Antisense oligonucleotide targeting of mRNAs encoding ENaC subunits α, β, and γ improves cystic fibrosis-like disease in mice

JCF-01683; No of Pages 10

Journal of Cystic Fibrosis xx (2018) xxx – xxx www.elsevier.com/locate/jcf

Original Article

Antisense oligonucleotide targeting of mRNAs encoding ENaC subunits α, β, and γ improves cystic fibrosis-like disease in mice Chenguang Zhao ⁎, Jeff Crosby, Tinghong Lv, Dong Bai, Brett P. Monia, Shuling Guo Ionis Pharmaceuticals, 2855 Gazelle Court, Carlsbad, CA 92010, USA Received 28 March 2018; revised 11 July 2018; accepted 20 July 2018 Available online xxxx

Abstract Background: The epithelial sodium channel ENaC consists of three subunits encoded by Scnn1a, Scnn1b, and Scnn1g and increased sodium absorption through this channel is hypothesized to lead to mucus dehydration and accumulation in cystic fibrosis (CF) patients. Methods: We identified potent and specific antisense oligonucleotides (ASOs) targeting mRNAs encoding the ENaC subunits and evaluated these ASOs in mouse models of CF-like lung disease. Results: ASOs designed to target mRNAs encoding each ENaC subunit or a control ASO were administered directly into the lungs of mice. The reductions in ENaC subunits correlated well with a reduction in amiloride sensitive channel conductance. In addition, levels of mucus markers Gob5, AGR2, Muc5ac, and Muc5b, periodic acid-Schiff's reagent (PAS) goblet cell staining, and neutrophil recruitment were reduced and lung function was improved when levels of any of the ENaC subunits were decreased. Conclusions: Delivery of ASOs targeting mRNAs encoding each of the three ENaC subunits directly into the lung improved disease phenotypes in a mouse model of CF-like lung disease. These findings suggest that targeting ENaC subunits could be an effective approach for the treatment of CF. © 2018 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved. Keywords: Cystic fibrosis; ENaC; Subunit; Antisense oligonucleotide

1. Introduction The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that regulates the epithelial sodium channel (ENaC) [1–3] and controls the transport of ions and water across the epithelial barrier. Mutations in the CFTR gene lead to cystic fibrosis, one of the most common lethal hereditary diseases. ENaC is a transmembrane protein composed of three subunits (α, β, and γ) that are expressed in absorptive epithelia, including the conducting airways and alveolar airspaces in the lung; ENaC mediates the limiting pathway for sodium uptake by epithelial cells [1]. Mutations in CFTR result in hyperactivity of ENaC, which results in reduced ⁎ Corresponding author at: Ionis Pharmaceuticals, 2855 Gazelle Ct., Carlsbad, CA 92010, USA. E-mail address: [email protected]. (C. Zhao).

airway surface liquid, dehydrated mucus, and an increased accumulation of mucus in the airways [4]. Only ENaC channels composed of all three subunits have optimal amiloride-sensitive channel activity [5]. When the α-ENaC subunit was expressed in Xenopus oocytes, a small amount of amiloride-sensitive current was generated suggesting this is the pore forming subunit, whereas expression of the β or γ-ENaC subunits resulted in no current [1]. The co-expression of all three together increased current over 100-fold [1] indicating that all three subunits expressed together is critical for maximal activity. It is thought that the α subunit forms functional channels, and the β and γ subunits are important for efficient trafficking of the heteromeric channel to the plasma membrane. Homozygous deletion of genes encoding any of the three ENaC subunits in mice results in death within 48 h of birth [6]. When Scnn1a, the gene encoding the α subunit, is

https://doi.org/10.1016/j.jcf.2018.07.006 1569-1993© 2018 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved. Please cite this article as: Zhao C, et al, Antisense oligonucleotide targeting of mRNAs encoding ENaC subunits α, β, and γ improves cystic fibrosis-like disease in mice, J Cyst Fibros (2018), https://doi.org/10.1016/j.jcf.2018.07.006

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deleted in mice, the amiloride-sensitive sodium transport and ability to clear liquid from the lungs is abolished; deletion of genes encoding either the β or the γ subunit (Scnn1b and Scnn1g, respectively) results in death from hyperkalemia [6] indicating that Scnn1a may be more crucial to proper sodium channel function in the lung. Antisense oligonucleotides (ASOs) and siRNAs targeting Scnn1a, Scnn1b, and Scnn1g were utilized to elucidate the roles of each subunit in respiratory tract cells including rat ATII cells [7], human bronchial epithelial cells [8, 9], X. laevis oocytes and human nasal epithelial cultures [10]. Only ASOs targeting Scnn1a resulted in a decrease in the apical membrane density of nonselective cation channels [7], or reduced amiloride sensitive current and conductance [10]. siRNAs targeting mRNAs encoding any of the subunits, however, resulted in reduction in ENaC activity in human bronchial epithelial cells while siRNA against β ENaC resulted in the strongest reduction at the mRNA and sodium current levels [8]. We have shown that reducing ENaC α subunit prevents and reverses CF lung diseases in mouse models [11]. However, the individual contribution of each ENaC subunit to its channel activity and CF lung pathogenesis in adult mice is not yet known. In this report, a comprehensive study defining the in vivo role of the individual ENaC subunits by specific inhibition of each followed by comparison in an animal CF model would help elucidate the potential of each subunit as a therapeutic target. We have previously shown that an ASO targeting the mRNA encoding the α subunit of ENaC was effective in mouse models of cystic fibrosis [11]. ASOs were tested by aerosol delivery in the βENaC transgenic mouse model [9], the Nedd4L KO mice [12], and in mice with ASO-mediated depletion of Nedd4L [11]. All models exhibit CF-like phenotypes and inhaled Scnn1a-specific ASO reduced channel activity, mucus production, inflammation, and airway hyper-responsiveness in each model [11]. ENaC ASO delivered locally to the lung resulted in an ~90% reduction of Scnn1a mRNA in the lung with no reduction in kidney and no resulting hyperkalemia [11]. In the present study, we compared ASO-mediated reductions of the α, β, and γ subunits of ENaC in an adult onset CF-like mouse model. Our results demonstrate that ASO-mediated inhibition of expression of any of the three ENaC subunits effectively improved CF-like lung disease in adult mice.

initial experiments and were therefore used in subsequent experiments are indicated in bold face type. Oligonucleotides were synthesized as described [11]. 2.2. Quantification of mRNA Whole lungs or fractioned lung cells were homogenized in RLT buffer (Qiagen). Total RNA was prepared using the Qiagen RNAeasy mini kit. 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 ASOs were suspended in 0.9% sodium chloride (Baxter Healthcare). ENaC and control ASOs were administered twice weekly by orotracheal instillation at a dose of 5 mg/kg. For the Nedd4L and house dust mite (HDM) prevention model, mice were treated with ENaC and control ASOs for 3 weeks before the challenge with Nedd4L ASO (8 mg/kg weekly orotracheal instillation for 3 weeks), or HDM (see Supplemental Methods for details). In the Nedd4L reversal model, after 3 weeks of Nedd4L ASO challenge, mice were given ENaC or control ASO orotracheally for 3 weeks, twice weekly at 5 mg/kg, while continuing the Nedd4L ASO challenge. 2.4. 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. Additional methods can be found in the supplemental section 3. Results 3.1. The specificity of ENaC subunit-targeting ASOs upon delivery to lung

2. Methods 2.1. Oligonucleotides All ASOs were 16 nucleotides in length with a phosphorothioate backbone and three constrained ethyl (cEt) residues at each termini (the 3–10-3 gapmer configuration). The sequences of oligonucleotides are: αENaC ASO1: AGCAACTCCG TTTCTT, αENaC ASO2: GAGCATCTAATACAGC, βENaC ASO1: GGATTATGCGATCAGG, βENaC ASO2: GTCGATAATGATCTCC, γENaC ASO1: GTCATAGGG AGACCAT, γENaC ASO2: ATGTCATAGGGAGACC, control ASO: GGCTACTACGCCGTCA, and the Nedd4L ASO: CCATTTTCAACCTCAA. The ASOs that were more potent in

ASOs have been successfully delivered by local administration to the lung where they distribute throughout the tissue and are able to reduce mRNA levels in multiple cell types including airway epithelial cells [11, 13]. cEt ASOs designed to specifically target mRNAs encoding each ENaC subunit, Scnn1a, Scnn1b, and Scnn1g, were identified in cell-based assays and in normal mice. To further evaluate the ASO activity and specificity in normal mice, the top two ASOs specific for each subunit were compared head to head by local delivery to the lungs twice a week for 3 weeks. ENaC levels have been demonstrated to be enriched in the 7% airway epithelial cell (AEC) fraction obtained by density gradient centrifugation [11]. Testing ENaC ASOs against the various subunits in naïve

Please cite this article as: Zhao C, et al, Antisense oligonucleotide targeting of mRNAs encoding ENaC subunits α, β, and γ improves cystic fibrosis-like disease in mice, J Cyst Fibros (2018), https://doi.org/10.1016/j.jcf.2018.07.006

C. Zhao et al. / Journal of Cystic Fibrosis xx (2018) xxx–xxx

mice resulted in greater than 90% reduction of the targeted mRNA in the 7% ENaC enriched fraction with little to no effect on mRNA of the other subunits (Fig. 1). Mice treated with ENaC subunit ASOs but not with Nedd4L ASOs have no lung pathology. The most potent ASO for each subunit was taken into subsequent experiments.

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3.2. ASOs targeting of ENaC subunits prevent Nedd4L ASOinduced CF-like lung disease Characterization of mouse models of CF demonstrated that both βENaC transgenic mice and Nedd4L KO mice may die within 3 weeks of birth [9, 12]. To evaluate the long-term

Fig. 1. ENaC ASOs specifically decrease levels of targeted ENaC subunit mRNAs. Wild-type mice were dosed orotracheally twice per week for 3 weeks with indicated ASO (5 mg/kg). Quantification of (A) Scnn1a, (B) Scnn1b, and (C) Scnn1g mRNA levels in fractions after density gradient centrifugation of dissociated lung cells are shown as a percentage of levels in 7% fraction, where ENaC is enriched, from untreated lungs. Please cite this article as: Zhao C, et al, Antisense oligonucleotide targeting of mRNAs encoding ENaC subunits α, β, and γ improves cystic fibrosis-like disease in mice, J Cyst Fibros (2018), https://doi.org/10.1016/j.jcf.2018.07.006

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effects of ASO-mediated reduction of ENaC subunits, we previously established an adult-onset model using ASOmediated reduction of Nedd4L. As with the Nedd4L KO mice [12], ASO-mediated depletion of Nedd4L resulted in reduction of Nedd4L mRNA and increases in ENaC protein, mucus markers (monitored at the mRNA level), PAS-positive goblet cells, inflammation, and airway hyper-responsiveness [11]. To evaluate whether ENaC subunit-targeted ASO treatment could prevent disease onset, adult C57Bl6 mice were given ENaC subunit ASOs twice a week for 3 weeks by orotracheal administration. After the 3 weeks of ASO dosing, Nedd4L ASO was administered by orotracheal administration once a week for 3 weeks. The ENaC ASO treatment was continued during the 3 weeks of Nedd4L ASO administration. ASOmediated depletion of Nedd4L in adulthood reduced survival significantly (Fig. 2A). Treatment with ASOs targeting any of the mRNAs encoding ENaC subunits almost completely prevented Nedd4L ASO-mediated death compared to the 40% mortality observed in the untreated and the control ASO-treated group (Fig. 2A). In addition, the body weight loss observed by Nedd4L ASO treatment was prevented in mice treated with ENaC subunit ASOs (Fig. 2B). Administration of Nedd4L ASO results in an increase in ENaC protein [11] although ENaC mRNA for all subunits is reduced (Fig. 2C). This is potentially due to a feedback response resulting in reduced ENaC transcription. ENaC ASOs reduced levels of Scnn1a, Scnn1b, or Scnn1g mRNA levels by 74%, 84% and 77%, respectfully (Fig. 2C). The ASOs were specific for the targeted ENaC mRNA (Fig. 2C). The expression levels of Scnn1g mRNA in naïve mice appears to be higher than Scnn1b which appears higher than Scnn1a. This is because the ENaC expression level of each subunit in lung is normalized to its respective level in the Nedd4L vehicle group and only suggests that the level of Scnn1g mRNA in naive is higher than that of the Nedd4L vehicle group, but not higher than levels of Scnn1a or Scnn1b mRNA. Nedd4L ASO treatment induces airway hyper-responsiveness (AHR) after methacholine (MCh) challenge [11]. Nedd4L ASO treatment increased the baseline value for AHR (Penh) compared to untreated (naïve) mice (Fig. 2D, 0 mg/ml Mch). In addition, all three ASOs targeting each ENaC subunits significantly reduced Penh to near naïve levels (Fig. 2D, E). Control ASO demonstrated no effect at reducing the increased AHR. To determine the effect of inhaled ENaC ASOs on sodium channel activity in the Nedd4L ASO-induced adultonset CF model, the tracheas of mice were removed to measure amiloride-sensitive conductance as previously described [11]. Nedd4L ASO treatment significantly increased sodium channel conductance compared to the untreated control group. We typically see an ~1.5 to 2-fold increase of lsc in the Nedd4L treated mice. ENaC ASOs specific for α, β or γ ENaC reduced Na+ conductance to near normal levels (Fig. 2F). Previous work showed that ENaC transgenic mice, Nedd4L KO mice and Nedd4L ASO treated mice have extensive regions of lungs infiltrated with neutrophils in the absence of a bacterial stimulus; this neutrophil infiltration can be detected in the bronchoalveolar lavage (BAL) [11, 12, 14]. All three ENaC

subunit ASOs effectively reduced the number of neutrophils recruited to the airways, whereas the control ASO had no effect (Fig. 2G). Eosinophil levels were not increased in the BAL of the Nedd4L ASO treated mice at time points when samples were analyzed. Nedd4L ASO treatment increased mucus markers at the mRNA level and increased PAS-positive mucus (Fig. 2H-L). Nedd4L ASO induces mucus in our model but because of the way we inflate mouse lungs with formalin to get the best histology sections, we typically don't see mucus plugging. In Nedd4L ASO-treated mice dosed with α, β or γ ENaC ASOs, levels of Muc5ac mRNA were reduced to the range observed in naive mice (Fig. 2H). In addition, each of the α, β and γ ENaC subunit ASOs decreased inflammation and mucus production to a similar level in these mice, whereas the control ASO did not (Fig. 2I-L).

3.3. ASO-mediated reduction of each ENaC subunit reverses Nedd4L ASO-induced CF-like lung disease We next evaluated whether ENaC ASOs could reverse CF-like disease after the Nedd4L ASO-induced phenotype had been established in adult mice. We previously showed that a 6-week Nedd4L ASO treatment elicited increased ENaC protein expression and CF-lung disease [11]. Upon subsequent careful examination, we found that a 3-week Nedd4L ASO treatment was sufficient to establish lung disease phenotypes (Supplemental Fig. 1). As a result, we use a 3-week Nedd4L ASO treatment regimen for the reversal study. After 3 weeks with Nedd4L ASO treatment, mice were dosed orotracheally with ENaC ASOs twice a week for 3 weeks at which time Nedd4L, Scnn1a, Scnn1b, and Scnn1g mRNA levels were measured. Nedd4L was reduced by approximately 50% in all Nedd4L ASO-treated groups (Fig. 3A) and the levels of mRNAs encoding ENaC subunits were specifically and significantly reduced by 63%, 84% and 69%, respectively, whereas the control ASO resulted in no reduction in Scnn1a, Scnn1b, or Scnn1g mRNA levels (Fig. 3B). In addition, α, β and γ ENaC ASO treatment increased survival (Fig. 3C). Nedd4L ASO treatment resulted in upregulation of Muc5ac (Fig. 3D). In addition, other mucus genes including AGR2, Gob5, and Muc5b were also upregulated (Supplemental Fig. 2A-C). Reduction in any of the ENaC subunits reduced levels of these mRNAs, whereas the control ASO had no effect (Fig. 3D and Supplemental Fig. 1A-C). PAS-positive mucus and inflammation were increased by Nedd4L ASO treatment compared to the naive mice (Fig. 3E, F). ENaC ASOs effectively reduced mucus and inflammation compared to the control ASO as shown by analysis of PAS-stained lung sections (Supplemental Fig. 1E-J). Quantitation of the number of PASpositive airways in lung sections demonstrated a significant improvement upon dosing of the Nedd4L ASO-treated mice with any of the ENaC ASOs (Fig. 3E). To examine neutrophil recruitment to the airways, BAL was obtained and percent neutrophils were determined. There was a large increase in the percent of BAL neutrophils in Nedd4L ASO-treated mice after 3 weeks of treatment (Supplemental Fig. 2A), and the percent

Please cite this article as: Zhao C, et al, Antisense oligonucleotide targeting of mRNAs encoding ENaC subunits α, β, and γ improves cystic fibrosis-like disease in mice, J Cyst Fibros (2018), https://doi.org/10.1016/j.jcf.2018.07.006

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Please cite this article as: Zhao C, et al, Antisense oligonucleotide targeting of mRNAs encoding ENaC subunits α, β, and γ improves cystic fibrosis-like disease in mice, J Cyst Fibros (2018), https://doi.org/10.1016/j.jcf.2018.07.006

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remained high after 6 weeks (Fig. 3F). ENaC subunit ASOs effectively reduced neutrophil recruitment (Fig. 3F). Mice treated for 3 weeks with Nedd4L ASO had increased AHR compared to naïve mice (Supplemental Fig. 2B, C). Treatment with ENaC ASOs after AHR had been established reduced AHR to levels observed in naïve mice (Fig. 3G-H). The control ASO had no effect. Nedd4L ASO-induced increases in sodium channel conductance were also reversed by ASOs targeting Scnn1a, Scnn1b, or Scnn1g (Fig. 3I). 3.4. ENaC ASOs improve AHR but not mucus in mouse asthma models ENaC may play a critical role in the pathogenesis of asthmatic exacerbations [15], and AHR has been demonstrated to be influenced by hydration of the airway surface layer. We therefore tested ENaC ASOs in a model of asthma induced by house dust mites (HDM) described previously [11]. In the HDM model, the levels of mRNAs encoding ENaC subunits were specifically and significantly reduced by 84%, 82% and 77% while the control ASO had no effect (Fig. 4A). The reduction of expression levels of each ENaC subunit correlated with a significant improvement of AHR with levels similar to that of untreated mice (Fig. 4B, C). HDM is known to cause a significant increase in eosinophils recruited to the airways. Although lung function was improved, eosinophil recruitment to the airways was not prevented by any of the ENaC subunit or control ASOs (Fig. 4D). Further, HDM is known to cause an increase in goblet cells and inflammation in treated lungs. PAS staining of lung sections demonstrated that ENaC subunit ASOs did not prevent HDM-induced goblet cell hyperplasia or mucus production in lungs or prevent inflammation (Fig. 4E–H). 4. Discussion It has been 30 years since experimental evidence first suggested that ENaC hyperactivity contributes to airway surface liquid depletion and reduced mucus clearance observed in cystic fibrosis patients [16, 17]. It has been shown that wildtype, but not F508del, CFTR inhibits ENaC [3, 18, 19], although the underlying mechanism is not yet fully understood. Sequencing of Scnn1a, Scnn1b, and Scnn1g, the genes encoding the three ENaC subunits, identified multiple

mutations in each subunit in patients with non-classical CF disease [20, 21]. Furthermore, mice that overexpress the β subunit of ENaC in the airway develop CF lung disease phenotypes further demonstrating the importance of ENaC in CF [9], supporting the role of ENaC hyperactivity in CF pathogenesis. Over the years, much effort has been made to identify therapies that rehydrate the airways of CF patients by targeting ENaC. The classical ENaC inhibitor amiloride was tested in clinical trials more than 20 years ago [22], but results were disappointing in long-term trials due to its short half-life and limited potency [23, 24]. Subsequent studies investigating more potent amiloride derivatives, such as phenamil and benzamil, showed little improvement [25]. A new ENaC analog, GS9411 (Parion sciences/Gilead) was discontinued due to induced hyperkalaemia in clinical trials [26]. Other ENaC-targeted strategies are being explored including inhibition of ENaC subunit expression with short interfering RNAs [8, 27, 28] and antisense oligonucleotides [7, 11] and ENaC inhibition by peptides from the SPLUNC1 inhibitory domain [4]. ENaC is composed of three homologous subunits (α, β, and γ) that form the channel [29]. Investigations that specifically inhibit each subunit utilizing siRNA or antisense oligonucleotides shed some light on the contribution of each subunit to this ion channel [7, 8, 10, 27, 28] but more effort is necessary to better understand the role of these subunits. Previous studies failed to compare targeting α, β, and γ ENaC subunits in vivo, partially because of the lack of animal models which allow for in vivo ENaC hyperactivity measurements. We previously described an adult-onset CF model induced using an ASO targeting Nedd4L. This treatment leads to a CF-like lung disease in mice, and our previous experiments showed improvement of CF endpoints in this model with ASOs targeting the mRNA encoding the α subunit of ENaC [11]. To differentiate the role of each of the ENaC subunits, we designed ASOs to induce targeted depletion of Scnn1a, Scnn1b, and Scnn1g mRNAs. In the Nedd4L ASO-induced CF mouse model, specific inhibition of expression of each of the subunits reduced CF phenotypes. Data from several groups suggests that the α subunit is the most abundant of the three ENaC subunits [8, 30]. The fact that the α subunit alone can form an active Na+ channel supports our initial work with an ASO targeting this subunit and explained the pharmacological effects we observed in the previous study. However, in experiments in which each ENaC

Fig. 2. ENaC ASOs prevent Nedd4L ASO-induced CF-like lung disease. Adult C57 mice were orotracheally dosed twice per week for 6 weeks with ENaC ASOs or a control ASO (10 mg/kg). After 3 weeks of ASO dosing, the Nedd4L ASO was administered orotracheally once a week for 3 weeks (8 mg/kg). At the end of the 6week treatment period, analyses were conducted. (A) Percent survival compared to naïve mice (n = 24–32 mice/group). (B) Body weights compared to naïve mice. Values are means ± s.e.m.; *P 0.05 (Student's t-test vs. vehicle-treated mice). (C) ENaC subunit mRNA levels (n = 4/group). Values are means ± s.e.m.; *P 0.05 (Student's t-test vs. vehicle-treated mice). (D) Penh as a function of MCh concentration (n = 8–12 mice/group). The area under the curves for the naïve and ENaC ASO treated groups are significantly different from the Nedd4L ASO treatment group and the control ASO treatment group. (E) Penh at 25 mg/ml MCh. Values are means ± s.e.m.; *P 0.01 (Student's t-test vs. vehicle-treated mice). (F) Amiloride-sensitive short-circuit current (Isc) (n = 6–8 mice/group). Data were normalized to data from naïve group. Values are means ± s.e.m.; *P 0.01 (Student's t-test vs. vehicle-treated mice). (G) Percent BAL neutrophils from naive mice and mice treated with Nedd4L ASO and vehicle or indicated ASOs (n = 6–8 mice/group). Values are means ± s.e.m.; *P 0.01 (Student's t-test vs. vehicle-treated mice). (H) Levels of Muc5ac (n = 4 mice/group). Values are means ± s.e.m.; *P 0.01 (Student's t-test vs. vehicle-treated mice). (I-L) PAS staining showed mucus accumulation and inflammation in representative Nedd4L ASO-treated mice that were treated with control ASO or with ENaC subunit specific ASOs.

Please cite this article as: Zhao C, et al, Antisense oligonucleotide targeting of mRNAs encoding ENaC subunits α, β, and γ improves cystic fibrosis-like disease in mice, J Cyst Fibros (2018), https://doi.org/10.1016/j.jcf.2018.07.006

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Please cite this article as: Zhao C, et al, Antisense oligonucleotide targeting of mRNAs encoding ENaC subunits α, β, and γ improves cystic fibrosis-like disease in mice, J Cyst Fibros (2018), https://doi.org/10.1016/j.jcf.2018.07.006

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Fig. 4. ENaC ASOs reverse lung function declines in an HDM-induced model of asthma. Adult AJ mice were dosed orotracheally twice per week for 3 weeks with ENaC ASOs or control ASO (10 mg/kg). After 1 week of ASO dosing, HDM was administered orotracheally once per week for 3 weeks. Lung function was evaluated at the end of the treatment period. (A) Levels of ENaC subunit-encoding mRNA (n = 4 mice/group). (B) AHR exposed to dose response of Mch (Penh) (n = 12 mice/group). (C) Penh at 25 mg/ml Mch. (D) Percent eosinophil in BAL (n = 8 mice/group). (E-H) PAS-stained lung sections from ENaC ASO and control ASO treatment groups.

subunit was overexpressed in mice, only overexpression of βENaC resulted in CF-like phenotypes suggesting that its low levels in the lower airway is the rate limiting factor for airway sodium absorption [9]. The ASO targeting of Scnn1b, which encodes the β subunit, was more effective than ASOs targeting mRNAs encoding the α and γ subunits as demonstrated by channel current, improvements in lung function, inflammation,

and mucus accumulation in both prevention and reversal studies (Figs. 2 and 3), which could be contributed by better target reduction induced by ASO against the β subunit. These findings are similar to that previously described in human bronchial cells [8], in which siRNA complementary to Scnn1b demonstrated more effective mRNA and Na+ channel activity reduction than siRNAs targeting mRNAs encoding the other

Fig. 3. ENaC ASOs reverse Nedd4L ASO-induced CF-like lung disease. Adult naïve mice were treated orotracheally once per week for 6 weeks with Nedd4L ASO (8 mg/kg). After 3 weeks of Nedd4L ASO treatment, ENaC ASOs or control ASO (10 mg/kg/dose) were given orotracheally twice a week for 3 weeks. At the end of the 6-week treatment period, analyses were conducted. (A) Nedd4L mRNA levels (n = 4 mice/group). (B) Levels of ENaC subunit-encoding mRNAs. (C) Survival compared to naïve mice (n = 20/group). (D) Levels of Muc5ac (n = 4 mice/group). (E) Quantitation of PAS-positive airways. (F) Percent neutrophils in BAL (n = 8 mice/group). (G) Airway hyper-responsiveness (Penh) (n = 12 mice/group). (H) Penh at 50 mg/ml MCh. (I) Amiloride-sensitive short-circuit current (Isc) (n = 6–8 mice/group). Please cite this article as: Zhao C, et al, Antisense oligonucleotide targeting of mRNAs encoding ENaC subunits α, β, and γ improves cystic fibrosis-like disease in mice, J Cyst Fibros (2018), https://doi.org/10.1016/j.jcf.2018.07.006

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subunits. The siRNAs used in this previous investigation caused simultaneous reduction in levels of Scnn1a and Scnn1g, which made it impossible to rule out the possibility that the observed channel activity reduction was due to the reduction of one of the subunits. It has been reported that γENaC plays a more potent role than βENaC in trafficking the channel complex to the plasma membrane [5]. The γENaC ASO had similar pharmacological effects to the αENaC ASO. Overall, our current work suggests that ASO-mediated reduction of any of the three ENaC subunits improve disease endpoints in a CF mouse model to a degree that correlates with reduction of the targeted mRNA. In the HDM study, as in the Nedd4L ASO-induced CF model, airway hyper-responsiveness was improved by reducing the expression of each ENaC subunit. ENaC may contribute to AHR through a variety of mechanisms. The mechanism that links overexpression of ENaC to AHR has not been defined, but AHR is improved by inhibiting the expression of each of the ENaC subunits. CFTR and ENaC are important channels for maintaining epithelial fluid and electrolyte transport and play a key role in AHR [15]. One component of AHR is linked to inflammation, although the precise mechanism is not well understood [31]. Neutrophil depletion has been demonstrated to reduce AHR [32]. In the CF mouse model, increased neutrophil levels are reduced by α, β and γ ENaC ASO treatment. In the HDM model, eosinophil and not neutrophil levels are increased which are not reduced by α, β or γ ENaC ASO treatment. Whereas lung inflammation and mucus were reduced in the CF mouse model after reduction of each ENaC subunit, these measures were not significantly improved in the HDM model. Mucus accumulation results from different mechanisms in these models. Mucus accumulation and plugging of airways occurs in the CF mouse models and in CF patients without goblet cell metaplasia [33]. In the HDM model, goblet cell metaplasia and inflammation occurs due to increased Th2 cytokine production [34]. Another difference between the CF mouse model studies and the HDM studies is the strain of mice used, which may also play some role in the differential effect on mucus and inflammation after ASO induced ENaC subunit reduction. The role of ENaC in increasing AHR needs to be further addressed. In conclusion, this study mainly sought to establish if it is possible to reduce ENaC channel activity and improve CF phenotypes in a mouse CF model by reducing individual ENaC subunit levels with antisense oligonucleotides. Our data demonstrate ASOs targeting of ENaC different subunits similarly improve Nedd4L ASO-induced CF-like lung disease, when treated before or after the establishment of the disease. Antisense oligonucleotides have improved with respect to their target specificity and stability since the first introduction of antisense technology almost 40 years ago [35]. Antisense technology offers advantages including high selectivity, long half-life thus less frequent administration, and lack of systemic exposure. The ability to effectively target ASOs in humans by inhalation has been previously demonstrated [36, 37]. In addition, ASO drugs are well suited for combination therapy and are expected to work well in combination with CFTR

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modulators. We are initiating a phase one clinical trial targeting the alpha ENaC subunit later this year with additional ENaC subunits an option for future trials. We expect that antisense therapy that reduce levels of the α, β, or γ subunit of ENaC would provide potent and safe treatment options for CF regardless of the underlying CFTR mutations. Declaration of interest and support Ionis employees and this research supported by Ionis Pharmaceuticals, Inc. Author contributions C.Z., J.C., and S.G. contributed to study design. C.Z., J.C., T.L. and D.B. contributed to in vivo studies and data analysis. C.Z., J.C, and S.G. wrote and/or edited the manuscript. Acknowledgments We thank T. Reigle for assistance with figures and manuscript preparation. We thank X. Xiao, B. DeBrosse-Serra and Y. Jiang for tissue processing. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.jcf.2018.07.006. References [1] Canessa CM, Schild L, Buell G, Thorens B, Gautschi I, Horisberger JD, et al. Amiloride-sensitive epithelial Na+ channel is made of three homologous subunits. Nature 1994;367(6462):463–7. [2] Anderson MP, Gregory RJ, Thompson S, Souza DW, Paul S, Mulligan RC, et al. Demonstration that CFTR is a chloride channel by alteration of its anion selectivity. Science 1991;253(5016):202–5. [3] Mall M, Hipper A, Greger R, Kunzelmann K. Wild type but not deltaF508 CFTR inhibits Na+ conductance when coexpressed in Xenopus oocytes. FEBS Lett 1996;381(1–2):47–52. [4] Hobbs CA, Da Tan C, Tarran R. Does epithelial sodium channel hyperactivity contribute to cystic fibrosis lung disease? J Physiol 2013; 591(18):4377–87. [5] Konstas AA, Korbmacher C. The gamma-subunit of ENaC is more important for channel surface expression than the beta-subunit. Am J Physiol Cell Physiol 2003;284(2):C447–56. [6] Hummler E, Barker P, Gatzy J, Beermann F, Verdumo C, Schmidt A, et al. Early death due to defective neonatal lung liquid clearance in alphaENaC-deficient mice. Nat Genet 1996;12(3):325–8. [7] Jain L, Chen XJ, Malik B, Al-Khalili O, Eaton DC. Antisense oligonucleotides against the alpha-subunit of ENaC decrease lung epithelial cation-channel activity. Am J Physiol 1999;276(6 Pt 1): L1046–51. [8] Caci E, Melani R, Pedemonte N, Yueksekdag G, Ravazzolo R, Rosenecker J, et al. Epithelial sodium channel inhibition in primary human bronchial epithelia by transfected siRNA. Am J Respir Cell Mol Biol 2009;40(2):211–6. [9] Mall M, Grubb BR, Harkema JR, O'Neal WK, Boucher RC. Increased airway epithelial Na+ absorption produces cystic fibrosis-like lung disease in mice. Nat Med 2004;10(5):487–93. [10] Sobczak K, Segal A, Bangel-Ruland N, Semmler J, Van Driessche W, Lindemann H, et al. Specific inhibition of epithelial Na+ channels by

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Please cite this article as: Zhao C, et al, Antisense oligonucleotide targeting of mRNAs encoding ENaC subunits α, β, and γ improves cystic fibrosis-like disease in mice, J Cyst Fibros (2018), https://doi.org/10.1016/j.jcf.2018.07.006