authors of the original paper to the use of DMSO and to improper handling. Here, we address these potential issues by demonstrating that Tα-1 changes induced by DMSO are fully reversible and that Tα-1 peptides prepared from different stock solutions have equivalent biological activity. Considering the negative results here reported, six independent laboratories failed to demonstrate F508del-CFTR correction by Tα-1. This study also calls into question the autophagy modulator cysteamine, since no rescue of mutant CFTR function was detected following treatment with cysteamine, while deleterious effects were observed when bronchial epithelia were exposed to cysteamine plus the antioxidant food supplement EGCG. Although these studies do not exclude the possibility of beneficial immunomodulatory effects of thymosin α-1, they do not support its utility as a corrector of F508del-CFTR.

Serial isolates of persistent Haemophilus influenzae in patients with chronic obstructive pulmonary disease express diminishing quantities of the HMW1 and HMW2 adhesins.

In patients with chronic obstructive pulmonary disease (COPD), the lower respiratory tract is commonly colonized by bacterial pathogens, including nontypeable Haemophilus influenzae. The H. influenzae HMW1 and HMW2 adhesins are homologous proteins that promote bacterial adherence to respiratory epithelium and are the predominant targets of the host immune response. These adhesins undergo graded phase variation, controlled by the numbers of 7-bp repeats upstream of the HMW1 and HMW2 structural genes (hmw1A and hmw2A, respectively). In this study, we examined the levels of HMW1 and HMW2 expressed by H. influenzae isolates collected serially from patients with COPD. We found that expression of HMW1 and HMW2 in a given strain decreased over time in a majority of patients, reflecting progressive increases in the numbers of 7-bp repeats and associated with high serum titers of HMW1/HMW2-specific antibodies. We speculate that the presence of high titers of antibodies against the HMW1 and HMW2 adhesins and other immune factors in the lower respiratory tracts of patients with COPD may result in gradual selection for bacteria with reduced levels of HMW1 and HMW2.

Recent progress in translational cystic fibrosis research using precision medicine strategies.

Significant progress has been achieved in developing precision therapies for cystic fibrosis; however, highly effective treatments that target the ion channel, CFTR, are not yet available for many patients. As numerous CFTR therapeutics are currently in the clinical pipeline, reliable screening tools capable of predicting drug efficacy to support individualized treatment plans and translational research are essential. The utilization of bronchial, nasal, and rectal tissues from individual cystic fibrosis patients for drug testing using in vitro assays such as electrophysiological measurements of CFTR activity and evaluation of fluid movement in spheroid cultures, has advanced the prediction of patient-specific responses. However, for precise prediction of drug effects, in vitro models of CFTR rescue should incorporate the inflamed cystic fibrosis airway environment and mimic the complex tissue structures of airway epithelia. Furthermore, novel assays that monitor other aspects of successful CFTR rescue such as restoration of mucus characteristics, which is important for predicting mucociliary clearance, will allow for better prognoses of successful therapies in vivo. Additional cystic fibrosis treatment strategies are being intensively explored, such as development of drugs that target other ion channels, and novel technologies including pluripotent stem cells, gene therapy, and gene editing. The multiple therapeutic approaches available to treat the basic defect in cystic fibrosis combined with relevant precision medicine models provide a framework for identifying optimal and sustained treatments that will benefit all cystic fibrosis patients.

Thymosin α-1 does not correct F508del-CFTR in cystic fibrosis airway epithelia.

In cystic fibrosis (CF), deletion of phenylalanine 508 (F508del) in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel causes misfolding and premature degradation. Considering the numerous effects of the F508del mutation on the assembly and processing of CFTR protein, combination therapy with several pharmacological correctors is likely to be required to treat CF patients. Recently, it has been reported that thymosin α-1 (Tα-1) has multiple beneficial effects that could lead to a single-molecule-based therapy for CF patients with F508del. Such effects include suppression of inflammation, improvement in F508del-CFTR maturation and gating, and stimulation of chloride secretion through the calcium-activated chloride channel (CaCC). Given the importance of such a drug, we aimed to characterize the underlying molecular mechanisms of action of Tα-1. In-depth analysis of Tα-1 effects was performed using well-established microfluorimetric, biochemical, and electrophysiological techniques on epithelial cell lines and primary bronchial epithelial cells from CF patients. The studies, which were conducted in 2 independent laboratories with identical outcome, demonstrated that Tα-1 is devoid of activity on mutant CFTR as well as on CaCC. Although Tα-1 may still be useful as an antiinflammatory agent, its ability to target defective anion transport in CF remains to be further investigated.

Efficacy of lumacaftor-ivacaftor for the treatment of cystic fibrosis patients homozygous for the F508del-CFTR mutation.

Cystic fibrosis (CF) results from mutations in the CF transmembrane conductance regulator (CFTR) gene, which codes for the CFTR channel protein. The most common mutation in CF is F508del, which produces a misfolded protein with diminished channel activity. The development of small-molecule CFTR-modulator compounds offers an exciting and novel approach for pharmacological treatment of CF. The corrector lumacaftor helps rescue F508del-CFTR to the cell surface, and potentiator ivacaftor increases F508del-CFTR channel activity. The combination of lumacaftor-ivacaftor (Vertex Pharmaceuticals Incorporated) represents the first FDA-approved therapy for CF patients with two copies of the F508del mutation. Although this combination therapy is the first treatment to directly target the F508del-CFTR mutation, patients taking this drug displayed only modest improvements in lung function. This article summarizes recent data from clinical trials and research discoveries relating to the lumacaftor-ivacaftor treatment, and considers options for identifying future therapies that will be most efficacious for all CF patients.

Potentiator ivacaftor abrogates pharmacological correction of ΔF508 CFTR in cystic fibrosis.

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR). Newly developed "correctors" such as lumacaftor (VX-809) that improve CFTR maturation and trafficking and "potentiators" such as ivacaftor (VX-770) that enhance channel activity may provide important advances in CF therapy. Although VX-770 has demonstrated substantial clinical efficacy in the small subset of patients with a mutation (G551D) that affects only channel activity, a single compound is not sufficient to treat patients with the more common CFTR mutation, ΔF508. Thus, patients with ΔF508 will likely require treatment with both correctors and potentiators to achieve clinical benefit. However, whereas the effectiveness of acute treatment with this drug combination has been demonstrated in vitro, the impact of chronic therapy has not been established. In studies of human primary airway epithelial cells, we found that both acute and chronic treatment with VX-770 improved CFTR function in cells with the G551D mutation, consistent with clinical studies. In contrast, chronic VX-770 administration caused a dose-dependent reversal of VX-809-mediated CFTR correction in ΔF508 homozygous cultures. This result reflected the destabilization of corrected ΔF508 CFTR by VX-770, markedly increasing its turnover rate. Chronic VX-770 treatment also reduced mature wild-type CFTR levels and function. These findings demonstrate that chronic treatment with CFTR potentiators and correctors may have unexpected effects that cannot be predicted from short-term studies. Combining these drugs to maximize rescue of ΔF508 CFTR may require changes in dosing and/or development of new potentiator compounds that do not interfere with CFTR stability.