Therapeutic benefit observed with the CFTR potentiator, ivacaftor, in a CF patient homozygous for the W1282X CFTR nonsense mutation

Abstract

Premature termination codons (PTCs) in cystic fibrosis transmembrane conductance regulator (CFTR) gene result in nonfunctional CFTR protein and are the proximate cause of ~11% of CF causing alleles. Aminoglycosides and other novel agents are known to induce translational readthrough of PTCs, a potential therapeutic approach. Among PTCs, W1282X CFTR is unique, as it is a C-terminal CFTR mutation that can exhibit partial activity, even in the truncated state. The potentiator ivacaftor (VX-770) is approved for treating CF patients with G551D and other gating mutations. Based on previous studies demonstrating beneficial effect of ivacaftor for PTC mutations following readthrough in vitro, we hypothesized that ivacaftor may enhance CFTR activity in CF patients expressing W1282X CFTR, and could be further enhanced by readthrough. Ivacaftor significantly increased CFTR activity in W1282X expressing cells compared to R1162X CFTR cells, and was further enhanced by readthrough with the aminoglycoside G418. Primary nasal epithelial cells from a W1282X homozygous patient showed improved CFTR function in the presence of ivacaftor. Upon ivacaftor administration to the same patient, there was significant improvement in pulmonary exacerbation frequency, BMI and, insulin requirement, whereas FEV₁ remained stable over 3 years. These studies suggest that ivacaftor may have moderate clinical benefit in patients with preserved expression of the W1282X CFTR mutation by stimulating residual activity of the truncated protein, suggesting the need for further studies including the addition of efficacious readthrough agents.

Patients with cystic fibrosis (CF) continue to have limited life expectancy and high morbidity. Their illness results from defective transepithelial transport of chloride and bicarbonate ions as a consequence of mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR).^1,2 Nonsense mutations that truncate CFTR protein by introducing a premature termination codon (PTC) into CFTR account for disease in ~11% of CF patients, and are generally associated with a particularly severe disease phenotype due to the absence of residual function. W1282X CFTR and other C-terminal CFTR mutants are unique among PTCs because they retain partial chloride channel function, even in their truncated form, due to partial preservation of CFTR activity once adequate surface expression is achieved.^3,4 Several clinical studies in patients with CFTR nonsense mutations have indicated moderate benefit of aminoglycoside antibiotics,^5-8 which induce translational readthrough of PTCs via near-cognate amino acid insertion, improving production of full-length protein and surface expression.^9,10 However, available aminoglycosides are not suitable for long-term administration. Other studies to develop novel molecules for treating patients with W1282X and other PTC mutations remain ongoing.

New therapeutic strategies in CF have focused on directly modulating the function of mutant CFTR. One such CFTR modulator, ivacaftor (formerly, VX-770), is a CFTR potentiator that acts to increase channel opening and, thus, chloride transport at the epithelial cell surface.¹¹ Ivacaftor treatment of CF patients with the G551D gating mutation demonstrated improved FEV₁, decreased exacerbations, and increased weight gain in both randomized studies^12,13 and post-approval observational cohorts.^14,15 Ivacaftor also has been demonstrated to confer benefit in patients with similar gating mutations (e.g., G1244E, G1349D, G178R, G551S, S1251N, S1255P, S549N, S549R),¹⁶ and in CF patients with R117H CFTR, which exhibits gating and conductance abnormalities.¹⁷ Further, ivacaftor increases chloride transport in vitro for multiple missense mutations responsible for a wide range of disease severity when adequate cell surface expression is preserved.¹⁸ Since truncated W1282X CFTR has residual function when expressed at the cell surface in sufficient quantity,⁴ we hypothesized that CFTR potentiators such as ivacaftor may have therapeutic benefit in patients harboring W1282X CFTR.

To explore this, we first tested the effect of CFTR potentiators, alone and in combination with PTC suppression, in W1282X CFTR-expressing cell lines.⁴ These studies were carried out in CFBE41o- and FRT cells stably transfected with a W1282X cDNA construct. The cells were pre-treated for 48 hrs with the aminoglycoside G418 (250 μg/mL), an agent with substantial in vitro evidence of readthrough efficacy,^4,9,19 followed by acute addition of ivacaftor (10 μM). To assess CFTR activity, short-circuit current (Isc) measurements under voltage clamp conditions and conductance (G_t) measurements by transepithelial chloride conductance assay were performed as previously described.¹⁹ In line with the observation that ivacaftor acts synergistically with readthrough agents to augment CFTR activity in W1282X-transduced CFBE41o- cells,²⁰ G418 significantly enhanced forskolin-induced CFTR activity in CFBE41o- W1282X (I_sc, Fig 1A, B) and FRT W1282X (Gt, Fig 1C, D) cells compared to DMSO vehicle. The synergistic effect of ivacaftor with G418 was significantly higher in FRT cells compared to CFBE1o- cells. In contrast, ivacaftor had negligible effects on cells expressing R1162X CFTR (Fig 1B,D)—which does not have function at the cell surface in the truncated state⁴—further supporting the rationale for targeting W1282X as a nonsense mutation for which CFTR potentiator strategies may be potentially efficacious.

Figure 1. Effect of Ivacaftor in vitro CFBE W1282X cells and W1282X homozygous primary nasal epithelial cells derived from the case subject.

(A) Short circuit (Isc) tracings of CFBE 41o- cells transduced with W1282X or R1162X cDNA, pretreated with DMSO control or G418 (250 μg/mL) for 48 hrs. FSK=forskolin. (B) Summary graph of A. (C) Raw Gt tracings of FRT cell monolayers transduced with R1162X and W1282X CFTR (D) Summary graph of C showing response to forskolin and VX-770. G418 treatment significantly enhanced forskolin mediated Isc in both R1162X and W1282X compared to vehicle and the synergistic effect of ivacaftor with G418 was significant in W1282X compared to R1162X cells. (E) Representative tracings of the short circuit current (Isc) in HNE W1282X/W1282X cells (n=3/each condition) incubated with either Ivacaftor (10 μM) or DMSO vehicle for 48 hrs, where Forskolin (20 μM; FSK) and ivacaftor (10 μM VX-770) were administered acutely to stimulate CFTR activity followed by the addition of CFTR specific inhibitor, CFTR_Inh-172 (10 μM). (F) Forskolin-induced CFTR activity was enhanced significantly in HNE cells incubated with ivacaftor compared to DMSO treated cells (G) CFTR mRNA levels detected by real time reverse transcriptase PCR, HNE W1282X/W1282X expressed 0.4 fold as compared to HNE donors expressing wild type CFTR and cultured with the same method. A second HNE donor heterozygous for nonsense mutations is shown for comparison. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Noting the responses seen with CFTR potentiator treatment in W1282X CFTR-expressing cells, even without translational readthrough, we considered treatment with ivacaftor in a 31 year-old female CF patient with genotype W1282X/W1282X who was approaching end-stage disease. The subject was diagnosed with pancreatic-insufficient CF as an infant and exhibited elevated sweat chloride (116 mEq/L). She was colonized with Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus (MRSA), and a CT scan performed demonstrated diffuse bronchiectasis. Despite aggressive pulmonary therapy with dornase alpha, hypertonic saline, azithromycin, and a rotating antibiotic regimen of aerosolized vancoymycin and oral doxycycline, her CF lung disease progressed (Fig 2A). Lung function parameters showed a decline in forced expiratory volume in 1 sec (FEV₁), from 1.46L (47% predicted) 5 years prior to ivacaftor to 1.11L (36%) at the time of ivacaftor administration in February 2013. She also suffered frequent pulmonary exacerbations requiring courses of intravenous and oral antibiotics (Fig 2C). Her course was complicated by poor nutrition, progressing from a body mass index (BMI) of 19.4 to below 17, 5 years prior to ivacaftor (Fig 2B). This could not be improved with intense nutritional support or treatment of CF-related diabetes with insulin therapy (3 years prior to ivacaftor administration). Given the progressive decline of this patient's health status and the continued advancement of her lung disease, the potential for ivacaftor therapy was evaluated.

Figure 2. Effect of Ivacaftor on lung function, BMI and exacerbation frequencies.

(A) Pre and post ivacaftor measurements of FEV₁% predicted from 2007 to 2016. Lines represent the regression analysis line for each treatment period. Following initiation of ivacaftor, there was no significant change in mean FEV₁% predicted or the rate of FEV₁% predicted decline. (B) BMI obtained during the study period. Rate of change in BMI improved significantly with ivacaftor administration (P<0.001). (C) The number of months with and without an exacerbation, for the treatment period before and after initiation of ivacaftor. Statistical comparison by Chi square. (D) Total daily insulin intake requirements before and after initiation of ivacaftor. Statistical comparisons by paired t-test. *P<0.05, ****P<0.0001.

Upon approval by the University of Alabama at Birmingham and University of Pennsylvania Institutional Review Boards, efficacy of ivacaftor was tested ex vivo using primary human nasal epithelial cells (HNE) harvested from this individual. Cells were collected by isolating nasal epithelial cells from the patient using the nasal brush method, expanded in culture using conditional reprogramming,²¹ and then grown as airway monolayers at air-liquid interface until well-differentiated. First-passage cells were incubated for 48 hrs with either ivacaftor (10 μM) or vehicle control (DMSO, 0.1%), after which monolayers were evaluated in modified Ussing chambers under voltage clamp conditions in the setting of a chloride secretory gradient (Fig 1E). CFTR was stimulated with forskolin (20 μM), followed by acute addition of ivacaftor (10 μM) and CFTR inhibition with CFTR_Inh-172 (10 μM). Ivacaftor generated a small but statistically significant increase in forskolin-dependent Isc as compared to DMSO-treated control (Ivacaftor 0.4 ± 0.04 μA/cm² vs. DMSO 0.08 ± 0.003 μA/cm², P<0.01), indicative of CFTR activity (Fig 1F). Acute addition of ivacaftor did not increase this signal further (Fig 1E). CFTR mRNA levels were ~40% (0.4±0.02) of that observed in normal wild type cells, probably indicative of nonsense-mediated decay as also observed in another HNE donor compound heterozygous for two other premature termination codons (Fig 1G).

Based in part on the above results, the patient initiated ivacaftor (150 mg twice daily) in February 2013. While no significant improvements were observed in FEV₁ (Fig 2A), the rate of FEV₁ decline, or sweat chloride (from 116 to 114 mEq/L) with ivacaftor treatment, FEV₁ remained stable over a period of 3 years after ivacaftor initiation. Further, the patient experienced significantly fewer exacerbations (0.5/year vs. 3.6/year, P<0.05), as based on the number of months with and without an exacerbation before and during the ivacaftor treatment period (Fig 2C). Following ivacaftor initiation, the patient also experienced a significant improvement in nutritional status (Fig 2B), as reflected by increased BMI (20.3) and body weight (50.3 kg), and rate of BMI change (P<0.001). In addition, average insulin requirement to manage diabetes decreased significantly (Fig 2D). The patient also reported subjectively less sputum production that was easier to clear and increased energy.

Taken together, our results provide evidence that ivacaftor had a modest favorable clinical benefit for a patient homozygous for the W1282X mutation. These findings are consistent with in vitro studies establishing activity of ivacaftor and other CFTR potentiators on the W1282X CFTR mutation. The results also demonstrate an important concept: ex vivo testing using primary human airway cells from the same individual may be able to predict patient-specific clinical benefit, and potentially accelerate the delivery of efficacious drugs to CF patients (particularly those with rare mutations who are not available in sufficient quantity to conduct placebo controlled studies). Overall, results suggest that CFTR potentiator therapy may be a valuable addition to selected patients with C-terminal CFTR truncation alleles, and indicate the need for additional evaluation of this approach.

Highlights.

1. In vitro findings demonstrated significant benefit of Ivacaftor in W1282X cells.

2. Ivacaftor improved exacerbation rates, BMI and insulin uptake in W1282X patient.

3. Our data suggest ivacaftor may have moderate clinical benefit in W1282X patients.