BACKGROUND:The triple combination of Elexacaftor/Tezacaftor/Ivacaftor (ETI) has revolutionized cystic fibrosis (CF) treatment; however, a subset of patients with eligible genotypes remains unresponsive. We previously identified the L467F-F508del complex allele as a cause of therapeutic failure, conferring a severe processing defect that renders the CFTR protein refractory to ETI. METHODS:To address this unmet need, we evaluated the efficacy of the next-generation combination comprising Vanzacaftor/Tezacaftor (Vnz/Tez). We utilized primary human nasal epithelial (HNE) cells derived from six patients carrying the L467F-F508del complex allele and a minimal function mutation and monitored the clinical response of a patient treated with Vanzacaftor/Tezacaftor/Deutivacaftor (VTD) via compassionate use. Mechanistic validation was performed in CFBE41o- cells. RESULTS:In patient-derived HNE models, treatment with Vnz/Tez resulted in a consistent and significant rescue of CFTR activity, overcoming the block previously observed with ETI. Of note, we report a patient treated with VTD who experienced a partial improvement in pulmonary function (FEV1 +220 mL) and exercise tolerance and a decrease of sweat Chloride concentration from 100 to 87 mmol/L. In heterologous expression systems, Vnz/Tez, but not Elx/Tez, successfully restored the processing of the L467F-F508del mutant, promoting the formation of the mature, complex-glycosylated CFTR form. CONCLUSIONS:Our findings demonstrate that the Vanzacaftor/Tezacaftor combination possesses superior corrective potency capable of rescuing the severe trafficking defect of the L467F-F508del complex allele. This study identifies a promising therapeutic solution for patients currently orphaned by standard-of-care modulators and highlights the utility of integrating ex vivo screening with clinical monitoring in defining precision medicine strategies.

Background and Purpose Cystic fibrosis (CF) is due to loss-of-function variants of the CF transmembrane conductance regulator (CFTR) channel. The most effective treatment for people with CF carrying the F508del mutation is the triple combination of elexacaftor-tezacaftor-ivacaftor (ETI). ETI can correct the underlying defect(s) in other CFTR mutants. The use of disease-relevant predictive models such as patient-derived human nasal epithelial cells allow to investigate the response to CFTR modulators of specific genotypes, possibly supporting patients' access to treatment.Experimental Approach Using computational, biochemical and functional methodologies, a detailed analysis of selected variants in the intracellular loop 4 (ICL4) to understand their impact on CFTR structure and function.Key Results Mutations affecting L1065, R1066 and L1077 compromise structural stability of CFTR. Analyses of single variants expressed heterologously in immortalized bronchial cells showed that, upon ETI, rescued activity for both L1065P and R1066C was close to 50% of the wild-type CFTR activity. Biochemical studies of ICL4 variants expression pattern in CFBE41o-cells, following treatment for 24 h, demonstrate the appearance of the mature, fully glycosylated band, with no changes in the immature band. Finally, our study provides evidence in primary nasal cells from a cohort of people with CF that L1065P and R1066C can be effectively rescued by ETI up to 25%-45% of the activity measured in non-CF epithelia.Conclusion and Implications Although the observed rescue for L1065P and R1066C was smaller than that of the F508del, it should fall in a range predicted, by various studies, to provide a clinical benefit.

BackgroundNotwithstanding guidance from the European Cystic Fibrosis (CF) Society (ECFS) neonatal screening (NBS) working group, significant variation persists in the evaluation and management of Cystic Fibrosis Screen Positive, Inconclusive Diagnosis (CFSPID) subjects, leaving many aspects of care under debate. This study reports the results of a national survey investigating management and treatment approaches of pre-school CFSPIDs in Italy.MethodsIn February 2024, a comprehensive questionnaire was distributed to all Italian CF centers. The survey explored various aspects of CFSPID management in the year 2023, including patient visit schedules, sweat tests (ST) timing, screening procedures, therapeutic interventions, and discharge criteria. Data on regional NBS protocols, number of CFSPID cases, and CF:CFSPID ratio were also collected.ResultsBy December 31, 2023, CF Italian centers were following 522 CFSPIDs. In 2023, CF NBS identified 85 CF and 68 CFSPID cases, resulting in a CF:CFSPID ratio of 1.25:1. Seven centers diagnosed more CFSPID than CF, with the lowest CF:CFSPID ratio being 0.20:1. A quarter of all centers reported management plans that deviated widely from ECFS guidelines. Respiratory cultures were performed in 16 (69.6%) centers in the absence of symptoms. Nine (38.9%) prescribed antibiotics in any case of positive Pseudomonas aeruginosa cultures, including first detections and asymptomatic subjects. Spirometries were performed by 14/23 centers (60.9%) in procedure-competent children at each visit. Follow up care continued after age 6 for all CFSPIDs in 15 (65.2%) centers regardless of age, genotype or ST results. A diagnosis of CF was established based on repeated pathological STs and/or multiorgan involvement. Children with STs in intermediate range and mono-organ involvement were classified as CFTR-related disorders (CFTR-RD).ConclusionsDespite available data on clinical course and recommendations on management of CFSPIDs, different approaches persist in clinical practice. Further efforts should be considered to disseminate and encourage adherence to international guidelines.

Rationale:Respiratory status of people with Cystic Fibrosis (pwCF) carrying N1303K is improved by Elexacaftor/Tezacaftor/Ivacaftor (ETI) but, contrary to other mutations, the impact on sweat test results is limited. Methods:To explore this discrepancy, we implemented new sweat gland and respiratory cell lines stably expressing Wild type (WT)-, F508del- and N1303K-CFTR. CFTR dependent chloride (Cl-) and bicarbonate (HCO3-) transport was measured by short circuit current in these new models and in primary Human Nasal Epithelial Cells (HNECs). CFTR expression was evaluated by Western blot. Results:In the airway and the sweat gland cells expressing F508del-CFTR, ETI induced maturation of CFTR and increased Cl- transport. In the respiratory cell lines and HNECs, N1303K-CFTR generated both immature and mature forms of CFTR. Correction by ETI increased CFTR amounts without promoting its maturation and improved Cl- secretion. N1303K-CFTR channel activity was markedly increased by co-potentiation of IVA with Apigenin. In the sweat gland, N1303K-CFTR was expressed as a globally misfolded protein, non-rescuable by ETI. API treatment to 2 patients improved FEV1 without lowering sweat Cl- content. Conclusion:N1303K-CFTR shows tissue specific correction and suboptimal response to ETI which can be improved by API.

Objective The objective of this study was to describe reported adverse events (AEs) associated with elexacaftor/tezacaftor/ivacaftor (ETI) in a pediatric sample with cystic fibrosis (CF) aged 6-18 years, with at least one F508del variant, followed at multiple Italian CF centers. Study design This was a retrospective, multicenter, observational study. All children receiving ETI therapy from October 2019 to December 2023 were included. We assessed the prevalence and type of any reported potential drug-related AEs, regardless of discontinuation necessity. Persistent AEs were defined as those continuing at the end of the observation period. Results Among 608 patients on ETI, 109 (17.9%) reported at least 1 AE. The majority (n = 85, 77.9%) were temporary, with a median duration of 11 days (range 1-441 days). Only 7 (1.1%) patients permanently discontinued treatment, suggesting good overall safety of ETI. The most common AEs leading to discontinuation were transaminase elevations (temporary 14.1%, persistent 25.9%) and urticaria (temporary 41.2%, persistent 7.4%). Creatinine phosphokinase elevation was uncommon. No significant differences in AEs were observed based on sex, age groups (6-11 vs 12-18 years), or genotype. Pre-existing CF-related liver disease was associated with an increased risk of transaminase elevations. We identified significant variability in the percentage of reported AEs (ANOVA P value .026). Conclusions This real-world study highlights significant variability in reported AEs. Our findings suggest that ETI is a safe and well-tolerated therapy in children and adolescents with CF. However, further long-term safety and effectiveness investigations are warranted.

Introduction N1303K is the fourth most frequent Cystic Fibrosis (CF) causing mutation. People with CF (pwCF) clinical status can be improved by Elexacaftor(ELX)/Tezacaftor(TEZ)/Ivacaftor (ETI) combotherapy. We investigated the mechanism underlying N1303K-CFTR rescue.Methods N1303K-CFTR expression and maturation was evaluated by Western Blot in cell lines and Human Nasal Epithelial Primary Cells (HNECs). Cell surface expression was studied by nanoluciferase complementation assay and TurboID proximity labeling. Functional rescue was tested in vitro by YFP-Based Assay and Short Circuit Current.Results Correction by ELX/TEZ increases N1303K-CFTR amounts, but not its maturation in CFTR-expressing HEK and 16HBEge cell lines and in HNECs. In control conditions, N1303K-CFTR is more distributed at the cell surface and significantly more surface partners are identified in the N1303K-CFTR interactome as compared to F508del-CFTR in HEK cells. ELX/TEZ induces a global stabilization of N1303K-CFTR without favoring its plasma membrane relocation in contrast to F508del-CFTR which is redistributed to the membrane. ETI increases N1303K-CFTR activity in HNECs and can be increased by API co-potentiation with a predicted increase in Forced Expiratory Volume in 1 second (ppFEV1) by respectively 13([2][1])% and 18%([3][2]). This is consistent with a gain in ppFEV1 reported in pwCF carrying the N1303K mutation and additional improvement by API in a patient.Conclusion These results support the expansion of ETI approval to N1303K mutation but highlight different mechanisms of action than for F508del.### Competing Interest StatementStefano Pantano declares Vertex pharmaceuticals support in sample collection without financial contribution. Stefano Costa declares payment or honoraria for speakers bureaus from Vertex pharmaceuticals. Sonia Volpi declares payment of honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events from Vertex pharmaceuticals and DMF Pharma FoodAR and support for attending meetings and/or travel from Chiesi. Stephanie Bui declares participation in the protocoles of Vertex pharmaceuticals studies as principal investigator. Clemence Martin reports payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events from Chiesi, Astra Zeneca, Boehringer Ingelheim, GSK. Support for attending meetings and/or travel from Chiesi, Boehringer Ingelheim. Nicoletta Pedemonte declares payment or honoraria from Vertex Pharmaceuticals for lectures, presentations, speakers bureaus, manuscript writing or educational events - Speaker for a lecture at the 45th European Cystic Fibrosis Society Conference, Rotterdam, June 2022. Pierre Regis Burgel reports grants from Vertex pharmaceuticals, GSK, outside the submitted work. Luis J.V. Galietta declares Patents planned, issued or pending. Compounds described are not present in the submitted paper. Isabelle Sermet-Gaudelus reports support for the present manuscript from Vaincre La Mucoviscidose and Mucoviscidose ABCF2. Isabelle Sermet-Gaudelus also reports, outside the submitted work, grants from Agence Nationale pour la Recherche, Assistance Publique Hopitaux de Paris, Vertex Innovation Award; consulting fees and travel support from Vertex therapeutics. [1]: #ref-2 [2]: #ref-3

Introduction: Evidence is currently lacking to guide the management of cystic fibrosis (CF) trans -membrane conductance regulator-related metabolic syndrome CF screen-positive inconclusive diagnosis (CRMS/CFSPID) with Pseudomonas aeruginosa (Pa)-positive respiratory culture. This study assessed the clinical data, management, and outcomes of an Italian cohort of CRMS/CFSPID infants with Pa isolated from their airways.Methods: Data of Pa-positive CRMS/CFSPID infants born between January 2011 and August 2018 and followed at five CF Italian centres were retrospectively extracted. Further data were collected until June 2021 to assess outcomes, prevalence of subjects treated with antimicrobials, and treatment type and duration.Results: Forty-three asymptomatic CRMS/CFSPID patients (median age on 30 June 2021, 82 months; in-terquartile range [IQR], 63-98 months) with at least one positive airway culture for non-mucoid Pa (me-dian age at first isolation, 18.7 months; IQR, 7-25 months) were enrolled. Of them, 24 (55.8%) underwent anti-Pa therapy. Pa clearance occurred in 22 (91.6%) of 24 patients versus spontaneous clearance in 16 of 19 (84.2%) untreated patients (chi-square, 0.5737; p = 0.44878). After a median follow-up of 6.2 years (IQR, 3.0-9.9), 7 (16.3%) were diagnosed with CF after a pathological sweat test (median age, 43 months; IQR, 28-77 months), 3 (7%) developed recurrent pancreatitis or isolated bronchiectasis consistent with CFTR-related disorder, and the CRMS/CFSPID classification remained in 33 (76.7%).Conclusions: Pa detection frequently occurs in asymptomatic infants with CRMS/CFSPID but tends to clear spontaneously. More studies are needed to determine if Pa isolation can predict evolution.(c) 2022 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Elexacaftor-tezacaftor-ivacaftor (ETI), a triple CFTR modulator combination, has proven to be highly effective in patients with cystic fibrosis (CF) carrying at least one Phe508del mutation. Since October 2022 triple therapy has been approved in Italy for 6 to 11-year-old children with this genotype. We report preliminary data on efficacy and safety of ETI in that patient population after one month of treatment.

The identification of cystic fibrosis screening-positive, inconclusive diagnosis (CFSPID) in infants is a controversial outcome of newborn screening for cystic fibrosis (CF). Today, despite improvements in the knowledge of CFSPID and the description of several cohorts, little data are available on cohorts with a follow-up period of more than 6 years. In this study, we report the outcomes of an Italian cohort of CFSPID individuals with CFSPID or formerly CFTR-related disorders (CFTR-RD) (CFSPID > CFTR-RD) or diagnosed with CF (CFSPID > CF). This was an observational and multicentre Italian study collecting clinical data on CFSPID born between the period January 1, 2011, and December 13, 2019. A total of 268 participants were included: 243 with persistent CFSPID, 7 with CFSPID > CFTR-RD, and 18 with CFSPID > CF. The trend of sweat chloride (SC) values, percentage of definitive diagnoses, lung function in school-aged children, and development of CF-related complications were evaluated. At the end of the observation period, almost 80

Objectives: A high number of CFSPID infants has been reported in Italy with a 0.64:1 CF:CFSPID rate. Our previous data showed that 5.3% of infants progressed to CF. Clinical data and outcomes of CFSPID after a longer follow up are missing. Aim of the study is to evaluate outcomes (CFSPID, CFTR-RD or CF) in a large series of subjects with a long follow up. Methods: We collected clinical data, trend of the sweat test (ST), definitive diagnoses of CFSPID subjects, from 5 CF centers. Results: Two hundred sixty-eight subjects born from 01/2011 to 12/2019 were enrolled in our study: after a follow-up of 1.8 y (range 1–7.2 y) 243 children remained CFSPID, 7 progressed to CFTR-RD and 18 to CF. At the end of the present study (243 CFSPID subjects), after a mean followup period of 4.9 y (range 1.5–10.5), 30/243 (12.3%) received a definitive diagnosis: 10 progressed to CF, 9 progressed to CFTR-RD, 10 were healthy carriers, and one a healthy subject. CFSPID progressed to CF due to pathological ST (7), or for related CF symptoms (3). CFTR-RD was diagnosed in 9 subjects with a mono-organ involvement. Subjects who progressed to CFTR-RD or CF, underwent microbiological or radiological examinations more frequently than subjects who remained CFSPID. Spirometry was available in 67 subjects, showing ppFEV1 value in the normal range in all. Thirty (11.1%) subjects had at least one hospitalization for CF related symptoms, 5 (16.7%) were CFTR-RD. During the follow up, two out of 7 subjects previously labeled as CFTR-RD progressed to CF due to multi-organ symptoms. At the end of the longer follow up, in 28/243 (11.5%) CF was confirmed, 16/243 (6.6%) received a CFTR-RD label, while 199/243 (81.9%) previously classified as CFSPID remained with an inconclusive diagnosis. Conclusions: In our population only a minority of CFSPID progressed to CF after a longer follow-up, rarely in presence of symptoms and with a normal lung function in school age.

There is limited information available on the clinical data, sweat test trends, and outcomes of individuals with cystic fibrosis (CF) who present with an isolated episode of hypoelectrolytemia with metabolic alkalosis (HMA). This study describes a cohort of Italian individuals with HMA as presenting symptom. The study is a retrospective multicenter analysis of individuals who presented with HMA as an initial symptom and was followed at 8 Italian CF Centers, from March 1988 to March 2022. Demographic, clinical, microbiological, biochemical, and genetic data were extracted from local health records. Ninety-three individuals were enrolled in the study. At first evaluation, 82 (88.2

In conclusion, our data suggest that subjects with genotype 5T;TG12/VVCC likely have a very low risk of progressing to CF, as compared to those with F508del/5T;TG12. This observation could lead to differentiate follow up in presence of at least one 5T;TG12. Knowing these data is crucial to offer a useful counseling for CRMS/CFSPID infants and for non‐CF adults with CBAVD alone. Anyway further data are needed to evaluate the outcomes after a longer follow up.

after initiation were used to determine the proportion of participants with one or more vitamin levels in the supratherapeutic or subtherapeutic range.Proportions were compared using the McNemar's chi-square test.A two-sided p < 0.05 was considered statistically significant, and all analyses were completed in R version 4.1.2.Results: Seventy-two participants met inclusion criteria for enrollment, and 54 (75%) had follow-up vitamin levels after ELX/TEZ/IVA was started.Median age at ELX/TEZ/IVA initiation was 15.9 (95% CI, 13.5-17.8),61% were homozygous for F508del, 54% were male, and 88% were Caucasian.Median baseline body mass index was 20.8 kg/m 2 (95% CI, 18.8-22.6),and median baseline percentage predicted forced expiratory volume in 1 second was 101% (95% CI, 93-110%).There were no significant differences in median vitamin A or D level or PT before and after ELX/TEZ/IVA initiation (Table 1).Median vitamin E level decreased from 8.8 mg/L to 6.7 mg/L ( p < 0.005).The percentage of participants with subtherapeutic vitamin D levels decreased from 80% to 63% afer ELX/TEZ/IVA initiation ( p = 0.007), and the percentage with high PT levels decreased from 20% to 7% ( p = 0.046).There was no significant difference in the proportion of participants with subtherapeutic vitamin A or E levels.There were no measured vitamin A, D, or E levels in the supratherapeutic range after ELX/TEZ/IVA initiation.Conclusions: Our results demonstrate a greater proportion of participants with vitamin D and PT within the normal range after ELX/TEZ/IVA initiation.By contrast, median vitamin E level decreased significantly, and there was no significant change in vitamin A level.No vitamin A, D, or E levels were in the supratherapeutic range after ELX/TEZ/IVA initiation.There are several possible explanations for our results.First, although it was assumed that participants continued vitamin supplementation after ELX/ TEZ/IVA initiation, we were unable to verify this.It is possible that, as participants' health improved on ELX/TEZ/IVA, they stopped taking vitamin supplements.We continue to investigate how vitamin intake has changed after ELX/TEZ/IVA initiation.Alternatively, greater exposure to sunlight or dietary changes could have contributed to our findings.Data collection and analysis at the second study site are ongoing and may help clarify these relationships.

BACKGROUND:In recent years, patients with cystic fibrosis (CF) conductance regulator (CFTR) variant poly(T) sequences have been increasingly reported with a wide spectrum of clinical severity. We describe the long-term clinical outcomes and progression to a CF diagnosis over time in a large Italian cohort of patients carrying the CFTR F508del/5T;TG12 genotype. METHODS:A retrospective analysis of subjects from 10 CF centres in Italy with the F508del/5T;TG12 genotype was performed. Demographic, clinical, microbiological, and biochemical data, as well as information about the follow-ups and complications of the enroled patients, were collected. RESULTS:A total of 129 subjects (54 females; median age: 15.0 years, range: 0-58 years; 59 older than 18 years) were included. In terms of initial diagnoses, 30 were CF (23.3%), 41 were CFTR-related disorder (CFTR-RD) (31.7%), and 58 were CF transmembrane conductance regulator-related metabolic syndrome/cystic fibrosis screen positive, inconclusive diagnosis (CRMS/CFSPID) (45.0%). After a median follow-up of 6.7 years (range 0.2-25 years), 15 patients progressed to CF, bringing the total number of CF diagnoses to 45/129 (34.9%). Most of these patients had mild lung diseases with pancreatic sufficiency and a low prevalence of CF-related complications. CONCLUSIONS:At the end of the study, 34.9% of subjects with the CFTR F508del/5T;TG12 genotype were diagnosed with CF. We suggest including patients with the F508del/5T;TG12 genotype in long-term follow-ups.