Elexacaftor/Tezacaftor/Ivacaftor Improved Clinical Outcomes in a Patient with N1303K-CFTR Based on In Vitro Experimental Evidence

To the Editor:

The new generation of CFTR (cystic fibrosis transmembrane conductance regulator) protein modulator, the triple combination consisting of elexacaftor/tezacaftor/ivacaftor (ETI), has been recognized as highly effective therapy for patients 12 years or older with cystic fibrosis (CF) and at least one F508del-CFTR allele. Recently, ETI has also shown clinically meaningful benefits both in patients with CF with advanced lung disease (baseline %FEV₁ < 40) (1–3) and in children 6 through 11 years with CF and at least one F508del allele (4). Similar results from different institutions have also been reported in conference abstracts (5–7). However, it is unclear if ETI is therapeutically beneficial to patients with CF with N1303K, another class II CFTR mutant. Using the whole-cell patch-clamp recording and biochemical approaches, we demonstrated that ETI increased maturation and function of N1303K-CFTR. After in vitro confirmation, we investigated if ETI would be beneficial to a patient with CF with two non-F508del-CFTR mutations, N1303K and E193X, and our findings are reported below.

Methods

To monitor CFTR function, HEK293 cells were transiently transfected with indicated CFTR variants. Cotransfection of GFP-expression plasmid was performed for positive selection. Cells were treated with 2 μM VX445/VX661 (elexacaftor/tezacaftor, MedChemExpress) for about 24 hours after overnight transfection. Whole-cell patch-clamp recordings were performed using an Axopatch-200B amplifier connected to Axon DigiData 1550B (Molecular Devices). Patch pipettes were prepared using a micropipette puller (P-1000; Sutter Instrument). To simultaneously obtain current traces at −60 mV and I/V curves of CFTR, whole-cell currents were consecutively recorded with a 1 s voltage ramp of ±100 mV applied every 10 s: hold at Vm (membrane voltage) = −60 mV and filtered at 1 kHz and sampled at 50 Hz. The pipette solution containing VX770 (ivacaftor, 2 μM, SelleckChem) was composed of (in mM) 116 N-methyl-D-glucamine chloride (NMDG-Cl⁻), 30 aspartic acid, 1 MgCl₂, 5 ethyleneglycol-bis-(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, 2.9 CaCl₂, 10 N-2-hydroxyethylpiperazine-N′-ethane sulfonic acid, and 3 MgATP, pH 7.4. Bath solution was composed of (in mM): 146 NMDG-Cl⁻, 1 CaCl₂, 1 MgCl₂, 10 glucose, and 10 N-2-hydroxyethylpiperazine-N′-ethane sulfonic acid, pH 7.4.

To determine the maturation status of CFTR, Western blot was performed. Briefly, HEK293 or CFBE parental cells were transfected with CFTR variants as indicated and treated as above. CFTR and Vinculin were probed using cell lysates.

Results

As shown in Figure 1, G551D-CFTR had immediate response to VX770 as previously reported (8), validating our in vitro experimental settings. N1303K-CFTR itself or treated with VX770 only showed little function, which is consistent with previous observations (9). In contrast, treatment with ETI enhanced N1303K-CFTR function to nearly 40% of the wild-type level, whereas F508del-CFTR function was almost fully restored. This functional improvement suggested that ETI may benefit patients with CF with N1303K with a less robust effect when compared with F508del-CFTR. Western blot data using both HEK293 and CFBE cells showed that VX445/VX661 significantly increased N1303K-CFTR expression levels, for both mature (band C) and immature (band B) CFTR (Figure 1D), which is consistent with previous reports (10, 11).

Figure 1.

N1303K-CFTR expression and function was rescued by VX445/VX661/VX770 (elexacaftor/tezacaftor/ivacaftor [ETI]). (A) Representation of individual recording of whole-cell patch-clamp using HEK293 cells transfected with WT-, G551D-, F508del (ΔF508)-, and N1303K-CFTR plus indicated treatments. CFTR function was activated by 20 μM FSK and inhibited by 20 μM CFTR_inh-172. (B) Representative I/V curves for indicated mutant plus treatment. (C) Summary bar graph for A and B. Each symbol represents one recording for one cell. (D) Western blot of CFTR variances transiently expressed in HEK293 (left) or CFBE (right) cells. Cells were transfected with indicated CFTR variances overnight and then treated with VX445/VX661 or DMSO for 24 hours. CFTR and vinculin were probed using anti-CFTR (596, CF Foundation) and antivinculin (Cell Signaling Technology) antibodies, respectively. Band C (mature form) and band B (immature form) were labeled. **P < 0.005 and ***P < 0.001. CFBE = cystic fibrosis bronchial epithelial cells; CFTR = cystic fibrosis transmembrane conductance regulator; CFTR_inh-172 = CFTR inhibitor-172; FSK = forskolin; IVA = ivacaftor; Par = parental cells; WT = wild-type.

Based on the above laboratory research data and after insurance approval, ETI was initiated in an 11-year-old patient with CF with N1303K and E193X in September 2020, about 9 months before the U.S. Food and Drug Administration approval of ETI for children 6 years and older. At baseline, the patient had a history of chronic cough with %FEV₁ of 80–87%, recurrent sinusitis that warranted multiple sinus surgeries, exocrine pancreatic insufficiency, and low body mass index (BMI) (16 kg/m²). The patient’s resilience against common viral infections was poor, with viral infections often progressing to acute CF exacerbations that needed enteral or intravenous antibiotics. Within a month of treatment with ETI, the patient reported significant improvement in cough and sinus symptoms, along with increased appetite and energy. Over the past 10 months, the patient has remained asymptomatic from a pulmonary, nutritional, and sinus perspective, with significant improvement in %FEV_1, chest and sinus imaging findings, and weight. Her %FEV₁ increased for the very first time to 108%, and BMI increased from the 34th to 45th percentile. She is also tolerating wean and discontinuation of routine CF medications and aerosols per CF center protocol. Although significant clinical benefit has been noted, she remains pancreatic insufficient and her sweat chloride (SwCl) levels are only marginally lowered (Figure 2). The patient is being monitored closely to ensure the sustenance of clinical benefit and safety.

Figure 2.

Clinical symptoms, pulmonary and nutritional parameters of patient with CF with N1303K/E193X-CFTR mutations before and after elexacaftor/tezacaftor/ivacaftor (ETI) treatment. (A) Top: A CT chest image taken in 2018 showed that mucus plugging and scattered ground-glass nodules had increased in the bilateral lower lobes and medial right middle lobe along with mild bronchial wall thickening and bronchiectasis. Bottom: A CT chest image taken in June 2021 showed the significant interval improvement compared with the top. Also, minimal areas of central bronchial dilatation and bronchial wall thickening were observed. No evidence for mucous plugging or focal airspace disease. (B) Top: Sinus CT imaging taken in July 2020 showed pansinus mucosal thickening and poorly pneumatized sinuses with inspissated secretions. Bottom: Sinus CT imaging taken in April 2021 showed moderate improvement in sinus mucosal thickening of bilateral maxillary and sphenoid sinuses and ethmoid air cells with partial opacified ethmoid air cells. Bilateral maxillary sinuses appear contiguous with nasal cavity. Mastoid air cells clear. (C) Summary of the patient symptoms before and after elexacaftor/tezacaftor/ivacaftor treatment. %FEF_25–75 = percent predicted forced expiratory flow between 25% and 75%; B/L = bilateral; BMI = body mass index; CF = cystic fibrosis; CFTR = cystic fibrosis transmembrane conductance regulator; CT = computed tomography; CXR = chest X-ray; GI = gastrointestinal; s/p = status post.

Discussion

N1303K-CFTR is one of the common class II CFTR mutations, accounting for 2.3% patients with CF (CF Foundation Patient Registry 2018 Annual Data Report). Consistent with its severe trafficking and gating defects, patients with N1303K are associated with severe CF symptoms (12) (Figure 2). Because it is unresponsive to single CFTR modulator, previous studies suggested a combination of corrector and potentiator to treat patients with CF with N1303K (9, 10). Indeed, two independent groups have recently demonstrated that ETI could increase N1303K-CFTR functional expression in primary CF patient cells (10, 11). However, no clinical data using this strategy has been reported.

Our study showed that treatment with ETI partially restored N1303K-CFTR expression and increased its function to nearly 40% of the wild-type level (Figure 1). Importantly, we have demonstrated clinical benefit of ETI in a child with N1303K (Figure 2), although the clinical response is slow and modest. It should be pointed out that the SwCl levels are only marginally reduced (-13 mmol/l) which recapitulates the clinical effect of lumacaftor/ivacaftor or tezacaftor/ivacaftor on F508del homozygous patients with CF with SwCl reduction of -17.1 or -9.9 mmol/l, respectively (13, 14), suggesting the correlation of SwCl levels with rescued CFTR levels. Possibly, our patient’s second CFTR allele, E193X, and other genetic and nongenetic factors may contribute to this slow improvement as well.

As with other case reports, the singularity of our patient report is limited by inability to generalize and establish definite evidence of benefit in other patients with similar mutations. However, reports from other groups using primary cells (10, 11) support that ETI is generally beneficial to patients with CF with N1303K.

We conclude that ETI could be a relatively effective treatment option to alleviate CF symptoms in patients with N1303K-CFTR while sustenance of clinical benefit and safety needs close monitoring.