Personalized CFTR Modulator Therapy for G85E and N1303K Homozygous Patients with Cystic Fibrosis

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.


Introduction
Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene [1]. As a consequence, the CFTR protein, which acts as an ion channel in various epithelia in the human body, is substantially reduced in quantity and/or quality, leading to an impaired function of the chloride channel [2]. This results in thickened secretions of multiple organs, including the airways, ultimately leading to damage of the affected organs and loss of their function. For a long time, therapeutic strategies aimed to reduce symptoms of organ dysfunction. The emergence of highly effective CFTR modulator therapies has transformed the clinical landscape of CF care. Novel triple combination elexacaftor/tezacaftor/ivacaftor (ETI) therapy is now available for~90% of people with CF carrying at least one allele of the most common mutation F508del [3][4][5][6][7][8][9]. ETI therapy leads to substantial clinical benefits and improvement in CFTR function to 40 to 50% of normal CFTR activity measured by nasal potential difference (NPD), intestinal current measurement (ICM), lung clearance index (LCI) and lung morphology in real world observational trials in patients with

Case Presentations
The G85E/G85E patient is a 15-year-old female diagnosed with CF at the age of two years secondary to recurrent respiratory infections and a failure to thrive. SCC at that time was clearly pathologic with 77 mmol/L and a genetic analysis that demonstrated homozygosity of G85E in the CFTR gene. Symptomatic therapies were initiated according to the European Cystic Fibrosis Society guidelines, including bronchodilator and mucolytics inhalation with airway clearance, oral pancreatic enzyme replacement therapy, and fatsoluble vitamin supplementation. This led to an improvement in thriving, but the girl soon experienced a first and then recurrent episodes of allergic bronchopulmonary aspergillosis (ABPA). Therefore, she required extended symptomatic therapy including recurrent cycles of systemic and inhaled glucocorticosteroids during childhood and adolescence. Recurring ABPA episodes were treated with anti-IgE antibodies without clear response in clinical parameters. Under this treatment, the patient suffered mild-to-moderate obstructive lung disease at age 14 years and had already shown advanced structural CF lung disease in chest MRI (Table 1). Continuous systemic antifungal therapy led to a deceleration of lung function decline; however, the patient still reported daily cough with both phases of persistent dry cough and extensive sputum production. In addition, bronchial tightness necessitated the frequent use of bronchodilators. In January 2021, our patient experienced a new significant decline in lung function during a pulmonary exacerbation that was only partly restored following intravenous antibiotic treatment.
The N1303K/N1303K patient is a 19-year-old male who was also diagnosed at preschool age due to recurrent pulmonary infections and failure to thrive. Issues with adherence to symptomatic therapy during adolescence added to the clear progression over time, especially of CF lung disease, with ultimately polymicrobial infection with Methicillin resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa and Mycobacterium abscessus, among other pathogens. Repeated Pseudomonas-effective antibiotic therapy cycles did not lead to stabilization of lung disease (Table 1). Besides the impaired pulmonary function, the N1303K/N1303K patient also suffered from an advanced CF liver disease with pronounced structural changes, but preserved function. However, infection with nontuberculous mycobacteria (NTM) could pose a problem if liver transplantation is required in the future. Therefore, we decided to start an antimycobacterial therapy in April 2021, taking into account the other pathogens present. The initial five-drug systemic NTM therapy with tigecycline, amikacin, cefoxitin, meropenem and clofazimine, in addition to colistin p.i., had to be adjusted in the first months due to limited tolerability, but was then carried out as a quadruple therapy over 9 months, which led to stabilization of the patient. However, with side effects increasing over time and a recurrence of pulmonary exacerbation under therapy, we evaluated the treatment with ETI for this patient.

Preclinical Testing of ETI Therapy In Vitro
To test the potential benefit of ETI therapy in vitro, we generated highly differentiated airway epithelial cultures from nasal brushings of both patients and performed transepithelial short-circuit current measurements in Ussing chambers ( Figure 1). The basal short circuit current (I sc ), amiloride-sensitive I sc and amiloride-insensitive I sc were not significantly different between vehicle control and ETI treatment in cultures derived from the G85E/G85E patient, although amiloride-sensitive I sc showed a trend towards a decrease with ETI treatment (Figure 1C,E-G). ETI treatment significantly enhanced CFTRmediated chloride currents compared with vehicle-treated control, as indicated by forskolin peak response (p < 0.05) and CFTRinhibitor-172 inhibited current (p < 0.01) in G85E/G85E pHNECs ( Figure 1H,J). Compared with our previously published data from pHNECs from healthy people (mean CFTRinhibitor-172 sensitive current −10.1 µA/mm 2 , n = 31) [22], rescue of CFTR function by ETI treatment, as determined from CFTRinhibitor-172 sensitive current (−1.1 µA/mm 2 ), was 11.0% of the normal CFTR activity for pHNECs of the G85E/G85E patient. In pHNECs derived from the N1303K/N1303K patient, basal I sc and amiloride-insensitive I sc were unchanged, whereas amiloride-sensitive I sc was significantly reduced by ETI treatment compared with dimethyl sulfoxide (DMSO) control (p < 0.05), suggesting decreased activity of the epithelial sodium channel (ENaC) ( Figure 1D,K-M). Forskolin-stimulated I sc showed a trend towards an increase upon ETI treatment and acute ivacaftor response was significantly increased with ETI treatment compared with vehicle control ( Figure 1D,N,O). Furthermore, ETI treatment significantly increased CFTRinhibitor-172 sensitive current (−1.1 µA/mm 2 ) compared with DMSO control (−0.44 µA/mm 2 ) in pHNECs from the N1303K/N1303K patient (p < 0.05) corresponding to 11.0% of the normal CFTR activity ( Figure 1D,P). current (−1.1 µA/mm 2 ) compared with DMSO control (−0.44 µA/mm 2 ) in pHNECs from the N1303K/N1303K patient (p < 0.05) corresponding to 11.0% of the normal CFTR activity ( Figure 1D,P).

Improvements in CFTR Function In Vivo
Based on the in vitro evidence, ETI was started as an off-label therapy in both patients. To evaluate the in vivo effect of ETI therapy, we measured CFTR function in three different tissues using the established CFTR biomarkers SCC, NPD and ICM at baseline and after initiation of ETI therapy ( Figure 2) [11,23,24].

Improvements in CFTR Function In Vivo
Based on the in vitro evidence, ETI was started as an off-label therapy in both patients. To evaluate the in vivo effect of ETI therapy, we measured CFTR function in three different tissues using the established CFTR biomarkers SCC, NPD and ICM at baseline and after initiation of ETI therapy ( Figure 2) [11,23,24]. patients treated with elexacaftor/tezacaftor/ivacaftor (n = 44) [11].
Both patients had a pathological SCC above 60 mmol/L and showed no CFTR function in the respiratory or rectal epithelium at baseline ( Figure 2). In the G85E/G85E patient, SCC was reduced by 40 mmol/L after initiation of ETI therapy ( Figure 2A). In the NPD, the basal potential was improved from -34.6 mV at baseline to -24.1 mV after initiation of ETI and the amiloride response improved from 20.8 mV to 13.9 mV ( Figure 2B,C). Furthermore, NPD total chloride response improved from 0.0 mV to −6.9 mV corresponding to 32.4% of CFTR activity in the nasal epithelium of healthy people ( Figure 2D). In the ICM, we observed a cAMP-dependent chloride secretory response of 32.0 µA/cm 2 after initiation of ETI therapy in the G85E/G85E patient, corresponding to rescue of CFTR function in the rectal mucosa to a level of 37.6% of normal ( Figure 2E). The total chloride secretory response increased from 1.1 µA/cm 2 at baseline to 82.4 µA/cm 2 after initiation of ETI, corresponding to a level of 29.4% of normal CFTR activity ( Figure 2F). In the N1303K/N1303K patient, SCC was reduced by 31 mmol/L after initiation of ETI therapy (Figure 2A). In the NPD, the basal potential was improved from −39.7 mV to -10.6 mV and the amiloride response from 14.1 mV to 3.9 mV ( Figure 2B,C). NPD total chloride response improved from 0.8 mV to −1.2 mV, corresponding to 5.5% of normal CFTR activity in the nasal epithelium ( Figure 2D). In the Both patients had a pathological SCC above 60 mmol/L and showed no CFTR function in the respiratory or rectal epithelium at baseline (Figure 2). In the G85E/G85E patient, SCC was reduced by 40 mmol/L after initiation of ETI therapy (Figure 2A). In the NPD, the basal potential was improved from -34.6 mV at baseline to -24.1 mV after initiation of ETI and the amiloride response improved from 20.8 mV to 13.9 mV ( Figure 2B,C). Furthermore, NPD total chloride response improved from 0.0 mV to −6.9 mV corresponding to 32.4% of CFTR activity in the nasal epithelium of healthy people ( Figure 2D). In the ICM, we observed a cAMP-dependent chloride secretory response of 32.0 µA/cm 2 after initiation of ETI therapy in the G85E/G85E patient, corresponding to rescue of CFTR function in the rectal mucosa to a level of 37.6% of normal ( Figure 2E). The total chloride secretory response increased from 1.1 µA/cm 2 at baseline to 82.4 µA/cm 2 after initiation of ETI, corresponding to a level of 29.4% of normal CFTR activity ( Figure 2F). In the N1303K/N1303K patient, SCC was reduced by 31 mmol/L after initiation of ETI therapy (Figure 2A). In the NPD, the basal potential was improved from −39.7 mV to -10.6 mV and the amiloride response from 14.1 mV to 3.9 mV ( Figure 2B,C). NPD total chloride response improved from 0.8 mV to −1.2 mV, corresponding to 5.5% of normal CFTR activity in the nasal epithelium ( Figure 2D). In the ICM, we observed a cAMP-dependent chloride secretory response of 14.0 µA/cm 2 after initiation of ETI therapy in the N1303K/N1303K patient, corresponding to rescue of CFTR function in the rectal mucosa to a level of 16.5% of normal ( Figure 2E). The total chloride secretory response increased from 1.0 µA/cm 2 to 40.8 µA/cm 2 , corresponding to a level of 14.6% of normal CFTR activity ( Figure 2F).

Clinical Improvements following Treatment with ETI
The initiation of therapy with ETI resulted in prompt stabilization of the clinical status of both patients, with a particular improvement in FEV 1 % predicted and LCI ( Figures 3A,B and 4A,B). The G85E/G85E patient stayed in the normal range for BMI after initiation of ETI ( Figure 3C). However, this patient showed a reduction in total serum IgE despite stopping all ABPA-related therapies ( Figure 3D). Furthermore, we observed substantial improvements in mucus plugging and bronchial wall thickening in MRI images of the lung after initiation of ETI ( Figure 3E). ICM, we observed a cAMP-dependent chloride secretory response of 14.0 µA/cm 2 after initiation of ETI therapy in the N1303K/N1303K patient, corresponding to rescue of CFTR function in the rectal mucosa to a level of 16.5% of normal ( Figure 2E). The total chloride secretory response increased from 1.0 µA/cm 2 to 40.8 µA/cm 2 , corresponding to a level of 14.6% of normal CFTR activity ( Figure 2F).

Clinical Improvements following Treatment with ETI
The initiation of therapy with ETI resulted in prompt stabilization of the clinical status of both patients, with a particular improvement in FEV1 % predicted and LCI ( Figures 3A,B and 4A,B). The G85E/G85E patient stayed in the normal range for BMI after initiation of ETI ( Figure 3C). However, this patient showed a reduction in total serum IgE despite stopping all ABPA-related therapies ( Figure 3D). Furthermore, we observed substantial improvements in mucus plugging and bronchial wall thickening in MRI images of the lung after initiation of ETI ( Figure 3E).  and stabilization in weight into the normal BMI range appeared that was not achieved in previous years ( Figure 1C). For safety monitoring, our patients received ophthalmology exams and routine hepatic function monitoring. In this context, transaminase levels showed improvements after initiation of ETI. Until now, neither the G85E/G85E patient who had been treated with ETI for 50 months, nor the N1303K/N1303K patient who had been treated for 16 months have shown signs of side effects and have reported no other adverse reactions after initiation of ETI.

Discussion
This study provides a comprehensive assessment of the effects of ETI on CFTR function in pHNECs of a G85E homozygous patient and an N1303K homozygous patient in vitro as well as of the improvement of CFTR function in vivo after initiation of ETI in both patients. We were able to show that ETI increased CFTR-mediated currents in pHNECs of both patients ( Figure 1). As predicted by the preclinical results, the in vivo CFTR function of the G85E/G85E patient improved to 30-40% of normal CFTR activity in the respiratory and rectal epithelium after initiation of ETI. The N1303K/N1303K patient showed an improvement to 5-20% in the respiratory and rectal epithelium after initiation of ETI ( Figure 2). In both patients, partial restoration of CFTR function led to improvements in clinical outcomes after initiation of ETI (Figures 3 and 4).
The G85E mutation is classified as a class II missense mutation, resulting in decreased quantity and function of CFTR protein. In the US, therapy with ETI is approved for patients with CF carrying the G85E mutation based on in vitro data in Fischer rat thyroid cells. In Europe, ETI is not approved for patients carrying the G85E mutation (without F508del on the other allele) due to the lack of clinical studies investigating efficacy and safety of this therapy in vivo. It has been previously shown that ETI improves CFTR function in rectal organoids of a G85E heterozygous patient with a stop codon on the other allele [25]. We observed a consistent rescue of CFTR function by ETI in the pHNEC of the In the N1303K/N1303K patient, we were able to stop the NTM therapy after start of ETI without a decline in respiratory status ( Figure 4A,B). Furthermore, an improvement and stabilization in weight into the normal BMI range appeared that was not achieved in previous years ( Figure 1C). For safety monitoring, our patients received ophthalmology exams and routine hepatic function monitoring. In this context, transaminase levels showed improvements after initiation of ETI. Until now, neither the G85E/G85E patient who had been treated with ETI for 50 months, nor the N1303K/N1303K patient who had been treated for 16 months have shown signs of side effects and have reported no other adverse reactions after initiation of ETI.

Discussion
This study provides a comprehensive assessment of the effects of ETI on CFTR function in pHNECs of a G85E homozygous patient and an N1303K homozygous patient in vitro as well as of the improvement of CFTR function in vivo after initiation of ETI in both patients. We were able to show that ETI increased CFTR-mediated currents in pHNECs of both patients ( Figure 1). As predicted by the preclinical results, the in vivo CFTR function of the G85E/G85E patient improved to 30-40% of normal CFTR activity in the respiratory and rectal epithelium after initiation of ETI. The N1303K/N1303K patient showed an improvement to 5-20% in the respiratory and rectal epithelium after initiation of ETI ( Figure 2). In both patients, partial restoration of CFTR function led to improvements in clinical outcomes after initiation of ETI (Figures 3 and 4).
The G85E mutation is classified as a class II missense mutation, resulting in decreased quantity and function of CFTR protein. In the US, therapy with ETI is approved for patients with CF carrying the G85E mutation based on in vitro data in Fischer rat thyroid cells. In Europe, ETI is not approved for patients carrying the G85E mutation (without F508del on the other allele) due to the lack of clinical studies investigating efficacy and safety of this therapy in vivo. It has been previously shown that ETI improves CFTR function in rectal organoids of a G85E heterozygous patient with a stop codon on the other allele [25]. We observed a consistent rescue of CFTR function by ETI in the pHNEC of the G85E/G85E patient. Of note, acute addition of ivacaftor did not further potentiate cAMP-stimulated chloride secretion in the G85E/G85E cultures and even tended to reduce the transepithelial current, indicating that the potentiator ivacaftor may not be essential for optimizing the chloride channel function of this specific trafficking mutation. However, to confirm this observation, the acute and chronic effects of ivacaftor on the G85E mutation need to be assessed in a larger sample size in future studies. Furthermore, by additionally measuring in vivo CFTR biomarkers, our study shows that ETI improves CFTR function in the G85E homozygous patient to levels comparable to ETI in F508del homozygous patients [11]. Overall, these data suggest that ETI is beneficial in patients with a G85E mutation.
N1303K, the fourth most common CFTR mutation, is also classified as a class II missense mutation. Due to a lack of improvement above a threshold of 10% WT CFTR function after treatment with ETI in Fischer rat thyroid cells [26,27], ETI is not approved for treatment in patients carrying the N1303K mutation (without another responsive mutation on the other allele) in any country worldwide. Recent evidence suggests that, in addition to impaired CFTR trafficking, the N1303K mutation also causes a severe gating defect in CFTR [16]. Consistent with these data, our results in pHNEC suggest that potentiation by ivacaftor is an important mechanism in rescuing N1303K-CFTR function. This observation is supported by a study in N1303K/N1303K rectal organoids showing that potentiation by ivacaftor is important for reaching therapeutically relevant levels of organoid swelling after pre-treatment with elexacaftor and tezacaftor [25]. Recently, it has been suggested that patients with an N1303K mutation could clinically benefit from ETI therapy [13,19,21]. This is further supported by our study showing improvement of CFTR function in the N1303K/N1303K patient in the range of tezacaftor/ivacaftor in F508del homozygous patients [11]. However, in previous studies, N1303K heterozygous and homozygous patients treated with ETI showed substantial improvements in lung function despite only a modest decrease in SCC [13,19,21]. The large decrease in SCC in the N1303K/N1303K patient in our study may partially be explained by the homozygous genotype. However, larger studies with N1303K homozygous and heterozygous patients are needed to address the question of a gene dosage effect for the N1303K mutation. Nevertheless, the relatively large improvement in lung function compared with the relatively modest improvement in sweat chloride in previous studies may suggest tissue-specific expression and therapeutic rescue of N1303K, indicating that SCC might not be the ideal biomarker to assess the response to therapy on the functional level therapy in patients carrying the N1303K mutation. This question needs to be addressed in future clinical trials and the in vivo CFTR biomarker NPD and ICM may help to solve this conundrum regarding N1303K. Interestingly, we further observed reduced amiloride-sensitive currents in ETI treated pHNECs of the N1303K/N1303K patient, suggesting decreased activity of ENaC. This observation may be explained by the restoration of a previously described functional interaction between CFTR and ENaC at the plasma membrane, leading to downregulation of ENaC-mediated sodium/fluid absorption and thus improved airway surface hydration and mucociliary clearance [28][29][30][31]. This is consistent with observations in the NPD of this patient showing a substantially lower basal transepithelial potential difference and a reduced amiloride response on ETI. In previous studies, improvements in basal potential difference and amiloride response were only observed for ETI in patients with at least one F508del allele, but not for tezacaftor/ivacaftor or lumacaftor/ivacaftor in F508del homozygous patients [11,23]. In pHNECs, we observed residual currents after inhibition of ENaC and CFTR at the end of the experiment. These residual currents were previously observed in freshly excised native nasal epithelium [32,33] and may be explained by (i) non-CFTR mediated anion conductance, such as SLC26A9 or TMEM16A; (ii) amiloride-insensitive sodium conductance; or (iii) passive chloride movement due to the chloride gradient applied under these experimental conditions (145 mM basolateral to 5 mM apical) [34]. Taken together, these data suggest that different preclinical models should be investigated, the cut-off in Fischer rat thyroid cells may need to be adjusted and the combination with in vivo testing may help to establish in vivo efficacy in patients with uncommon CFTR mutations.
In pHNECs from the G85E/G85E and the N1303K/N1303K patient, we observed an improvement of in vitro CFTR function to about 10% of CFTR activity of healthy controls. However, in vivo CFTR function was improved to 30-40% of normal CFTR activity in the G85E/G85E patient and to 5-20% in the N1303K/N1303K patient. This finding shows that patient-derived models such as pHNECs enable detection of functional restoration of CFTR variants in vitro but cannot predict the degree of the in vivo response in an individual patient. This has also previously been observed for other preclinical models, such as intestinal organoids, where no correlation between in vitro organoid swelling and in vivo improvement in CFTR function was observed in F508del homozygous patients treated with lumacaftor/ivacaftor [35]. We therefore suggest that in vivo biomarkers provide a valuable tool to confirm findings in preclinical in vitro models in individual patients.
Our study has several limitations: First, the small sample size may limit generalizability of the data. In n-of-1 studies, the variability of clinical endpoints could affect or disguise potential therapeutic effects. The intraindividual variability of repeated short-term measurements has been previously reported at 6.3% for FEV 1 % predicted and 7.4% for LCI [36,37]. The G85E/G85E patient showed an improvement in lung function in repeated measurements after initiation of ETI with a mean improvement of +6.6% in FEV 1 % predicted and −2.1 (20.2%) in LCI when comparing the mean values before and after initiation of ETI. In the N1303K/N1303K patient, FEV 1 % predicted improved initially by +10.3% and LCI improved by −2.3 (15.7%). Although FEV 1 % predicted decreased and LCI increased again, both parameters were still improved compared with the repeated measurements before initiation of ETI. Previous studies in N1303K patients have reported higher increases in FEV 1 % predicted after initiation of ETI treatment than observed in our study [13,19,21]. Clinical trials and real-world studies assessing the effects of ETI in patients with at least one F508del mutation have also observed a high heterogeneity in the response in FEV 1 % predicted, which may be explained by the fact that FEV 1 is influenced by numerous factors independent of CFTR function, such as fixed airflow limitation due to irreversible structural lung damage or adherence to concomitant therapies. We used multiple repeated clinical outcome measures and independent measurements of the in vitro and in vivo CFTR function to determine the response to ETI in both patients, suggesting that the improvement in lung function is related to the therapy rather than variability of the measurements. In addition, other recent reports support that ETI shows clinical benefit in patients with a G85E and an N1303K mutation [13,19,21,38]. Second, pHNECs have not been validated in large patient cohorts receiving CFTR modulator therapy and it is unknown if a lack of response in pHNECs predicts a lack of clinical response. Third, for the in vitro experiments, we used a concentration of tezacaftor that was previously used in the pre-clinical development of the triple combination therapy [39], although it is unknown if these concentrations are reached in patients. Finally, the elexacaftor used in the in vitro experiments is a racemic mixture of two enantiomers which may have different effects than those of ETI in patients and therefore potentially underestimate the in vitro effects [18,40].
In summary, our results show an improvement of CFTR function and clinical benefit in patients homozygous for G85E or N1303K after initiation of ETI therapy. Furthermore, these results support that patient-derived pHNECs may have the potential to indicate clinical response of rare CFTR mutations to ETI therapy. We suggest that the sequential use of in vitro testing of CFTR function in pHNECs and subsequent in vivo testing using biomarkers of CFTR function, in combination with clinical outcome measures, is a promising approach to facilitate access to CFTR modulator therapy and to enhance precision medicine for patients with CF carrying rare CFTR mutations.

Study Design and Participants
Patients participated in an ongoing multicenter longitudinal study on the effects of CFTR modulators (ClinicalTrials.gov Identifier: NCT04732910). The study was approved by the ethics committee of the Charité-Universitätsmedizin Berlin (EA2/220/18). Written informed consent was obtained from both patients included in the study and the parents of the G85E/G85E patient. Patients provided nasal brushes for preclinical testing in primary nasal epithelial cell cultures. After proof of a significant preclinical response, the patients were treated with the standard dose of ELX 200 mg and TEZ 100 mg every 24 h in combination with IVA 150 mg every 12 h. Anthropometrics, spirometry, multiple-breath washout, SCC, NPD and ICM were assessed at baseline and after initiation of ETI. In the G85E/G85E patient, total IgE levels and lung MRI were additionally performed at baseline and after initiation of ETI.

Nasal Epithelial Cell Cultures
Primary human nasal epithelial cells were cultured as previously described [22,41]. Briefly, cells were obtained by nasal brushing and expanded in co-culture with irradiated mouse 3T3 fibroblasts in the presence of RhoA kinase inhibitor Y-27632 (Stemcell Technologies, Cologne, Germany). Epithelial cells were seeded at passage 2 at a density of 200,000 cells/cm 2 on human placental type IV collagen-coated Snapwell supports (Corning, Glendale, NY, USA) in UNC-ALI medium and differentiated at the air-liquid interface (ALI) for at least 3 weeks.

Lung Function and Multiple-Breath Washout (MBW) Measurements
Spirometry was performed according to the standards endorsed by the American Thoracic Society and European Respiratory Society and values were expressed as percent predicted according to published normative data [43,44]. MBW testing was performed with the Exhalyzer D system (Eco Medics, Duernten, Switzerland) and 100% oxygen was used to wash out resident nitrogen from the lungs with a mouthpiece as interface [45,46]. All measurements were evaluated using spiroware 3.3.1 (Eco Medics, Duernten, Switzerland) [10,47].

Morpho-Functional Chest Magnetic Resonance Imaging (MRI)
A standardized MRI protocol was used on a 1.5 Tesla MRI scanner (Magnetom Avanto, Magnetom Aera; Siemens Healthineers, Erlangen, Germany) at baseline and after initiation of therapy to acquire T1-and T2-weighted sequences. Lung perfusion was assessed by contrast-enhanced imaging [48]. Images were assessed for abnormalities in lung morphology and perfusion using a dedicated morpho-functional MRI score as previously described [49]. In this MRI score, structural findings are assessed by the MRI morphology score that comprises subscores for (i) bronchial wall abnormalities (wall thickening and/or bronchiectasis), (ii) mucus plugging, (iii) abscesses and/or sacculations, (iv) consolidations, and (v) special findings such as pleural effusion. The extent of these structural findings, as well as of changes in lung perfusion, are rated in each lobe (right upper lobe, right middle lobe, right lower lobe, left upper lobe, lingula, left lower lobe) as 0 (no abnormality), 1 (<50% of the lobe involved), or 2 (≥50% of the lobe involved) resulting in a range of the MRI global score from 0 (normal) to 72 (severely abnormal) with each score of ≥1 being rated as abnormal [48,49].

Sweat Chloride
Sweat tests were performed according to the German national diagnostic guideline [50] and the guidelines of the Clinical and Laboratory Standards Institute [51]. Sweating was stimulated by pilocarpine iontophoresis and samples were collected with the Macroduct ® system (Model 3700, Wescor, Logan, UT, USA). SCC was measured in a minimum volume of 30 µL using a chloridometer (KWM 20 Chloridometer, Kreienbaum, Langenfeld, Germany).

NPD Measurements
NPD measurements were performed as previously described [23,52,53]. The percentage of normal CFTR activity in the nasal epithelium was defined as the ratio of the total chloride conductance (TCC) of the individual CF patient to the median TCC in age-matched non-CF controls as previously described [23].

Intestinal Current Measurements
ICM was performed as previously described [11,23,24,54]. In brief, biopsies of the rectal mucosa were collected by endoscopic forceps biopsy, immediately stored in tissue medium (medium 199 containing Hank's salts, L-glutamine and 25 mM HEPES complemented with 5 mM glycine and 0.5 mM sodium-DL-β-hydroxybutyrate or RPMI-1640 medium with L-glutamine and sodium bicarbonate) and mounted in custom-made perfused micro-Ussing chambers. The luminal and basolateral compartments were perfused continuously with a buffer solution of the following composition: 145 mM NaCl, 0.4 mM KH 2 PO 4 , 1.6 mM K 2 HPO 4 , 5 mM D-glucose, 1 mM MgCl 2 , 1.3 mM Ca-gluconate, pH 7.4, at 37 • C. All reagents were obtained from Sigma-Aldrich at the highest level of available purity. Experiments were performed under open circuit conditions. Transepithelial voltage (V te ) was recorded and transepithelial resistance (R te ) was determined by applying intermittent (1 s) current pulses (∆I = 0.5 µA). The equivalent short-circuit current (I sc ) was determined from continuous V te and R te recordings according to Ohm's law (I sc = V te /R te ) after appropriate correction for fluid resistance [34,[55][56][57]. The percentage of normal CFTR function for each CF patient was calculated by dividing the individual cAMP-induced Isc of the patient by the median cAMP-induced Isc of age-matched non-CF controls as previously described [11,23,24].

Statistical Analysis
Data were analyzed using GraphPad Prism version 9.4.1 (GraphPad Software, San Diego, CA, USA) and R 3.6.2 [58]. Data were normally distributed and are presented as mean and standard deviation. Differences between the ETI and vehicle group were tested by Student's t-test, p < 0.05 was accepted to indicate statistical significance.  Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The data that support the findings of this study are available on request from the corresponding author M.S. The data are not publicly available due to legal restrictions, e.g., they contain information that could compromise the privacy of research participants.