Nanomolar-potency 'co-potentiator' therapy for cystic fibrosis caused by a defined subset of minimal function CFTR mutants.

Available CFTR modulators provide no therapeutic benefit for cystic fibrosis (CF) caused by many loss-of-function mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, including N1303K. We previously introduced the concept of 'co-potentiators' (combination-potentiators) to rescue CFTR function in some minimal function CFTR mutants. Herein, a screen of ~120,000 drug-like synthetic small molecules identified active co-potentiators of pyrazoloquinoline, piperidine-pyridoindole, tetrahydroquinoline and phenylazepine classes, with EC50 down to ~300 nM following initial structure-activity studies. Increased CFTR chloride conductance by up to 8-fold was observed when a co-potentiator (termed 'Class II potentiator') was used with a classical potentiator ('Class I potentiator') such as VX-770 or GLPG1837. To investigate the range of CFTR mutations benefitted by co-potentiators, 14 CF-associated CFTR mutations were studied in transfected cell models. Co-potentiator efficacy was found for CFTR missense, deletion and nonsense mutations in nucleotide binding domain-2 (NBD2), including W1282X, N1303K, c.3700A > G and Q1313X (with corrector for some mutations). In contrast, CFTR mutations G85E, R334W, R347P, V520F, R560T, A561E, M1101K and R1162X showed no co-potentiator activity, even with corrector. Co-potentiator efficacy was confirmed in primary human bronchial epithelial cell cultures generated from a N1303K homozygous CF subject. The Class II potentiators identified here may have clinical benefit for CF caused by mutations in the NBD2 domain of CFTR

New era of cystic fibrosis: full mutational analysis and personalized therapy

Despite its apparently simple genetics, cystic fibrosis (CF) is a rather complex genetic disease. A lot of variability in the steps of the path from the cystic fibrosis transmembrane conductance regulator (CFTR ) gene to the clinical manifestations originates an uncertain genotype - phenotype relationship. A major determinant of this uncertainty is the incomplete knowledge of the CFTR mutated genotypes, due to the high number of CFTR mutations and to the higher number of their combinations in trans and in cis. Also the very limited knowledge of functional effects of CFTR mutated alleles severely impairs our diagnostic and prognostic ability. The final phenotypic modulation exerted by CFTR modifier genes and interactome further complicates the framework. The next generation sequencing approach is a rapid, lowcost and high-throughput tool that allows a near complete structural characterization of CFTR mutated genotypes, as well as of genotypes of several other genes cooperating to the final CF clinical manifestations. This powerful method perfectly complements the new personalized therapeutic approach for CF. Drugs active on specific CFTR mutational classes are already available for CF patients or are in phase 3 trials. A complete genetic characterization has been becoming crucial for a correct personalized therapy. However, the need of a functional classification of each CFTR mutation potently arises. Future big efforts towards an ever more detailed knowledge of both structural and functional CFTR defects, coupled to parallel personalized therapeutic interventions decisive for CF cure can be foreseen

Mechanisms of endothelial cell dysfunction in cystic fibrosis

Although cystic fibrosis (CF) patients exhibit signs of endothelial perturbation, the functions of the cystic fibrosis conductance regulator (CFTR) in vascular endothelial cells (EC) are poorly defined. We sought to uncover biological activities of endothelial CFTR, relevant for vascular homeostasis and inflammation. We examined cells from human umbilical cords (HUVEC) and pulmonary artery isolated from non-cystic fibrosis (PAEC) and CF human lungs (CF-PAEC), under static conditions or physiological shear. CFTR activity, clearly detected in HUVEC and PAEC, was markedly reduced in CF-PAEC. CFTR blockade increased endothelial permeability to macromolecules and reduced trans‑endothelial electrical resistance (TEER). Consistent with this, CF-PAEC displayed lower TEER compared to PAEC. Under shear, CFTR blockade reduced VE-cadherin and p120 catenin membrane expression and triggered the formation of paxillin- and vinculin-enriched membrane blebs that evolved in shrinking of the cell body and disruption of cell-cell contacts. These changes were accompanied by enhanced release of microvesicles, which displayed reduced capability to stimulate proliferation in recipient EC. CFTR blockade also suppressed insulin-induced NO generation by EC, likely by inhibiting eNOS and AKT phosphorylation, whereas it enhanced IL-8 release. Remarkably, phosphodiesterase inhibitors in combination with a β2 adrenergic receptor agonist corrected functional and morphological changes triggered by CFTR dysfunction in EC. Our results uncover regulatory functions of CFTR in EC, suggesting a physiological role of CFTR in the maintenance EC homeostasis and its involvement in pathogenetic aspects of CF. Moreover, our findings open avenues for novel pharmacology to control endothelial dysfunction and its consequences in CF

Mucin glycosylation and sulphation in airway epithelial cells is not influenced by cystic fibrosis transmembrane conductance regulator expression

Abnormalities in mucus properties and clearance make a major contribution to the pathology of cystic fibrosis (CF). Our aim was to test the hypothesis that the defects in CF mucus are a direct result of mutations in the CF transmembrane conductance regulator (CFTR) protein. We evaluated a single mucin molecule MUC1F/5ACTR that carries tandem repeat sequence from MUC5AC, a major secreted airway mucin, in a MUC1 mucin vector. To establish whether the presence of mutant or normal CFTR directly influences the O-glycosylation and sulphation of mucins in airway epithelial cells, we used the CFT1-LC3 (DeltaF508 CFTR mutant) and CFT1-LCFSN (wild-type CFTR corrected) human airway epithelial cell lines. MUC1F/5ACTR mucin was immunoprecipitated, centricon purified, and O-glycosylation was evaluated by Matrix-assisted laser desorption ionization and electrospray tandem mass spectrometry to determine the composition of different carbohydrate structures. Mass spectrometry data showed the same O-glycans in both CFTR mutant and wild-type CFTR corrected cells. Metabolic labeling assays were performed to evaluate gross glycosylation and sulphation of the mucins and showed no significant difference in mucin synthesized in six independent clones of these cell lines. Our results show that the absence of functional CFTR protein causes neither an abnormality in mucin O-glycosylation nor an increase in mucin sulphation

Cystic fibrosis mice carrying the missense mutation G551D replicate human genotype phenotype correlations

We have generated a mouse carrying the human G551D mutation in the cystic fibrosis transmembrane conductance regulator gene (CFTR) by a one-step gene targeting procedure. These mutant mice show cystic fibrosis pathology but have a reduced risk of fatal intestinal blockage compared with 'null' mutants, in keeping with the reduced incidence of meconium ileus in G551D patients. The G551D mutant mice show greatly reduced CFTR-related chloride transport, displaying activity intermediate between that of cftr(mlUNC) replacement ('null') and cftr(mlHGU) insertional (residual activity) mutants and equivalent to approximately 4% of wild-type CFTR activity. The long-term survival of these animals should provide an excellent model with which to study cystic fibrosis, and they illustrate the value of mouse models carrying relevant mutations for examining genotype-phenotype correlations

Microparticle-mediated transfer of the viral receptors CAR and CD46, and the CFTR channel in a CHO cell model confers new functions to target cells

Cell microparticles (MPs) released in the extracellular milieu can embark plasma membrane and intracellular components which are specific of their cellular origin, and transfer them to target cells. The MP-mediated, cell-to-cell transfer of three human membrane glycoproteins of different degrees of complexity was investigated in the present study, using a CHO cell model system. We first tested the delivery of CAR and CD46, two monospanins which act as adenovirus receptors, to target CHO cells. CHO cells lack CAR and CD46, high affinity receptors for human adenovirus serotype 5 (HAdV5), and serotype 35 (HAdV35), respectively. We found that MPs derived from CHO cells (MP-donor cells) constitutively expressing CAR (MP-CAR) or CD46 (MP-CD46) were able to transfer CAR and CD46 to target CHO cells, and conferred selective permissiveness to HAdV5 and HAdV35. In addition, target CHO cells incubated with MP-CD46 acquired the CD46-associated function in complement regulation. We also explored the MP-mediated delivery of a dodecaspanin membrane glycoprotein, the CFTR to target CHO cells. CFTR functions as a chloride channel in human cells and is implicated in the genetic disease cystic fibrosis. Target CHO cells incubated with MPs produced by CHO cells constitutively expressing GFP-tagged CFTR (MP-GFP-CFTR) were found to gain a new cellular function, the chloride channel activity associated to CFTR. Time-course analysis of the appearance of GFP-CFTR in target cells suggested that MPs could achieve the delivery of CFTR to target cells via two mechanisms: the transfer of mature, membrane-inserted CFTR glycoprotein, and the transfer of CFTR-encoding mRNA. These results confirmed that cell-derived MPs represent a new class of promising therapeutic vehicles for the delivery of bioactive macromolecules, proteins or mRNAs, the latter exerting the desired therapeutic effect in target cells via de novo synthesis of their encoded proteins

Modulator Therapy for Cystic Fibrosis: An Exploration of Current Research

Developing a drug therapy that addresses the root cause of cystic fibrosis (CF) by increasing CFTR protein levels has long been a research challenge. After genetic therapy failed because a suitable delivery system could not be found, researchers began searching for small organic molecules that could act as chaperones for CFTR. These molecules, known as modulators, allowed CFTR to be assembled correctly and function similarly to wild type CFTR. Since 2012, four modulator drugs have been developed, tested, and approved by the FDA. In October 2019, Trikafta was approved as the first triple-combination modulator drug and has completely revolutionized CF therapy. This paper details the research challenges, successes, and failures that led to the development of modulator therapies

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

A Genotypic-oriented View of CFTR Genetics Highlights Specific Mutational Patterns Underlying Clinical Macro-categories of Cystic Fibrosis.

Cystic Fibrosis (CF) is a monogenic disease caused by mutations of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene. The genotype-phenotype relationship in this disease is still unclear, and diagnostic, prognostic and therapeutic challenges persist. We enrolled 610 patients with different forms of CF and studied them from a clinical, biochemical, microbiological and genetic point of view. Overall, 125 different mutated alleles (11 of which with novel mutations and 10 of which complex) and 225 genotypes were found. A strong correlation between mutational patterns at the genotypic level and phenotypic macro-categories emerged. This specificity appears to be largely dependent on rare and individual mutations, as well as on the varying prevalence of common alleles in different clinical macro-categories. However, 19 genotypes appeared to underlie different clinical forms of the disease. The dissection of the pathway from the CFTR mutated genotype to the clinical phenotype allowed to identify at least two components of the variability usually found in the genotype - phenotype relationship. One component seems to depend on the genetic variation of CFTR, the other component on the cumulative effect of variations in other genes and cellular pathways independent from CFTR. The experimental dissection of the overall biological CFTR pathway appears to be a powerful approach for a better comprehension of the genotype - phenotype relationship. However, a change from an allele-oriented to a genotypic-oriented view of CFTR genetics is mandatory, as well as a better assessment of sources of variability within the CFTR pathway