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p.Asn1303Lys (N1303K) is a common missense variant of the CFTR gene, causing cystic fibrosis (CF). In this study, we initially evaluated the influence of CFTR modulators on the restoration of N1303K-CFTR function using intestinal organoids derived from four CF patients expressing the N1303K variant. The forskolin-induced swelling assay in organoids offered valuable insights about the beneficial effects of VX-770 + VX-661 + VX-445 (Elexacaftor + Tezacaftor + Ivacaftor, ETI) on N1303K-CFTR function restoration and about discouraging the prescription of VX-770 + VX-809 (Ivacaftor + Lumacaftor) or VX-770 + VX-661 (Ivacaftor + Tezacaftor) therapy for N1303K/class I patients. Then, a comprehensive assessment was conducted on an example of one patient with the N1303K/class I genotype to examine the ETI effect on the restoration of N1303K-CFTR function using in vitro the patient's intestinal organoids, ex vivo the intestinal current measurements (ICM) method and assessment of the clinical status before and after targeted therapy. All obtained results are consistent with each other and have proven the effectiveness of ETI for the N1303K variant. ETI produced a significant positive effect on forskolin-induced swelling in N1303K/class I organoids indicating functional improvement of the CFTR protein; ICM demonstrated that ETI therapy restored CFTR function in the intestinal epithelium after three months of treatment, and the patient improved his clinical status and lung function, increased his body mass index (BMI) and reduced the lung pathogenic flora diversity, surprisingly without improving the sweat test results. [ABSTRACT FROM AUTHOR]

Cystic fibrosis (CF) is caused by a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Thousands of CFTR mutations have been identified, but only a fraction are known to cause CF, with the most common being the prototypical class II CFTR mutation F508del. Elexacaftor-Tezacaftor-Ivacaftor (ETI) is a CFTR modulator that significantly increases ppFEV1 and reduces exacerbation frequencies. It is indicated for people with CF (pwCF) 2 years or older with at least one copy of F508del or one copy of the other 177 CFTR mutations that are responsive to ETI based on clinical or in vitro data. N1303K is the second most common class II mutation in the U.S. but is not yet FDA-approved for CFTR modulator therapy. However, N1303K is very similar to the F508del mutation and reveals variable in vitro responses to ETI.Theratyping provides an opportunity to consider ETI therapy for pwCF with mutations currently not approved by the FDA. We describe the case of an adult CF patient with W1282X and N1303K CFTR mutations and advanced CF lung disease (ACFLD) and declining lung function in which ETI was started after theratyping of nasal cells showed a meaningful response to ETI (current enhanced to over 10% of WT CFTR). The patient experienced clinical improvement with a 5% improvement in ppFEV1 and 10% increase in weight. However, there was no change in sweat chloride and the increase in ppFEV1 was less than what has been described for ACFLD patients with more typical ETI-amenable mutations. However, the response was in line with a few other cases described in the literature. This suggests a partial functional CFTR rescue like first-generation modulators for F508del. Thus, pwCF with N1303K CFTR variant could be considered for ETI eligibility.

CFTR modulator therapy with elexacaftor/tezacaftor/ivacaftor (ETI) has been approved for people with CF and at least one F508del allele in Europe. In the US, the ETI label has been expanded to 177 rare CFTR mutations responsive in Fischer rat thyroid cells, including G85E, but not N1303K. However, knowledge on the effect of ETI on G85E or N1303K CFTR function remains limited. In vitro effects of ETI were measured in primary human nasal epithelial cultures (pHNECs) of a G85E homozygous patient and an N1303K homozygous patient. Effects of ETI therapy in vivo in these patients were assessed using clinical outcomes, including multiple breath washout and lung MRI, and the CFTR biomarkers sweat chloride concentration (SCC), nasal potential difference (NPD) and intestinal current measurement (ICM), before and after initiation of ETI. ETI increased CFTR-mediated chloride transport in G85E/G85E and N1303K/N1303K pHNECs. In the G85E/G85E and the N1303K/N1303K patient, we observed an improvement in lung function, SCC, and CFTR function in the respiratory and rectal epithelium after initiation of ETI. The approach of combining preclinical in vitro testing with subsequent in vivo verification can facilitate access to CFTR modulator therapy and enhance precision medicine for patients carrying rare CFTR mutations. [ABSTRACT FROM AUTHOR]

p.Asn1303Lys (N1303K) is a common missense variant of the CFTR gene, causing cystic fibrosis (CF). In this study, we initially evaluated the influence of CFTR modulators on the restoration of N1303K-CFTR function using intestinal organoids derived from four CF patients expressing the N1303K variant. The forskolin-induced swelling assay in organoids offered valuable insights about the beneficial effects of VX-770 + VX-661 + VX-445 (Elexacaftor + Tezacaftor + Ivacaftor, ETI) on N1303K-CFTR function restoration and about discouraging the prescription of VX-770 + VX-809 (Ivacaftor + Lumacaftor) or VX-770 + VX-661 (Ivacaftor + Tezacaftor) therapy for N1303K/class I patients. Then, a comprehensive assessment was conducted on an example of one patient with the N1303K/class I genotype to examine the ETI effect on the restoration of N1303K-CFTR function using in vitro the patient’s intestinal organoids, ex vivo the intestinal current measurements (ICM) method and assessment of the clinical status before and after targeted therapy. All obtained results are consistent with each other and have proven the effectiveness of ETI for the N1303K variant. ETI produced a significant positive effect on forskolin-induced swelling in N1303K/class I organoids indicating functional improvement of the CFTR protein; ICM demonstrated that ETI therapy restored CFTR function in the intestinal epithelium after three months of treatment, and the patient improved his clinical status and lung function, increased his body mass index (BMI) and reduced the lung pathogenic flora diversity, surprisingly without improving the sweat test results.

BackgroundCurrent modulator therapies for some cystic fibrosis-causing CFTR mutants, including N1303K, have limited efficacy. We provide evidence here to support combination potentiator (co-potentiator) therapy for mutant CFTRs that are poorly responsive to single potentiators.MethodsFunctional synergy screens done on N1303K and W1282X CFTR, in which small molecules were tested with VX-770, identified arylsulfonamide-pyrrolopyridine, phenoxy-benzimidazole and flavone co-potentiators.ResultsA previously identified arylsulfonamide-pyrrolopyridine co-potentiator (ASP-11) added with VX-770 increased N1303K-CFTR current 7-fold more than VX-770 alone. ASP-11 increased by ~65% of the current of G551D-CFTR compared to VX-770, was additive with VX-770 on F508del-CFTR, and activated wild-type CFTR in the absence of a cAMP agonist. ASP-11 efficacy with VX-770 was demonstrated in primary CF human airway cell cultures having N1303K, W1282X and G551D CFTR mutations. Structure-activity studies on 11 synthesized ASP-11 analogs produced compounds with EC50 down to 0.5 μM.ConclusionsThese studies support combination potentiator therapy for CF caused by some CFTR mutations that are not effectively treated by single potentiators.

CFTR modulator therapy with elexacaftor/tezacaftor/ivacaftor (ETI) has been approved for people with CF and at least one F508del allele in Europe. In the US, the ETI label has been expanded to 177 rare CFTR mutations responsive in Fischer rat thyroid cells, including G85E, but not N1303K. However, knowledge on the effect of ETI on G85E or N1303K CFTR function remains limited. In vitro effects of ETI were measured in primary human nasal epithelial cultures (pHNECs) of a G85E homozygous patient and an N1303K homozygous patient. Effects of ETI therapy in vivo in these patients were assessed using clinical outcomes, including multiple breath washout and lung MRI, and the CFTR biomarkers sweat chloride concentration (SCC), nasal potential difference (NPD) and intestinal current measurement (ICM), before and after initiation of ETI. ETI increased CFTR-mediated chloride transport in G85E/G85E and N1303K/N1303K pHNECs. In the G85E/G85E and the N1303K/N1303K patient, we observed an improvement in lung function, SCC, and CFTR function in the respiratory and rectal epithelium after initiation of ETI. The approach of combining preclinical in vitro testing with subsequent in vivo verification can facilitate access to CFTR modulator therapy and enhance precision medicine for patients carrying rare CFTR mutations.

Due to progress in infertility etiology, several genetic bases of infertility are revealed today. This study aimed to investigate the distribution of mutations in the CFTR gene, M470V polymorphism, and IVS8 poly T. Furthermore, we aimed to examine the hotspot exons (4, 7, 9, 10, 11, 20, and 21 exons) to find a new mutation in cystic fibrosis transmembrane conductance regulator (CFTR) gene among infertile Iranian men very severe oligozoospermia (<1 million sperm/mL ejaculate fluid). In the present case-control study, 200 very severe oligozoospermia (20--60s) and 200 fertile men (18--65s) were registered. Five common CFTR mutations were genotyped using the ARMS-PCR technique. The M470V polymorphism was checked out by real-time PCR, and poly T and exons were sequenced. The F508del was the most common (4.5%) CFTR gene mutation; G542X and W1282X were detected with 1.5% and 1%, respectively. N1303K and R117H were detected in 0.5% of cases. F508del was seen as a heterozygous compound with G542X in one patient and with W1282X in the other patient. Also, in the case of M470V polymorphism, there are differences between the case and control groups (p=0.013). Poly T assay showed statistical differences in some genotypes. The study showed no new mutation in the exons mentioned above. Our results shed light on the genetic basis of men with very severe oligozoospermia in the Iranian population, which will support therapy decisions among infertile men. [ABSTRACT FROM AUTHOR]

Abstract Background N1303K, one of the common, severe disease-causing mutations in the CFTR gene, causes both defective biogenesis and gating abnormalities of the CFTR protein. The goals of the present study are to quantitatively assess the gating defects associated with the N1303K mutation and its pharmacological response to CFTR modulators including potentiators VX-770 and GLPG1837 and correctors VX-809, and VX-661. Methods Gating behavior and pharmacological responses to CFTR potentiators were assessed using patch-clamp technique in the excised, inside-out mode. We also examined the effects of GLPG1837, VX-770, VX-809 and VX-661 on N1303K-CFTR surface expression using Western blot analysis. Results Like wild-type (WT) CFTR, N1303K-CFTR channels were activated by protein kinase A-dependent phosphorylation, but the open probability ( P o ) of phosphorylated N1303K-CFTR was extremely low (~0.03 vs ~0.45 in WT channels). N1303K mutants showed abnormal responses to ATP analogs or mutations that disrupt ATP hydrolysis and/or dimerization of CFTR's two nucleotide-binding domains (NBDs). However, the P o of N1303K-CFTR was dramatically increased by GLPG1837 (~17-fold) and VX-770 (~8-fold). VX-809 or VX-661 enhanced N1303K-CFTR maturation by 2–3 fold, and co-treatment with GLPG1837 or VX-770 did not show any negative drug-drug interaction. Conclusion N1303K has a severe gating defect, reduced ATP-dependence and aberrant response to ATP analogs. These results suggest a defective function of the NBDs in N1303K-CFTR. An improvement of channel function by GLPG1837 or VX-770 and an increase of Band C protein by VX-809 or VX-661 support a therapeutic strategy of combining CFTR potentiator and corrector for patients carrying the N1303K mutation. [ABSTRACT FROM AUTHOR]

Abstract: The CFTR genotype N1303K/IVS8-5T can cause very mild cystic fibrosis (CF) and congenital bilateral absence of the vas deferens (CBAVD). We report one family consisting of five affected patients in two generations, presenting minor symptoms of CF at different ages, segregating the CFTR mutations N1303K and IVS8-T5-TG13 in trans. Common features were chronic sinopulmonary symptoms and borderline or slightly elevated sweat chloride values. One patient had CBAVD. [Copyright &y& Elsevier]