RESUMEN
Positive results in pre-clinical studies of the triple combination of elexacaftor, tezacaftor and ivacaftor, performed in airway epithelial cell cultures obtained from patients harbouring the class II cystic fibrosis transmembrane conductance regulator (CFTR) mutation F508del-CFTR, translated to impressive clinical outcomes for subjects carrying this mutation in clinical trials and approval of Trikafta.Encouraged by this correlation, we were prompted to evaluate the effect of the elexacaftor, tezacaftor and ivacaftor triple combination on primary nasal epithelial cultures obtained from individuals with rare class II CF-causing mutations (G85E, M1101K and N1303K) for which Trikafta is not approved.Cultures from individuals homozygous for M1101K responded better than cultures harbouring G85E and N1303K after treatment with the triple combination with respect to improvement in regulated channel function and protein processing. A similar genotype-specific effect of the triple combination was observed when the different mutations were expressed in HEK293 cells, supporting the hypothesis that these modulators may act directly on the mutant proteins. Detailed studies in nasal cultures and HEK293 cells showed that the corrector, elexacaftor, exhibited dual activity as both corrector and potentiator, and suggested that the potentiator activity contributes to its pharmacological activity.These pre-clinical studies using nasal epithelial cultures identified mutation genotypes for which elexacaftor, tezacaftor and ivacaftor may produce clinical responses that are comparable to, or inferior to, those observed for F508del-CFTR.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística , Fibrosis Quística , Aminofenoles , Benzodioxoles , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Combinación de Medicamentos , Células HEK293 , Humanos , Indoles , Mutación , Pirazoles , Piridinas , Pirrolidinas , Quinolinas , QuinolonasRESUMEN
It has been suggested that in vitro studies of the rescue effect of CFTR modulator drugs in nasal epithelial cultures derived from people with cystic fibrosis have the potential to predict clinical responses to the same drugs. Hence, there is an interest in evaluating different methods for measuring in vitro modulator responses in patient-derived nasal cultures. Commonly, the functional response to CFTR modulator combinations in these cultures is assessed by bioelectric measurements, using the Ussing chamber. While this method is highly informative, it is time-consuming. A fluorescence-based, multi-transwell method for assaying regulated apical chloride conductance (Fl-ACC) promises to provide a complementary approach to theratyping in patient-derived nasal cultures. In the present work, we compared Ussing chamber measurements and fluorescence-based measurements of CFTR-mediated apical conductance in matching, fully differentiated nasal cultures derived from CF patients, homozygous for F508del (n = 31) or W1282X (n = 3), or heterozygous for Class III mutations G551D or G178R (n = 5). These cultures were obtained through a bioresource called the Cystic Fibrosis Canada-Sick Kids Program in Individual CF Therapy (CFIT). We found that the Fl-ACC method was effective in detecting positive responses to interventions for all genotypes. There was a correlation between patient-specific drug responses measured in cultures harbouring F508del, as measured using the Ussing chamber technique and the fluorescence-based assay (Fl-ACC). Finally, the fluorescence-based assay has the potential for greater sensitivity for detecting responses to pharmacological rescue strategies targeting W1282X.
Asunto(s)
Fibrosis Quística , Humanos , Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fluorescencia , Mutación , GenotipoRESUMEN
Previous studies reported the influence of cis variants in F508del cystic fibrosis (CF) patients in their responses to CFTR modulators. The current study is a prospective, observational study involving three patients with CF and pancreatic insufficiency, carrying a complex allele including F508del with A238V, I1027T, or L467F. We report clinical data before and after 4 weeks of treatment with tezacaftor (TEZ)/ivacaftor (IVA), elexacaftor (ELX)/TEZ/IVA, and lumacaftor (LUM)/IVA for patients with complex alleles A238V, I1027T, and L467F, respectively. The 50-year-old patient bearing F508del;A238V/D1152H showed a normal sweat test (13 mEq/L) and improvements in forced expiratory volume in the first second (FEV1) (+7 points), body mass index (BMI) (+0.85), and respiratory CF Questionnaire-Revised (CFQ-R) domain (+22.2 points). The 12-year-old patient bearing F508del;I1027T/R709X showed an improvement in a sweat test (-40 mEq/l), FEV1 (+9 points) and the respiratory CFQ-R domain (+16.7 points). No changes in outcomes were observed for the 6-year-old patient F508del;L467F/F508del. Our data highlight that the reported variants do not modify the phenotypic expression of F508del. Searching L467F is crucial in CF patients with F508del nonresponsive to ELX/TEZ/IVA. Further data are needed to evaluate the clinical effect of these variants after a longer follow up.
RESUMEN
BACKGROUND: F508del is prototypical of Class 2 CFTR mutations associated with protein misprocessing and reduced function. Corrector compounds like lumacaftor partially rescue the processing defect of F508del-CFTR whereas potentiators like ivacaftor, enhance its channel activity once trafficked to the cell surface. We asked if emerging modulators developed for F508del-CFTR can rescue Class 2 mutations previously shown to be poorly responsive to lumacaftor and ivacaftor. METHODS: Rescue of mutant CFTRs by the correctors: AC1, AC2-1 or AC2-2 and the potentiator, AP2, was studied in HEK-293 cells and in primary human nasal epithelial (HNE) cultures, using a membrane potential assay and Ussing chamber, respectively. RESULTS: In HEK-293 cells, we found that a particular combination of corrector molecules (AC1 plus AC2-1) and a potentiator (AP2) was effective in rescuing both the misprocessing and reduced function of M1101K and G85E respectively. These findings were recapitulated in patient-derived nasal cultures, although another corrector combination, AC1 plus AC2-2 also improved misprocessing in these primary tissues. Interestingly, while this corrector combination only led to a modest increase in the abundance of mature N1303K-CFTR it did enable its functional expression in the presence of the potentiator, AP2, in part, because the nominal corrector, AC2-2 also exhibits potentiator activity. CONCLUSIONS: Strategic combinations of novel modulators can potentially rescue Class 2 mutants thought to be relatively unresponsive to lumacaftor and ivacaftor.
Asunto(s)
Aminofenoles/uso terapéutico , Aminopiridinas/uso terapéutico , Benzodioxoles/uso terapéutico , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/genética , Mutación , Quinolonas/uso terapéutico , Células Cultivadas , Combinación de Medicamentos , Evaluación Preclínica de Medicamentos , Resistencia a Medicamentos , HumanosRESUMEN
The combination therapies ORKAMBITM and TRIKAFTATM are approved for people who have the F508del mutation on at least one allele. In this study we examine the effects of potentiator and corrector combinations on the rare mutation c.3700A>G. This mutation produces a cryptic splice site that deletes six amino acids in NBD2 (I1234-R1239del). Like F508del it causes protein misprocessing and reduced chloride channel function. We show that a novel cystic fibrosis transmembrane conductance regulator CFTR modulator triple combination (AC1, corrector, AC2-2, co-potentiator and AP2, potentiator), rescued I1234-R1239del-CFTR activity to WT-CFTR level in HEK293 cells. Moreover, we show that although the response to ORKAMBI was modest in nasal epithelial cells from two individuals homozygous for I1234-R1239del-CFTR, a substantial functional rescue was achieved with the novel triple combination. Interestingly, while both the novel CFTR triple combination and TRIKAFTATM treatment showed functional rescue in gene-edited I1234-R1239del-CFTR-expressing HBE cells and in nasal cells from two CF patients heterozygous for I1234-R1239del/W1282X, nasal cells homozygous for I1234-R1239del-CFTR showed no significant response to the TRIKAFTATM combination. These data suggest a potential benefit of CFTR modulators on the functional rescue of I1234-R1239del -CFTR, which arises from the rare CF-causing mutation c.3700A>G, and highlight that patient tissues are crucial to our full understanding of functional rescue in rare CFTR mutations.
RESUMEN
The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene variant, c.3453G > C (D1152H), is associated with mild Cystic Fibrosis (CF) disease, though there is considerable clinical variability ranging from no detectable symptoms to lung disease with early acquisition of Pseudomonas aeruginosa. The approval extension of ivacaftor, the first CFTR modulator drug approved, to include D1152H was based on a positive drug response of defective CFTR-D1152H chloride channel function when expressed in FRT cells. Functional analyses of primary human nasal epithelial cells (HNE) from an individual homozygous for D1152H now revealed that while CFTR-D1152H demonstrated normal, wild-type level chloride conductance, its bicarbonate-selective conductance was impaired. Treatment with ivacaftor increased this bicarbonate-selective conductance. Extensive genetic, protein and functional analysis of the nasal cells of this D1152H/D1152H patient revealed a 90% reduction of CFTR transcripts due to the homozygous presence of the 5T polymorphism in the poly-T tract forming a complex allele with D1152H. Thus, we confirm previous observation in patient-derived tissue that 10% normal CFTR transcripts confer normal, wild-type level chloride channel activity. Together, this study highlights the benefit of patient-derived tissues to study the functional expression and pharmacological modulation of CF-causing mutations, in order to understand pathogenesis and therapeutic responses.