Inhaled ENaC antisense oligonucleotide ameliorates cystic fibrosis-like lung disease in mice

JCF-01489; No of Pages 10

Journal of Cystic Fibrosis xx (2017) xxx – xxx www.elsevier.com/locate/jcf

Original Article

Inhaled ENaC antisense oligonucleotide ameliorates cystic fibrosis-like lung disease in mice Jeff R. Crosby a,⁎, Chenguang Zhao a , Chong Jiang b , Dong Bai a , Melanie Katz a , Sarah Greenlee a , Hiroshi Kawabe c , Michael McCaleb a , Daniela Rotin b , Shuling Guo a , Brett P. Monia a b

a Ionis Pharmaceuticals, 2855 Gazelle Court, Carlsbad, CA 92010, USA The Hospital for Sick Children, 686 Bay Street, Toronto, Ontario M5G 0A4, Canada c Max Planck Institute of Experimental Medicine, Göttingen, Germany

Received 23 November 2016; revised 26 April 2017; accepted 8 May 2017

Abstract Background: Epithelial sodium channel (ENaC, Scnn1) hyperactivity in the lung leads to airway surface dehydration and mucus accumulation in cystic fibrosis (CF) patients and in mice with CF-like lung disease. Methods: We identified several potent ENaC specific antisense oligonucleotides (ASOs) and tested them by inhalation in mouse models of CF-like lung disease. Results: The inhaled ASOs distributed into lung airway epithelial cells and decreased ENaC expression by inducing RNase H1-dependent degradation of the targeted Scnn1a mRNA. Aerosol delivered ENaC ASO down-regulated mucus marker expression and ameliorated goblet cell metaplasia, inflammation, and airway hyper-responsiveness. Lack of systemic activity of ASOs delivered via the aerosol route ensures the safety of this approach. Conclusions: Our results demonstrate that antisense inhibition of ENaC in airway epithelial cells could be an effective and safe approach for the prevention and reversal of lung symptoms in CF and potentially other inflammatory diseases of the lung. © 2017 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved. Keywords: Cystic fibrosis; ENaC; Antisense oligonucleotide; Aerosol

1. Introduction Cystic fibrosis (CF) is a disease caused by mutations in the CFTR gene, which encodes an epithelial chloride channel [1–3]. Mutations in CFTR are associated with decreased chloride secretion and increased sodium absorption, at least partially due to regulatory effects of CFTR on the epithelial sodium channel (ENaC) [4–6]. In mice, amiloride-sensitive ENaC [7] includes three subunits encoded by Scnn1a, Scnn1b, and Scnn1c [8]. This complex controls the absorption of sodium and is localized to the apical membranes of polarized epithelial cells in kidney, colon, lung, and sweat glands [9]. Although somewhat controversial ⁎ Corresponding author. E-mail address: [email protected] (J.R. Crosby).

[10], numerous reports have demonstrated that the loss of CFTR function leads to hyperactive ENaC, resulting in reduced airway surface liquid, mucus dehydration, and mucus accumulation in airways [11]. Increased ENaC levels also result in CF-like disease in the lungs of mice that overexpress ENaC or that are depleted of the ENaC suppressor Nedd4L [12–14]. Therapies such as hypertonic saline improve lung function in CF patients [15], and small-molecule inhibitors such as amiloride, an ENaC blocker that acts as a potassium-sparing diuretic, result in some benefit in both animal studies and clinical trials [16,17]. The efficacy of amiloride is severely limited due to its short half-life. The longer-lasting ENaC inhibitor GS-9411 caused hyperkalemia in a phase 1 clinical trial in healthy volunteers [18]; this use-limiting side-effect was believed to be due to ENaC inhibition in the kidney.

http://dx.doi.org/10.1016/j.jcf.2017.05.003 1569-1993/© 2017 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved. Please cite this article as: Crosby JR, et al, Inhaled ENaC antisense oligonucleotide ameliorates cystic fibrosis-like lung disease in mice, J Cyst Fibros (2017), http:// dx.doi.org/10.1016/j.jcf.2017.05.003

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We have recently shown that antisense oligonucleotides improved disease symptoms when delivered locally to the lung in mouse models of asthma [19]. ASOs delivered via aerosol are well tolerated in rodents, non-human primates, and human subjects [19–21]. Importantly, aerosolized ASOs delivered to the lung result in negligible systemic exposure [22]. In the present study, we evaluated an antisense strategy for inhibition of ENaC activity in two existing mouse models that result in a CF-like lung disease. One involving airway-specific overexpression of βENaC and a second involving airwayspecific knockout (KO) of Nedd4L, the gene encoding a ubiquitin ligase that controls cell-surface levels of ENaC [12,13]. In addition, we developed a mouse model of adult-onset CF-like disease by treating mice with ASOs designed to specifically reduce levels of Nedd4L mRNA in the lung. Our results demonstrate that ASO-mediated inhibition of ENaC is an efficacious and safe approach for the treatment of lung disease in CF. 2. Methods 2.1. Oligonucleotides All oligonucleotides were 16 nucleotides in length (3-10-3 gapmers) with a phosphorothioate backbone and constrained ethyl (cEt) modifications in the wings. The sequences of oligonucleotides are: ENaC ASO #1: AGCAACTCCGTTTCTT, ENaC ASO #2: GAGCATCTAATACAGC, control ASO: GGCTACTACGC CGTCA, and the Nedd4L ASO: CCATTTTCAACCTCAA. Oligonucleotides were synthesized as described [23]. 2.2. Quantification of mRNA Whole lungs or fractioned lung cells were homogenized in RLT buffer. Total RNA was prepared using the RNAeasy mini kit (Qiagen). RT-PCR was performed using the StepOnePlus Real-Time PCR System (Applied Biosystems). The sequences of primers and probes are given in Supplementary Table 1. The amount of each mRNA was normalized to the amount of total RNA determined by Ribogreen (Invitrogen). 2.3. Animals and ASO dosing For the Nedd4L ASO induced model, ASO was administered once weekly by orotracheal instillation at a dose of 10 mg/kg. For aerosol dosing, ASOs were suspended in 0.9% sodium chloride (Baxter Healthcare) and delivered via inhalation using a noseonly delivery system as described previously [21]. 2.4. Ion transport measurements Mice were euthanized under IACUC approved guidelines and tracheal tissue was removed and incubated in cold Krebs Ringers with 10 μM Indomethacin (Sigma-Aldrich) for 15 min. Trachea were then mounted on Ussing chambers (Physiologic Instrument) using the P2308 Slider. After an equilibration period of 30 min, the following drugs were added sequentially: forskolin (10 μM, apical and basolateral), bumetanide (100 μM, apical), and

amiloride (100 μM, apical). All the reagents were purchased from Sigma-Aldrich. 2.5. Measurement of airway hyper-responsiveness (AHR) AHR was determined by inducing bronchoconstriction with methacholine aerosol [24]. Total pulmonary airflow in unrestrained mice was estimated using a whole body plethysmograph (Buxco Electronics). Pressure differences between a chamber containing an individual mouse and a reference chamber were used to extrapolate the enhanced pause (Penh). Penh is a dimensionless parameter that is a function of total pulmonary airflow in mice during each respiratory cycle. 2.6. Statistical analysis Analysis of group differences in Penh response was performed using two-way repeated measures ANOVA using Prism software. Analysis of group differences in all other endpoints was performed using a Student's t-test. P values less than 0.05 were considered significant. 3. Results 3.1. Aerosolized delivery of ENaC ASOs reduces Scnn1a mRNA levels in lungs of normal mice ASOs delivered by aerosol have been demonstrated to distribute throughout the lung and reduce target RNA in a variety of cell types, including airway epithelial cells (AECs) [21]. Two cEt ASOs designed to target Scnn1a, which encodes the ENaC α subunit, demonstrated IC50 values for mRNA reduction of ~ 0.2 μM in a cell-based assay and were selected for further evaluation. Wild-type mice were treated with aerosolized ASO at three different doses over the course of two weeks. ENaC is expressed in lung epithelial cells [25]. To enrich for this lung cell population, we performed density gradient fractionation of perfused lungs. We observed that ENaC and epithelial cell markers (CAV1, AGER, HOPX, PDPN) were enriched in the 7% fraction relative to levels in total lung cell lysate (Fig. 1A). Treatment with both ASOs resulted in a dose-dependent reduction of Scnn1a mRNA levels of up to ~ 90% in the epithelial fraction relative to untreated controls (Fig. 1B). ENaC protein levels were also reduced upon ASO treatment (Fig. 1C). To evaluate the duration of action of inhaled ENaC ASOs, 3 doses of ENaC ASO2 (0.5, 1.0 and 2.0 mg/kg) were administered via the aerosol route and ASO concentration and Scnn1a mRNA levels in the lung were determined over a four-week period. ENaC ASO had a half-life of approximately 13 days in lung tissue, and Scnn1a levels were suppressed by ~ 80% by 48 h after a single treatment with recovery at ~ 28 days for the low dose (Supplementary Fig. 1). Since airway mucus accumulation is a symptom of CF, we investigated whether the ENaC ASO was effective in the presence of mucus. Wild-type mice were treated with house dust mite (HDM) to induce goblet cell metaplasia and mucus production (Fig. 1D). To test ASO activity in the presence of

Please cite this article as: Crosby JR, et al, Inhaled ENaC antisense oligonucleotide ameliorates cystic fibrosis-like lung disease in mice, J Cyst Fibros (2017), http:// dx.doi.org/10.1016/j.jcf.2017.05.003

J.R. Crosby et al. / Journal of Cystic Fibrosis xx (2017) xxx–xxx

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Fig. 1. Aerosolized ENaC ASO treatment decreases ENaC levels in mouse lung. (A) Quantification of AEC markers (Cav1, Ager, Hopx, and Pdpn) in fractions after density gradient centrifugation of dissociated lung cells as a percentage of levels in total cell lysate. (B) Scnn1a mRNA quantification in lungs of wild-type mice treated with aerosolized ENaC ASOs as a percentage of levels in untreated lungs. *, P b 0.05 compared with untreated naive (n = 4/group). (C) Representative images of ENaC protein detected by IHC in untreated and ENaC ASO-treated wild-type mice (n = 8/group). (D) Orotracheal administration of a house dust mite preparation was used to induce goblet cell hyperplasia and mucus production in lung airways as indicated by PAS-staining of mucus. GC indicates goblet cell. (E) Representative staining with an antibody recognizing the ASO demonstrated that ENaC ASOs administered via aerosol at 0.33 mg/kg twice a week for 3 weeks penetrated established mucus and was taken up by lung cells. (F) Control IgG demonstrates specificity of staining. Scale bars, 200 μm (C, D, E, F). (G) Scnn1a mRNA levels in the lungs of naïve or HDM-treated mice *, P b 0.05 compared with ASO treatment (n = 8/group). Values are means ± s.e.m. (B, G).

mucus, naive or HDM mice were treated with ENaC ASO by aerosol after mucus was established. The inhaled ENaC ASOs penetrated the mucus as indicated by IHC (Fig. 1E) and staining was specific for ENaC as no staining was observed with control IgG (Fig. 1F). Moreover, ENaC ASO treatment

induced similar Scnn1a mRNA reduction in naive mice and mice with HDM-induced mucus (Fig. 1G). In a phase 1 clinical trial, intranasal administration of a smallmolecule inhibitor of ENaC resulted in clinically significant hyperkalemia [18]. This prompted us to investigate the effects of

Please cite this article as: Crosby JR, et al, Inhaled ENaC antisense oligonucleotide ameliorates cystic fibrosis-like lung disease in mice, J Cyst Fibros (2017), http:// dx.doi.org/10.1016/j.jcf.2017.05.003

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the ENaC ASOs delivered via the aerosol route on systemic sodium and potassium homeostasis. Extracts of lungs and kidneys of wild-type mice given ENaC ASO or a control ASO delivered by aerosol administration were analyzed by qRT-PCR to determine the extent of Scnn1a mRNA reduction. With a dosing regimen that achieved greater than 90% Scnn1a reduction in the lung airway epithelial cells as compared to control-treated animals, no Scnn1a reduction was observed in the kidney (Fig. 2A). Further, urine and blood potassium or sodium levels were the same within experimental variation in mice treated with the ENaC ASO and mice treated with the control ASO (Fig. 2B and C). In contrast, mice treated intranasally with amiloride had lower potassium levels and higher sodium levels in the urine than naive control mice (Fig. 2D), consistent with previous reports [17,18]. In agreement with our previous findings [20], these data indicate that there is negligible systemic exposure when mice are treated with ASO via aerosol delivery. In addition, cells in which ENaC is expressed in the kidney (the collecting duct and distal convoluted tubule) are generally refractory to ASO treatment. Even with systemic delivery of the ENaC ASO, which resulted in significant ASO accumulation in kidney and approximately 80% reduction in Scnn1a in colon, there was minimal effect on renal Scnn1a (~ 25% decrease in Scnn1a mRNA) (Supplementary Fig. 2). These experiments demonstrate that an antisense therapy

against ENaC has the advantage of potent inhibition in the lung with no effect on renal ENaC activity.

3.2. ENaC ASOs reduce mucus accumulation in Nedd4L-KO mice Ablation of the gene encoding the ubiquitin ligase Nedd4L in lung epithelia of mice results in a CF-like lung disease due to elevated ENaC protein expression in the lung. Increased ENaC activity results in airway mucus obstruction, goblet cell hyperplasia, massive inflammation, fibrosis, and death by three weeks of age [13]. We treated Nedd4L-KO mice with two ENaC-specific ASOs or a control ASO intranasally (10 mg/kg) once per day for 11 days beginning on postnatal day 1. ASOs distributed broadly in the lung as shown by IHC analyses (Supplementary Fig. 3 A–C). Because of the difficult nature of intranasal delivery in newborn pups, only 40–60% suppression of Scnn1a mRNA levels in total lung was achieved (Supplementary Fig. 3D); however, this relatively moderate level of ASO activity significantly reduced the number of mucus positive airways in parasagittal lung sections (Supplementary Fig. 3E). Furthermore, both goblet cell hyperplasia and inflammation were reduced by ENaC ASO treatment of the Nedd4L-KO pups (Supplementary Fig. 3F–H).

Fig. 2. ENaC ASOs do not decrease target mRNA levels in kidney after aerosol delivery. (A) Scnn1a mRNA levels in the lung and kidney after a high dose of aerosolized ENaC ASO (1 mg/kg/week) for 3 weeks plotted as a percentage of naive control (n = 8 mice/group). (B) Urine Na+ and K+ levels (mmol/L) in mice treated with aerosolized ENaC ASO (n = 8 mice/group). (C) Blood Na+ and K+ levels (mmol/L) in mice treated with aerosolized ENaC ASO and a control ASO (n = 8 mice/group). (D) Urine Na+ to K+ ratios in mice treated with amiloride (intranasal dosing 3 times a day for 2 weeks) or naïve control (n = 4 mice/group). Values are means ± s.e.m. *, P b 0.05 by Student's t-test versus untreated naïve mice (A, D). Please cite this article as: Crosby JR, et al, Inhaled ENaC antisense oligonucleotide ameliorates cystic fibrosis-like lung disease in mice, J Cyst Fibros (2017), http:// dx.doi.org/10.1016/j.jcf.2017.05.003

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3.3. Establish an adult-onset CF mouse model with Nedd4L-targeted ASO Nedd4L-deficient mice die within 3 weeks, which makes it difficult to evaluate long-term effects of ASO treatment or to detect the reversal of established lung disease. We therefore developed an ASO against Nedd4L to generate an adult-onset CF-like lung disease model. To determine the optimal time point for delivery of the ASO, adult mice were treated orotracheally with Nedd4L ASO (10 mg/kg) weekly for 2, 4, and 6 weeks. The 6-week treatment period resulted in the highest level of inflammation and mucus accumulation and was selected for future studies. After a 6-week treatment period with Nedd4L ASO, Nedd4L mRNA levels were reduced by approximately 70% in lung compared to levels in naive untreated mice (Fig. 3A). Mice treated with Nedd4L ASO had higher levels of α and γ ENaC proteins in the lung than did untreated mice (Fig. 3B–C). Further, treatment with Nedd4L ASO resulted in a dramatic up-regulation of mRNAs encoding mucus markers Gob5 (Fig. 3D), Foxa3, and Agr2 (data not shown) and higher levels of goblet cell hyperplasia, mucus production, and inflammation (Fig. 3E–F). In addition, there was a significant increase of neutrophil recruitment to the airways in the Nedd4L ASO-treated mice which correlated well with an increase in the neutrophil chemoattractant CXCL1 (Fig. 3G). These phenotypes are similar to those of Nedd4L-deficient mice and in transgenic mice that overexpress βENaC [12,13]. Airway hyper-responsiveness (AHR) in Nedd4L ASO-treated mice was also observed after methacholine challenge (Fig. 3H). AHR, which is a characteristic CF phenotype, persisted for at least two weeks after ASO treatment was halted (data not shown). This was expected due to the long duration of action of antisense compounds. 3.4. Aerosolized ENaC ASO prevents Nedd4L ASO-induced mucus accumulation and AHR To determine whether ENaC ASOs would prevent induction of CF-like disease in the adult-onset Nedd4L ASO-induced CF model, mice were first given inhaled ENaC ASO for two weeks (twice a week). After 4 ENaC ASO doses, orotracheal dosing with Nedd4L ASO was initiated. Compared to the Nedd4L ASOtreated mice given vehicle alone, Scnn1a mRNA levels were reduced by 80% in mice treated with ENaC ASO (Fig. 4A). ENaC ASO treatment also reduced ENaC protein levels to that of control mice not given the Nedd4L ASO (Fig. 4B–C). To determine the effect of inhaled ENaC ASO on sodium channel activity in the adult-onset CF model, amiloride sensitive conductance was assessed in the tracheas of mice. The tissue was initially treated with Forskolin to confirm the functionality of CFTR followed by amiloride treatment to test for ENaC activity. Nedd4L ASO treatment increased sodium channel conductance by 225% compared with saline-treated controls (Fig. 4D). ENaC ASO treatment of the Nedd4L ASO-treated mice reduced Na+ conductance to near normal levels, approximately 113% of saline-treated controls (Fig. 4D). Treatment of the Nedd4L ASOtreated mice also significantly reduced levels of Gob5 and Agr2 mRNAs (Fig. 4E–G). In addition, the goblet cell metaplasia and

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inflammation induced by Nedd4L ASO treatment were reduced in ENaC ASO treated lungs (Fig. 4H–I). The increase in BAL neutrophil levels and increase in AHR observed following Nedd4L ASO treatment were also prevented by treatment with the ENaC ASO (Fig. 4J–K). 3.5. Aerosolized ENaC ASO reverses CF-like symptoms In order to evaluate the efficacy of the ENaC ASO on CF-like symptoms after disease was established, mice were treated for six weeks with Nedd4L ASO to induce disease. Mice were then treated with the ENaC ASO or a control ASO for three weeks (3 doses/wk at 0.33 mg/kg/dose). The ENaC ASO treatment reduced Scnn1a mRNA levels compared to untreated mice, whereas the control ASO resulted in no reduction in Scnn1a mRNA levels (Fig. 5A). Upregulation of the mucus marker Gob5 induced by Nedd4L ASO was reversed by treatment with the ENaC ASO but not by the control ASO (Fig. 5B). Histopathology analyses showed that ENaC ASO treatment decreased the thickness of the previously established mucus layer (Fig. 5C–D). All mice treated with the Nedd4L ASO had increased AHR relative to untreated mice before ENaC ASO treatment started (Fig. 5E). The treatment with aerosolized ENaC ASO reversed AHR while the control ASO had no effect (Fig. 5F). The effects of aerosolized ENaC ASOs were confirmed in βENaC transgenic mice (Supplementary Fig. 4). 4. Discussion Despite improved care and recent progress in the identification of new therapies to treat CF, the predicted median survival of CF patients still remains in the low 40-year range in developed countries [26]. Novel therapies, alone or in combination with established therapies, are needed to prolong survival and improve the quality of life for CF patients. The antisense oligonucleotide drug platform is a proven approach for drug discovery [27]. Delivery of ASOs by aerosol results in repression of target gene expression in the lung with minimal systemic exposure [22]. In the present study, we demonstrated that ASOs designed to Scnn1a mRNA reduced activity of ENaC in airway epithelial cells, resulting in the prevention of CF-like disease and in the reversal of CF-like lung phenotype in multiple mouse models. A number of mouse models have been tested for relevance to CF. The Cftr KO mice have severe intestinal disease but fail to develop lung disease [28]. Mouse models were therefore needed that replicate the lung disease observed in human patients with CF. Two models have been established: one by overexpression of an ENaC subunit in lung AECs [12] and one by deletion of the gene encoding the ubiquitin ligase Nedd4L in AECs [13]. The Nedd4L family of ubiquitin ligases binds to the cytoplasmic pY motif of ENaC subunits, leading to their ubiquitination, endocytosis and subsequent degradation [29]. The Nedd4L-KO CF mouse model was established by deletion of Nedd4L specifically in the airway epithelium; this results in a phenotype comparable to that of the βENaC transgenic mouse [13]. Nedd4L-deficient mice have features characteristic of cystic fibrosis lung disease but

Please cite this article as: Crosby JR, et al, Inhaled ENaC antisense oligonucleotide ameliorates cystic fibrosis-like lung disease in mice, J Cyst Fibros (2017), http:// dx.doi.org/10.1016/j.jcf.2017.05.003

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Fig. 3. A late-onset cystic fibrosis-like model established by treatment of mice with Nedd4L-ASO. (A–H) Adult naïve mice were treated orotracheally once per week for 6 weeks with Nedd4L ASO (10 mg/kg, n = 4 mice/group). (A) Nedd4L mRNA levels. (B) Quantification of Western blots for α and γ ENaC subunits in whole lungs of mice treated with Nedd4L ASO. (C) Western blot showing both the α and γ subunits of ENaC after Nedd4L ASO treatment. (D) Gob5 mRNA was quantified by RT-PCR in fractionated lung. (E–F) Mucus accumulation measured by PAS-staining in Nedd4L ASO treated mice (n = 8 mice/group). Scale bars, 600 μm. (G) Increase in BAL neutrophils and CXCL1 levels (n = 8 mice/group). (H) Increase of airway hyper-responsiveness (Penh) in Nedd4L ASO treatment group (n = 48) compared to naïve mice (n = 16). The area under the curves for the naïve group is significantly different from the Nedd4L ASO treatment group. *, P b 0.0001. Values are means ± s.e.m. *, P b 0.05 (A, D) *, P b 0.01 (B) *, P b 0.0001 (G) by Student's t-test versus naïve mice.

Please cite this article as: Crosby JR, et al, Inhaled ENaC antisense oligonucleotide ameliorates cystic fibrosis-like lung disease in mice, J Cyst Fibros (2017), http:// dx.doi.org/10.1016/j.jcf.2017.05.003

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Fig. 4. Aerosolized ENaC ASOs prevent Nedd4L-ASO-induced CF-like lung disease. (A–K) Adult naïve mice were aerosol dosed twice a week for 8 weeks with ENaC ASO (0.333 mg/kg). After two weeks of ENaC ASO dosing, the Nedd4L model was initiated by orotracheal administration of Nedd4L ASO once a week for 6 wks (10 mg/kg/dose). (A) ENaC mRNA levels compared to vehicle control mice treated with Nedd4L ASO (n = 4/group). (B) Representative Western analysis (3 independent studies with independent cohorts of 3 animals each) and quantitation of Western bands (C) for both α and γ ENaC subunits compared to tubulin for load control. (D) Measurements of ENaC ASO effect on amiloride-sensitive short-circuit current (Isc) in adult Nedd4L ASO-treated mice (n = 8–16 mice/group). Data are normalized to untreated (naïve) littermate controls. (E–G) Nedd4L ASO induces mRNA of mucus markers (gob5, AGR2 and FOXa3) which is prevented by ENaC ASO (n = 4 mice/ group). (H) Mucus accumulation (PAS staining) and inflammation in Nedd4L ASO treated mice (I) is prevented with aerosolized ENaC ASO (n = 8 mice/group). Scale bar, 800 μm (H) and 300 μm (I). (J) Increase in recruitment of BAL neutrophils with Nedd4L ASOs is prevented with ENaC ASO (n = 8 mice/group). (K) Nedd4L ASO induced airway hyper-responsiveness (Penh) is prevented with aerosolized ENaC ASO treatment (n = 8 mice/group).The area under the curves for the naïve and ENaC ASO treated groups are significantly different from the Nedd4L ASO treatment group. *, P b 0.0001.Values are means ± s.e.m. *, P b 0.05 (A, C, J) and P b 0.01 (D–G) by Student's t-test versus Nedd4L ASO treated vehicle mice.

Please cite this article as: Crosby JR, et al, Inhaled ENaC antisense oligonucleotide ameliorates cystic fibrosis-like lung disease in mice, J Cyst Fibros (2017), http:// dx.doi.org/10.1016/j.jcf.2017.05.003

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Fig. 5. Aerosolized ENaC ASO reverses Nedd4L-ASO-induced CF-like lung disease. (A–H) Adult naïve mice were treated orotracheally once per week for 9 weeks with Nedd4L ASO (10 mg/kg). After 6 weeks of Nedd4L ASO treatment, ENaC ASO or control ASO (0.333 mg/kg/dose) were aerosol dosed 3 times a week for 3 weeks. (A) ENaC mRNA and (B) Gob5 mRNA reduction after ENaC ASO treatment (n = 4 mice/group) compared to a control ASO at the same dose. Data presented as mean ± s.e.m. P b 0.01 (A) and P b 0.05 (B) by Student's t-test. (C–D) PAS stained lung mucus after ASO treatment (n = 8 mice/group). Scale bar, 200 μm. (E) Nedd4L ASO-induced airway-hyper-responsiveness (Penh) after 6 weeks of Nedd4L ASO. (F) Reversal of Nedd4L ASO induced airway hyper-responsiveness (Penh) by aerosolized ENaC ASO treatment at 9 wks (3 wks of ENaC ASO treatment) (n = 12 mice/group). A control ASO shows no effect of reversing the AHR. The area under the curves for the naïve (E) or naïve and ENaC ASO treated groups (F) are significantly different from the Nedd4L and control ASO treatment groups. *, P b 0.0001.

typically die by 3 weeks of age. The ability of amiloride to reverse the CF lung defects that occur in Nedd4L-KO pups suggests that increased ENaC levels and/or activity in AECs is responsible for the CF-like phenotype in this model [13,17]. This conclusion is supported by our results demonstrating that treatment of Nedd4L-KO mouse pups with intranasally

administered ENaC ASOs reduced airway mucus accumulation and inflammation. Since Nedd4L-KO mice typically die before reaching adulthood, we sought to create an adult-onset model of CF. Orotracheal administration of Nedd4L-specific ASOs into the lungs of mice for 6 weeks reduced Nedd4L mRNA levels,

Please cite this article as: Crosby JR, et al, Inhaled ENaC antisense oligonucleotide ameliorates cystic fibrosis-like lung disease in mice, J Cyst Fibros (2017), http:// dx.doi.org/10.1016/j.jcf.2017.05.003

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upregulated ENaC protein levels, induced goblet cell hyperplasia and sterile neutrophilic inflammation in both lungs and airways, and increased airway hyper-responsiveness to a methacholine challenge (non-invasive lung function, Penh, measured by Buxco). Subsequent treatment of the Nedd4L ASO-treated mice with aerosolized ENaC ASO resulted in a substantial reduction in Scnn1a mRNA levels. In both models induced by Nedd4L ASO and in the βENaC transgenic mouse the levels of ENaC protein are elevated, while ENaC mRNA levels are reduced compared to normal mice possibly due to a feedback response resulting in reduced ENaC transcription. The Nedd4L ASO induced an adultonset CF-like disease, and this model will be useful for evaluating the long-term effects of pharmacological therapies and determining whether a pharmacological agent can reverse the CF-like phenotype. Although the exact mechanisms of ENaC hyperactivity in CF airways is still unclear [30], it has been suggested that the upregulation of ENaC activity observed in CF patient airways may be due to the inability of SPLUNC1 to inhibit ENaC in the acidic environment of the CF airways [31]. ENaC is an attractive target for CF drug development as its inhibition should improve airway surface hydration and mucus clearance in CF patients independent of the mutations leading to their disease [32]. Rehydrating airway surfaces with hypertonic saline has been shown to improve lung function and decrease the rate of pulmonary exacerbations by 50% [15], confirming that targeting ENaC could ameliorate the CF symptoms. The sodium channel inhibitor amiloride, which works by directly blocking ENaC, given via aerosol improves mucociliary clearance and slows the loss of lung function in CF patients [16]. In order to establish proof of concept for an aerosolized antisense approach against ENaC for the treatment of CF, we designed and screened ASOs to induce targeted depletion of Scnn1a mRNA, which encodes the ENaC α subunit. In mouse CF models, ENaC ASO delivery to the lungs resulted in a significant reduction in Scnn1a levels and improvement in markers of disease. Importantly, no Scnn1a reductions occurred in the kidney when the ASO was delivered by inhalation. In fact, even with direct systemic delivery of ENaC ASOs, minimal Scnn1a reduction occurred in the kidney, although ~ 80–90% reduction in Scnn1a mRNA levels were observed in lung and colon compared to levels in control mice. In addition, no effects on urine or serum electrolytes were observed with either aerosol or systemic (data not shown) dosing of ENaC ASO, confirming the lack of ENaC inhibition in kidney. Several antisense or siRNA inhibitors designed to inhibit the expression of the ENaC α subunit have been tested in cell culture to validate the feasibility of this therapeutic approach for treating CF patients [33–35]. These studies were conducted in vitro or in normal animals lacking the characteristic mucus observed in CF, which impedes the delivery of hydrophobic drugs to the lung [36]. The size and charge of cEt ASOs, which are delivered in simple saline solution, independent of any special formulation, and the long duration of action of the cEt ASOs make them an ideal choice for inhaled drug delivery. Here we show that cEt ASOs dissolved in saline solution traversed mucus and distributed throughout mouse lung. We have demonstrated that a 10 μg/g lung concentration of ENaC

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ASO results in ~ 70% mRNA target knockdown in AECs in mice (Supplemental Fig. 1). Based on prior experience with the IL-4Rα program [20], we estimate the human dose of ASO to give us an approximately 70% reduction of ENaC mRNA is ~ 12 mg delivered to the lung. In a previous study with inhaled ASOs in human, a dose of 30 mg was successfully administered with no safety issues [37]. In addition, ASO aerosol delivery has been shown to be well tolerated in humans as well as rodents and non-human primates [19–21]. An antisense ENaC inhibitor offers several important advantages over other ENaC therapeutic approaches currently in development, such as small molecule and peptide inhibitors. By targeting RNA sequences, ASOs can achieve high selectivity, including the ability to specifically inhibit the different ENaC subunits. ASO drugs can be delivered to the lungs at low doses and low volumes in simple water-based solutions and, due to the long tissue half-life and duration of action, dosing can be as infrequent as monthly. Lack of systemic exposure for inhaled ENaC ASOs and their inability to reduce ENaC message in the kidney is another advantage of this therapeutic approach. That ASO drugs are well suited for combination therapy with other classes of drugs, together with previously mentioned advantages, supports the conclusion that antisense oligonucleotide therapy targeting ENaC could serve as an effective alternative to current approaches. Recently approved CF drugs ivacaftor (Kalydeco) and lumacaftor/ivacaftor (Orkambi), which improve CFTR protein trafficking to the plasma membrane and/or improve CFTR channel function, are effective in patients with specific CFTR mutations [32,38]. In contrast, ENaC antisense therapy is expected to benefit CF patients regardless of the CFTR mutations they carry. Furthermore, ENaC ASOs are expected to work well in combination with CFTR modulators. Our data demonstrate that an ENaC antisense therapy may provide a novel potent and safe treatment option for CF. Declaration of interest and support J.C., C.Z., D.B., M.K., S.G., M.M., S.G. and B.P.M. are Ionis employees, and this research is supported by Ionis Pharmaceuticals, Inc. Author contributions J.C., M.M., S.G. and D.R. contributed to the study design. J.C., C.Z., C.J. and D.B. contributed to in vivo studies and data analysis. D.R. and H.K. provided unique reagents. M.K. and S.Gr. contributed to in vitro and PK studies. J.C, H.K., M.M., D.R., S.G. and B.P.M. wrote and/or edited the manuscript. Acknowledgments We thank T. Reigle and M. David for assistance with figures and manuscript preparation. We thank X. Xiao, B. DeBrosseSerra and Y. Jiang for tissue processing. We thank Ken Luu for PK discussions.

Please cite this article as: Crosby JR, et al, Inhaled ENaC antisense oligonucleotide ameliorates cystic fibrosis-like lung disease in mice, J Cyst Fibros (2017), http:// dx.doi.org/10.1016/j.jcf.2017.05.003

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Please cite this article as: Crosby JR, et al, Inhaled ENaC antisense oligonucleotide ameliorates cystic fibrosis-like lung disease in mice, J Cyst Fibros (2017), http:// dx.doi.org/10.1016/j.jcf.2017.05.003