Isabelle Fajac

Ataluren for the treatment of nonsense-mutation cystic fibrosis: a randomised, double-blind, placebo-controlled phase 3 trial

BACKGROUND Ataluren was developed to restore functional protein production in genetic disorders caused by nonsense mutations, which are the cause of cystic fibrosis in 10% of patients. This trial was designed to assess the efficacy and safety of ataluren in patients with nonsense-mutation cystic fibrosis. METHODS This randomised, double-blind, placebo-controlled, phase 3 study enrolled patients from 36 sites in 11 countries in North America and Europe. Eligible patients with nonsense-mutation cystic fibrosis (aged ≥ 6 years; abnormal nasal potential difference; sweat chloride >40 mmol/L; forced expiratory volume in 1 s [FEV1] ≥ 40% and ≤ 90%) were randomly assigned by interactive response technology to receive oral ataluren (10 mg/kg in morning, 10 mg/kg midday, and 20 mg/kg in evening) or matching placebo for 48 weeks. Randomisation used a block size of four, stratified by age, chronic inhaled antibiotic use, and percent-predicted FEV1. The primary endpoint was relative change in percent-predicted FEV1 from baseline to week 48, analysed in all patients with a post-baseline spirometry measurement. This study is registered with ClinicalTrials.gov, number NCT00803205. FINDINGS Between Sept 8, 2009, and Nov 30, 2010, 238 patients were randomly assigned, of whom 116 in each treatment group had a valid post-baseline spirometry measurement. Relative change from baseline in percent-predicted FEV1 did not differ significantly between ataluren and placebo at week 48 (-2.5% vs -5.5%; difference 3.0% [95% CI -0.8 to 6.3]; p=0.12). The number of pulmonary exacerbations did not differ significantly between treatment groups (rate ratio 0.77 [95% CI 0.57-1.05]; p=0.0992). However, post-hoc analysis of the subgroup of patients not using chronic inhaled tobramycin showed a 5.7% difference (95% CI 1.5-10.1) in relative change from baseline in percent-predicted FEV1 between the ataluren and placebo groups at week 48 (-0.7% [-4.0 to 2.1] vs -6.4% [-9.8 to -3.7]; nominal p=0.0082), and fewer pulmonary exacerbations in the ataluern group (1.42 events [0.9-1.9] vs 2.18 events [1.6-2.7]; rate ratio 0.60 [0.42-0.86]; nominal p=0.0061). Safety profiles were generally similar for ataluren and placebo, except for the occurrence of increased creatinine concentrations (ie, acute kidney injury), which occurred in 18 (15%) of 118 patients in the ataluren group compared with one (<1%) of 120 patients in the placebo group. No life-threatening adverse events or deaths were reported in either group. INTERPRETATION Although ataluren did not improve lung function in the overall population of nonsense-mutation cystic fibrosis patients who received this treatment, it might be beneficial for patients not taking chronic inhaled tobramycin. FUNDING PTC Therapeutics, Cystic Fibrosis Foundation, US Food and Drug Administrations Office of Orphan Products Development, and the National Institutes of Health.

Potocytosis and cellular exit of complexes as cellular pathways for gene delivery by polycations.

Although polycations are among the most efficient nonviral vectors for gene transfer, the gene expression they allow is still too low for in vivo applications. To engineer more potent polycationic vectors, the factors governing the intracellular trafficking of a plasmid complexed with current polycations need to be identified.

Sugar-Mediated Uptake of Glycosylated Polylysines and Gene Transfer into Normal and Cystic Fibrosis Airway Epithelial Cells

We have examined the membrane lectin expressed by immortalized normal and cystic fibrosis (CF) airway epithelial cells, using fluorescein-labeled neoglycoproteins; the uptake of plasmid DNA using fluoresceinylated glycoplexes (plasmid/glycosylated polylysine complexes); and the efficiency of gene transfer when glycosylated polylysines and glycosylated, partially gluconoylated polylysines were used as vectors. The most efficient uptake of neoglycoproteins by normal and CF cells was obtained with mannosylated BSA (bovine serum albumin). Similarly, the most efficient uptake of plasmid DNA was obtained with glycoplexes bearing alpha-D-Man residues. Surprisingly, glycoplexes bearing alpha-D-Man residues were poorly efficient for gene transfer into normal and CF cells. The highest luciferase activity was achieved with lactosylated polylysine- and beta-D-GlcNAc-substituted gluconoylated polylysine as vectors. Gene transfer efficiency obtained with gluconoylated polylysine bearing beta-D-GlcNAc residues was similar to that observed with polyethylenimine (PEI; 25 and 800 kDa) and 10-fold higher than that observed with lipofectin and LipofectAMINE. These results suggest that the transfection efficiency with glycoplexes is not determined only by the specificity of the lectin expressed at the cell surface membrane but also by intracellular trafficking of the glycoplexes, which could be mediated by lectins present inside the cells.

Lactosylated polyethylenimine for gene transfer into airway epithelial cells: role of the sugar moiety in cell delivery and intracellular trafficking of the complexes.

As we have previously shown that lactosylated polyethylenimine (PEI) is the most efficient glycosylated PEI for gene transfer into human airway epithelial cells in primary culture, we have studied here the role of the lactose residue in the enhancement of gene transfer efficiency observed with lactosylated PEI as compared with unsubstituted PEI in immortalized (ΣCFTE29o‐ cells) and primary human airway epithelial cells.

Uptake of plasmid/glycosylated polymer complexes and gene transfer efficiency in differentiated airway epithelial cells.

We have studied gene transfer efficiency of glycosylated polylysines and glycosylated polyethylenimines as vectors in immortalized differentiated airway gland serous cells and primary cultures of human airway surface epithelial cells.

Glycofection: facilitated gene transfer by cationic glycopolymers.

Abstract. Mammalian cells express several types of lectins involved in intracellular trafficking, including endocytosis, interorganelle routing and putatively nuclear import. In order to enhance the gene transfer efficiency, glycosylated cationic polymers have been used as nonviral vectors. We developed a simple method to convert reducing saccharides into glycosynthons. Glycosynthons are used to synthesize cationic glycopolymers, called Glycofectins. Glycofectins interact with a plasmid to give a glycoplex, a compacted form of a polymer/DNA complex. The high glycoplex efficiency depends on the sugar involved in the uptake and in the intracellular trafficking of glycoplexes. The present paper deals with glycoplexes, with gene transfer into cystic fibrosis airway epithelial and gland serous cells, and with some of the problems that have to be solved before clinical trials.

Cystic Fibrosis Transmembrane Conductance Regulator Channel Dysfunction in Non–Cystic Fibrosis Bronchiectasis

RATIONALE Although in patients with diffuse bronchiectasis (DB) and a normal sweat test the presence of one mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene is frequently observed, its pathogenic role in the development of DB remains unclear. OBJECTIVES To evaluate the association between CFTR heterozygosity and CFTR protein dysfunction in the airways of patients with DB. METHODS Nasal potential difference was measured in 122 patients with DB of unknown origin and with a normal sweat test (Cl(-) < 60 mmol/L). They were classified according to the presence of CFTR mutations: zero (85 patients), one (22 patients), or two mutations (15 patients). Control groups comprised 26 healthy subjects, 38 obligate heterozygotes for CFTR, and 92 patients with classic cystic fibrosis (CF) with an abnormal sweat test (Cl(-) > or = 60 mmol/L). Patients classified as mild-CF were carrying at least one mild mutation and patients classified as severe-CF were homozygous for the F508del mutation. MEASUREMENTS AND MAIN RESULTS There was a continuum of airway CFTR dysfunction in the study population as shown by nasal potential difference measurements, ranging from normal values in healthy subjects, to intermediate values in subjects with DB, to highly abnormal values in subjects classified as severe-CF. This continuum of airway CFTR dysfunction was thus strongly associated with defects in the CFTR gene. Moreover, among patients with DB, a similar continuum in intermediate nasal potential difference was identified that was associated with the bearing of zero, one, or two CFTR mutations. These electrophysiological phenotypes and CFTR genotypes were also associated with the clinical phenotype, as shown by the frequency of Staphylococcus aureus and Pseudomonas aeruginosa bronchial colonization. CONCLUSIONS Our study supports the hypothesis that a unique CFTR mutation may have pathogenic consequences in patients with DB.

Clinical phenotype and genotype of children with borderline sweat test and abnormal nasal epithelial chloride transport.

RATIONALE The diagnosis of cystic fibrosis (CF) is based on a characteristic clinical picture in association with a sweat chloride (Cl(-)) concentration greater than 60 mmol/L or the identification of two CF-causing mutations. A challenging problem is the significant number of children for whom no definitive diagnosis is possible because they present with symptoms suggestive of CF, a sweat chloride level in the intermediate range between 30 and 60 mmol/L, and only one or no identified CF-causing mutation. OBJECTIVES To investigate the function of the cystic fibrosis transmembrane conductance regulator (CFTR) protein in the airways of children with intermediate sweat tests and inconclusive genetic findings in correlation with clinical phenotype and genotype. METHODS We developed a composite nasal potential difference (NPD) diagnostic score to discriminate patients with CF from non-CF patients. We tested NPD in 50 children (age, 6 mo to 18 yr) with equivocal diagnoses and correlated the NPD diagnostic score with clinical phenotypes and genotypes. MEASUREMENTS AND MAIN RESULTS Fifteen of the 50 children had NPD scores in the CF range. Eight of the 15 carried two CFTR mutations compared with only 5 of the 35 children with normal NPD scores (P = 0.01). They were significantly younger at evaluation and had recurrent lower respiratory tract infections, chronic productive coughs, and chronic Staphylococcus aureus colonization significantly more often than the 35 children with normal NPD results. CONCLUSIONS Evaluation of CFTR function in the nasal epithelium of children with inconclusive CF diagnoses can be a useful diagnostic tool and help clinicians to individualize therapeutic strategy.

Histidylated polylysine as a synthetic vector for gene transfer into immortalized cystic fibrosis airway surface and airway gland serous cells.

We recently designed a cationic polymer called histidylated polylysine made of polylysine partially substituted with histidyl residues which become protonated at slightly acidic pH. This polymer is thought to induce the leakage of acidic vesicles containing plasmid/histidylated polylysine complexes.

Nasal airway ion transport is linked to the cystic fibrosis phenotype in adult patients.

Background: This study was conducted to determine whether the major nasal airway ion transport abnormalities in cystic fibrosis (that is, defective cAMP regulated chloride secretion and basal sodium hyperabsorption) are related to the clinical expression of cystic fibrosis and/or to the genotype. Methods: Nasal potential difference was measured in 79 adult patients with cystic fibrosis for whom clinical status, respiratory function, and CFTR genotype were determined. Results: In univariate and multivariate analysis, patients with pancreatic insufficiency were more likely to have low responses to low chloride (odds ratio (OR) 8.6 (95% CI 1.3 to 58.5), p = 0.03) and isoproterenol (OR 11.2 (95% CI 1.3 to 93.9), p = 0.03) solutions. Similarly, in univariate and multivariate analysis, patients with poor respiratory function (forced expiratory volume in 1 second <50% of predicted value) were more likely to have an enhanced response to amiloride solution (OR 3.7 (95% CI 1.3 to 11.0), p = 0.02). However, there was no significant relationship between nasal potential difference and the severity of the genotype. Conclusions: Nasal epithelial ion transport in cystic fibrosis is linked to the clinical expression of the disease. The pancreatic status appears to be mostly related to the defect in epithelial chloride secretion whereas the respiratory status is mostly related to abnormal sodium transport and the regulatory function of the CFTR protein.