Restoration of CFTR function in patients with cystic fibrosis carrying the F508del-CFTR mutation

Abstract

These authors equally contributed to this work.Keywords: cystic fibrosis, CFTR, autophagy, cysteamine, epigallocatechin gallate, sweat chloride Abbreviations: BECN1/Beclin 1, autophagy-related; CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; CHX, cycloheximide; CSNK2, casein kinase 2; CXCL2, chemokine (C-X-C motif) ligand 2; CXCL8, chemokine (C-X-C motif) ligand 8; EGCG, epigallocatechin gallate; FEV, forced expiratory volume; PM, plasma membrane; RPD, rectal potential difference; SQSTM1, sequestosome 1; TGM2, transglutaminase 2; TNF, tumor necrosis factor.Restoration of BECN1/Beclin 1-dependent autophagy and depletion of SQSTM1/p62 by genetic manipulation or autophagy-stimulatory proteostasis regulators, such as cystamine, have positive effects on mouse models of human cystic fibrosis (CF). These measures rescue the functional expression of the most frequent pathogenic CFTR mutant, F508del, at the respiratory epithelial surface and reduce lung inflammation in Cftr F508del homozygous mice. Cysteamine, the reduced form of cystamine, is an FDA-approved drug. Here, we report that oral treatment with cysteamine greatly reduces the mortality rate and improves the phenotype of newborn mice bearing the F508del-CFTR mutation. Cysteamine was also able to increase the plasma membrane expression of the F508del-CFTR protein in nasal epithelial cells from F508del homozygous CF patients, and these effects persisted for 24 h after cysteamine withdrawal. Importantly, this cysteamine effect after washout was further sustained by the sequential administration of epigallocatechin gallate (EGCG), a green tea flavonoid, both in vivo, in mice, and in vitro, in primary epithelial cells from CF patients. In a pilot clinical trial involving 10 F508del-CFTR homozygous CF patients, the combination of cysteamine and EGCG restored BECN1, reduced SQSTM1 levels and improved CFTR function from nasal epithelial cells in vivo, correlating with a decrease of chloride concentrations in sweat, as well as with a reduction of the abundance of TNF/ TNF-alpha (tumor necrosis factor) and CXCL8 (chemokine [C-X-C motif] ligand 8) transcripts in nasal brushing and TNF and CXCL8 protein levels in the sputum. Altogether, these results suggest that optimal schedules of cysteamine plus EGCG might be used for the treatment of CF caused by the F508del-CFTR mutation.

Erratum: A novel treatment of cystic fibrosis acting on-target: cysteamine plus epigallocatechin gallate for the autophagy-dependent rescue of class II-mutated CFTR

“…The pharmacokinetic profile of cysteamine was as described in our pilot trial (Supplementary Figure S3). 20 The incidence of adverse events was comparable to that observed in cysteaminetreated cystinosis (Supplementary Tables S7-S11). …”

“…In vitro studies before in vivo treatment: Before in vivo treatment, primary nasal epithelial cells freshly collected by nasal brushing at baseline were challenged in vitro with cysteamine and/or EGCG, and then kept in culture with medium alone or EGCG up to 48 h. 20 The combination treatment in vitro restored both CFTR function and CFTR band C protein in PM fractions up to 450% of non-CF controls in all but one patient bearing at least one class II mutation. F508del homozygous patients were responsive, as described, 20 whereas all subjects bearing two class I mutations were unresponsive. The effects of combination treatment in vitro in class II patients persisted up to 48 h following cysteamine washout, provided that EGCG was maintained in the culture medium ( Figure 4, Supplementary Table S6).…”

“…17 Combined inhibition of both TG2 by the repurposed cysteamine, FDA-approved for the treatment of cystinosis, 18,19 (which reestablishes autophagy) and overactive CK2 by the over-the-counter green-tea flavonoid epigallocatechin gallate (EGCG) synergize in vitro and thereby rescue and stabilize, respectively, a functional F508del-CFTR protein at the PM, both in mice and in primary nasal cells from F508del-CFTR homozygotes. 20 This prompted a pilot trial combining cysteamine and EGCG in ten F508del-CFTR homozygotes, showing that the combination treatment was well tolerated, reverted sweat chloride toward normal and significantly attenuated biomarkers of airway inflammation. 20 Here, we provide new insights from newly generated transgenic mice and patients primary cells and investigate in a phase-2 clinical trial the individual patient responsiveness to a combination of cysteamine plus EGCG in CF patients bearing different classes of CFTR mutation.…”

“…20 This prompted a pilot trial combining cysteamine and EGCG in ten F508del-CFTR homozygotes, showing that the combination treatment was well tolerated, reverted sweat chloride toward normal and significantly attenuated biomarkers of airway inflammation. 20 Here, we provide new insights from newly generated transgenic mice and patients primary cells and investigate in a phase-2 clinical trial the individual patient responsiveness to a combination of cysteamine plus EGCG in CF patients bearing different classes of CFTR mutation. Moreover, we evaluate the feasibility of using both functional and mechanistic biomarkers as a prediction test of patient responsiveness to facilitate a future personalized approach to precision CF therapy.…”

Synthesis of diacylated γ-glutamyl-cysteamine prodrugs, and in vitro evaluation of their cytotoxicity and intracellular delivery of cysteamine

“…Cysteamine is also of interest for its potential in treating other diseases, such as Huntington's disease [11,12], cystic fibrosis [13][14][15], chronic kidney disease [16], and non-alcoholic fatty liver disease (NAFLD), including non-alcoholic steatohepatitis (NASH) [17]. A current clinical trial is evaluating the potential of Procysbi ® as a disease-modifying treatment for Huntington's disease.…”