# Restoring Chloride Efflux in Cystic Fibrosis with TMEM16a Antisense Oligonucleotides

Christie Mitri, Nathalie Rousselet, Pauline Bardin, Madara Dias

Wickramanayaka, Tobias Foussignière, Gabrielle Dupuis, Marion Leblanc,

Victoire Gournet, Florence Sonneville, Harriet Corvol, et al.

► To cite this version:

Christie Mitri, Nathalie Rousselet, Pauline Bardin, Madara Dias Wickramanayaka, Tobias Foussignière, et al.. Restoring Chloride Efflux in Cystic Fibrosis with TMEM16a Antisense Oligonucleotides. Molecular Therapy, 2025, Online ahead of print. 10.1016/j.ymthe.2025.08.045. hal-05379256

HAL Id: hal-05379256

https://hal.science/hal-05379256v1

Submitted on 24 Nov 2025

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1 Restoring Chloride Efflux in Cystic Fibrosis with TMEM16a Antisense

2 Oligonucleotides

3 Christie Mitri¹, Nathalie Rousselet¹, Pauline Bardin¹, Madara Dias Wickramanayaka¹, Tobias

4 Foussignière¹, Gabrielle Dupuis¹, Marion Leblanc¹, Victoire Gournet¹, Florence Sonneville¹,

5 Harriet Corvol¹,², Olivier Tabary¹*

6

7 ¹Sorbonne Université, Inserm, Centre de Recherche Saint-Antoine, CRSA, F-75012 Paris, France

8

²Département de Pédiatrie Respiratoire, Hôpital Trousseau, AP-HP, Paris, France

10 *Correspondence and requests for reprints should be addressed to Olivier Tabary

11 Email: olivier.tabary@inserm.fr

12 **Short title:** New treatment for all cystic fibrosis patients

13
14 ABSTRACT

Patients with cystic fibrosis (CF) who are non-responsive to treatments due to specific mutations need alternative CFTR-independent therapies. This study aims to assess the impact of TMEM16a expression by a specific oligonucleotide (TMEM16a ASO) on dysregulated parameters in CF, which will help prepare for preclinical studies. In this study, we analyzed the effects of TMEM16a oligonucleotide within a CF context by evaluating the impact, optimal administration route, toxicity, and specificity in primary cells and various mouse models. The oligonucleotide enhances TMEM16a activity, increases Cl⁻-efflux, and improves mucociliary clearance in cells from all individuals tested with CF. TMEM16a antisense oligonucleotide (ASO) effect is detectable in mice 30 days after subcutaneous injection, enhances TMEM16a mRNA expression, and significantly extends the lifespan of CF mice. Acute administration of 50 times the effective dose showed no toxicity. Importantly, TMEM16a ASO is highly specific, not inducing inflammation or altering intracellular calcium mobilization and cell proliferation, instilling confidence in its potential effectiveness. Our study demonstrates that TMEM16a ASO can compensate for CFTR deficiency in CF models. Additionally, it's important to note that this strategy could apply to all CF patients, regardless of their CFTR mutation, thereby broadening the scope of treatment options for CF.
30 INTRODUCTION

Cystic fibrosis (CF) is an autosomal recessive genetic disease caused by mutations in the CF
Transmembrane conductance Regulator (CFTR) gene.¹ More than 2,100 mutations affecting
protein production or activity have been identified. In CF, mutations are categorized into different
classes based on their effects on CFTR protein function, ranging from class I mutations, which
result in the absence of protein production, to class VI mutations, characterized by the production
of a protein with a modified function. In CF, impaired Cl⁻ ion efflux coupled with sodium (Na⁺)
ion hyperabsorption results in epithelial surface dehydration and the production of persistent, thick
mucus that obstructs the lungs, pancreatic ducts, biliary tract, intestines, and male reproductive
tract.¹ The most common clinical manifestations of CF include chronic lung infections and
pulmonary inflammation, which result in progressive tissue damage and loss of function, leading
to increased morbidity and mortality.

Over the past decade, new treatments have revolutionized CF clinical management by introducing
innovative therapies that target and activate CFTR at the apical membrane, especially with
Elexacaftor/Tezacaftor/Ivacaftor (ETI).² Unfortunately, these approaches are mutation-dependent
and still not effective for 10-15% of people with CF due to class I mutations or drug intolerance.³
Therefore, it is necessary to develop alternative therapies that are CFTR-independent to enhance
chloride activity and/or improve mucociliary clearance. Sodium channel (ENaC) blockers have
been under development for some time to decrease ENaC hyperactivity in CF, although none have
yet progressed through pivotal trials to licensing.⁴

In 2008, three research teams identified TMEM16a (Anoctamin-1 or ANO1) as the gene encoding
the calcium-activated chloride channel (CaCC),⁵⁻⁷, and it has been proposed as an alternative
strategy to compensate for CFTR deficiency.⁸ Therefore, a strategy to enhance TMEM16a
expression or activity could bypass CFTR deficiency and benefit all people with CF (pwCF), especially those ineligible for current curative treatments. This strategy is highly controversial because multiple studies indicate that overexpression may play a role in regulating mucus secretion and is present in many types of cancer, including those of the digestive system such as esophageal squamous cell carcinoma, gastric, colorectal, and pancreatic cancers.⁹⁻¹¹ Therefore, our primary approach is not to excessively express TMEM16a but to return its activity to normal levels, which have been disrupted in pwCF. We initially demonstrated the specific inhibitory role of microRNA-9 (miR-9) in regulating TMEM16a expression using a luciferase activity assay in the presence of a mimic or antagomiR, a finding that other research groups across various pathologies have since corroborated.¹²⁻¹⁵

Building on this insight, we developed a targeted approach based on antisense oligonucleotides (ASOs) to specifically prevent only miR-9 from binding to the 3' UTR of TMEM16a mRNA through competitive interaction, thereby enhancing translation and its expression.¹⁶ ASOs are synthetically modified single-stranded nucleic acid sequences, typically ranging from 12 to 30 nucleotides in length, that selectively bind to specific complementary RNA targets through Watson-Crick base pairing.¹⁷ The sequence of TMEM16a ASO was designed to be specific to human and mouse sequences. Locked nucleic acids (LNA) and phosphorothioate modifications were introduced to enhance the stability, affinity, and specificity of the oligonucleotides. We selected this strategy due to ASO's reported benefits: specificity, versatility, and therapeutic potential. In this article, we strengthened the initial observation published on F508del cells by incorporating additional cell models, electrophysiological data, and toxicity studies, and we broadened this research to include various cells and mice with CFTR class I mutations.
Our research suggests that this strategy can be implemented in pwCF to restore Cl⁻ efflux and mucociliary clearance without causing inflammation or toxicity. Furthermore, our research has demonstrated that this strategy can treat various organs affected by cystic fibrosis, increasing the life expectancy of CF mice and enhancing the fertility of CF male mice. Our findings support the concept that an increase in TMEM16a Cl⁻ activity may represent a promising avenue for drug development for all pwCF.

## 81 RESULTS

### 82 TMEM16a ASO potentiates CaCC chloride efflux in class II CF cells

To determine whether TMEM16a ASO induces TMEM16a expression by binding to the TMEM16a 3'UTR, HEK cells were stably transfected with a luciferase reporter vector containing the TMEM16a 3' UTR sequence. Cells were treated for 24 h with TMEM16a ASO at the specified concentration. The results confirmed that under basal conditions, TMEM16a increases the activity of the 3' UTR TMEM16a, as visualized by the expression of luciferase (Figure 1, A). Initially, TMEM16a chloride activity was assayed using a halide-sensitive YFP probe. We demonstrated that TMEM16a ASO greatly enhanced the TMEM16a Cl⁻ channel activity in a dose-dependent manner compared to the control ASO, starting at 25 mM (Figure 1, B-C). These results were confirmed using various F508del cell types, including tracheal CF gland serous cells (KM4), bronchial epithelial cells (CUFI and IB3-1), primary human bronchial glandular cells (hBGC), and pancreatic cell line (CFPAC) (Figure 1, D). Compared to the cells treated with control ASO, the effects observed in Cl⁻ efflux assays are significant across all tested CF cells, even though the degree of correction varies. In CF cells treated with TMEM16a ASO, the Cl⁻ efflux increased by 20-25% in KM4, CUFI, and IB3-1, reaching nearly 50% in the most responsive cells, such as
hBGC and CFPAC (Figure 1, D). These results appear proportional to the basal expression of TMEM16a in the various cells tested (data not shown).

The transepithelial Cl⁻ current, driven by cAMP for CFTR or Ca²⁺ mediated signaling pathways for TMEM16a, was measured using the Ussing chamber method. In primary human bronchial epithelial cells (hBEC), we observed a strong CFTR-dependent Cl⁻ efflux in non-CF hBEC (Fig. 1, E-G). This activation was abolished by the specific FTR inhibitor Inh-172 compared to CF hBEC cells. UTP-induced Ca²⁺-mediated Cl⁻ efflux was significantly higher in both ASO TMEM16a-treated CF and non-CF cells compared to the control group (Figure 1, E-G). The TMEM16a-mediated Cl⁻ efflux was strongly inhibited by the specific inhibitor of TMEM16a, Ani9,¹⁸ demonstrating the specificity of the TMEM16 ASO activation (Figure 1, H).

## TMEM16a ASO increases Cl⁻ activity in class I CF cells

On CF primary cells cultured in ALI with class I mutations (2184delA/W1282X) treated with TMEM16a ASO, we observed a strong and significant increase in the UTP response (P<0.01) (Figure 2, A-B). TMEM16a ASO also induced significant and robust activation of mucociliary clearance, as shown by the movement of fluorescent beads across all tested mutations (Figure 2, C-F). The effects of ETI (Elexacaftor, Tezacaftor, and Ivacaftor) on CFTR were significantly positive in cells with at least one F508del mutation (Figure 2, D-E) but not in cells carrying two class I mutations (Figure 2, F). Interestingly, the combination of TMEM16a ASO and ETI showed additive effects on F508del/F508del cells compared to cells treated with either TMEM16a ASO or ETI alone. However, a significant increase in Cl⁻ efflux was observed in cells treated with TMEM16a ASO (45.5±0.3 vs. 96.3±0.3) (Figure 2, F).

In addition, TMEM16a ASO had no direct or intrinsic bacteriostatic or bactericidal effect on *Pseudomonas aeruginosa* (*P. aeruginosa*), the most prevalent bacteria present in CF lungs
(Figure 2, G). Similar results were observed in the presence of cells (Figure 2, H), indicating that the treatment was not involved in cellular defense (Figure 2, I).

## TMEM16a ASO is specific and not toxic

We conducted an *in vitro* pharmaceutical profiling as described by Bowes *et al*. to identify undesirable off-target interactions.¹⁹ No notable adverse drug binding of TMEM16a ASO was observed (Figure 3, A and Table S1). *In vitro* pharmacological profiling involves screening compounds against various targets (receptors, ion channels, enzymes, and transporters) that differ from the intended therapeutic target(s) to identify specific molecular interactions that could lead to adverse human drug reactions. We also evaluated TMEM16a ASO toxicity after 24 hours of treatment on CFBE410⁻ cells by measuring LDH levels and identified no differences up to 200 mM (Figure 3, B). Similar results were obtained on hBEC (data not shown). No significant induction of cytokines, including IL-8, the most activated cytokine in CF lungs, was observed in cell lines with different mutations (Figure 3, C and Figure S1, A). Our study also demonstrated that ASO TMEM16a does not activate TMEM16a by increasing intracellular Ca²⁺ (Figure 3, D). Similarly, we didn’t observe a significant change in cell proliferation compared to cells treated with the control ASO (Figure 3, E).

To confirm the specificity of the ASO TMEM16a, we conducted a transcriptomic analysis on primary cells cultured in an air-liquid interface with a class II mutation (F508del/F508del) and a class I mutation (2184delA/W1282X). Cells were treated with 50 nM for 24 h (Figure 3, F-G), 48 h (Figure S1, B-C), and 200 nM (data not shown). TMEM16a mRNA is overexpressed in all cases and ranks in the TOP10 genes when cells are treated with TMEM16a ASO compared to the control ASO (Table 1 left & right), with a log fold change between 3.2 and 3.8. Consistent with
the results above, our transcriptomic analysis showed no significant increase in RNAs associated
with inflammation or evidence of cellular toxicity, even at a concentration of 200 nM for 48 hours.

## Subcutaneous administration allows for optimal distribution and is not toxic to mice

To determine the optimal route of administration, fluorescent TMEM16a ASO was administered to nude mice (10 mg/kg) through intravenous, subcutaneous, intraperitoneal injections, or intranasal instillation (Figure 4, A). Fluorescence in mice was recorded weekly for one month (Figure 4, B-E). With the intraperitoneal injection, the drug only dispersed to a limited extent, even 30 days after administration. In contrast, intravenous, subcutaneous, and intranasal routes showed better distribution in all tissues as early as 7 days (Figure 4, C-D). After day 21, we observed a decline in fluorescence levels for all conditions (Figure 4, E). At 30 days, the mice were euthanized, and the fluorescence of TMEM16a ASO in the major organs was quantified (Figure 4, F). For all routes of administration, as expected for other studies with ASOs, we observed a significant accumulation in the kidney and liver. A more effective distribution of fluorescence is noted with intranasal or subcutaneous administration (Figure 4, F). On day 30, we observed a significant increase in TMEM16a mRNA expression in the lung after intravenous, intranasal, and subcutaneous administration compared to the control ASO (Figure 4, G). The same results were found in the liver, kidney, and intestine. Pharmacokinetic and pharmacodynamic experiments using mass spectrometry were performed. Mice were administered TMEM16a ASO subcutaneously (10 mg/kg), and we demonstrated rapid diffusion of the ASO to the organs. We observed a peak at 4 hours, followed by a gradual decrease in the presence of ASO over time. We detected the presence of TMEM16 ASO at a very low concentration on day 30 in various organs (Figure 4, H). Since the primary target organs in humans are the lungs and, in mice, the intestines, we have chosen subcutaneous injection, a common method of ASO administration. In plasma,
TMEM16a ASO was undetectable after 24 hours (Fig. 4, I). Toxicity in mice was evaluated in vivo by observing general signs (morbidity and mortality, clinical symptoms, body weight, food, and water intake) three times a week for 14 days. Mice were injected subcutaneously with six doses ranging from 0 to 500 mg/kg. At 50 times the active dose (500 mg/kg), we observed no significant differences in the mice's behavior, macroscopic autopsy, or histological analysis (data not shown). More we complete this study by analyzing the tissue after one year of treatment. A portion of the tissue was embedded in paraffin, and the remainder was stored at -80°C for subsequent examination of myeloperoxidase (MPO). Sections 4µm thick were stained with hematoxylin and eosin before histological examination (Figure S2, A-B). Tissue injuries were scored according to the following criteria: hemorrhage (+1), cell infiltration (+1), and remodeling (+1). The data were then compared to those of the WT mice tissues. After one year of treatment, we observed no significant differences in the mice's behavior, macroscopic autopsy, histological analysis (Figure S2, C), or in MPO quantitation that reflected neutrophil infiltration (Figure S2, D).

In blood samples, parameters reflecting liver toxicity (ALT and AST), kidney toxicity (urea), and pancreas toxicity (amylase) remained consistent across all tested doses (Figure S3, A-D), and no differences were found in the hematological analysis (data not shown). In CF mice receiving repeated injections every 15 days for over 200 days, the levels of transaminases, urea, and creatinine (Figure S3, E-F) were comparable to those in control mice. The histopathological observations of the liver, kidney, intestine, and lung were also similar (data not shown).
TMEM16a ASO decreases the physiopathology of CF mice

In the following experiments, we used CF F508del mice (Figure 5) and CF G542X mice (Figure 6).
We treated CF mice before weaning, on days 11 and 18 after birth, with subcutaneous injections
of control ASO or TMEM16a ASO (10 mg/kg). We continued treatment every 15 days after
weaning, as illustrated in Figures 5, A and 6, A. With ASO TMEM16a treatment, median survival
ranged from 36 days in control mice to 196 days in F508del mice treated with TMEM16a ASO
(p<0.0001) (Figure 5, B). As previously reported, CF mice died from severe intestinal
obstructions, especially in the ileum. Therefore, after only two injections on days 11 and 18, the
treatment appeared to provide temporary protection to the F508del CF mice (Figure S3, G). In
F508del mice, there was no significant difference in weight gain compared to WT (+/+) mice and
CF mice treated with TMEM16a ASO (Figure 5, C). Comparing these results to those of the
control mice is challenging due to the high mortality rate in younger mice and the small number
of mice surviving after 60 days or more (Figure 5, B). Macroscopic observation reveals no
difference in the intestines of CF-treated adult mice compared to those of normal mice (data not
shown). In contrast, untreated mice or those treated with control ASO exhibit significant intestinal
occlusion, as previously described (data not shown). After at least four weeks of treatment, we
also assessed gut inflammation in mice using two fecal biomarkers: lipocalin (Figure 5, D) and
calprotectin (Figure 5, E), which are secreted by neutrophils and monocytes, respectively. We
observed notably higher fecal lipocalin levels in F508del CF mice. Following treatment with
TMEM16a ASO, we observed a significant decrease in values compared to the value obtained
(p<0.01) (Fig. 5, D), returning to levels seen in WT mice. The same results were obtained with
fecal calprotectin; however, the difference between wild-type and TMEM16a ASO mice remained
significant (Figure 5E). In CF-treated mice, less ileal obstruction with more fluid feces was observed compared to non-treated mice (data not shown).

As previously reported, macroscopic analysis of the vas deferens showed that male -/- mice treated with Ctl ASO displayed numerous malformations with significant variation.²⁰⁻²² In most cases, we observed a 30% unilateral or bilateral shortening of the vas deferens in mice treated with the Ctl ASO, compared to 6.5% in TMEM16 ASO mice. In 79% of cases, the vas deferens narrowed at specific locations, a phenomenon not observed in wild-type mice (Table 2). These observations may elucidate the vas deferens atresia in these mice, rendering them non-functional and leading to infertility (Table 2 and Figure S4, A, 2nd generation, left). Most TMEM16a ASO-treated male mice were able to reproduce during the study, unlike the majority of mice treated with control ASO.

In our study, homozygous mice (F508del/F508del) chronically treated on day 11 after birth (Figure S4, A, 2nd generation right) were able to reproduce, and the pups were viable (Figure S4, A, 3rd generation, B-C). These third-generation CF mice, born to parents treated with TMEM16a ASO, survived the weaning stage and had a longer lifespan than CF mice born to untreated parents, even though these third-generation mice received ASO control treatment (control mice median survival: 55 vs. 41) (Figure 5, F).

We monitored fluorescence in newborn pups by injecting fluorescent TMEM16a ASO into gestating female mice before delivery. We observed a significantly higher level of fluorescence (Figure 5, G), confirming ASO transmission from the pregnant female to the pups. Additionally, we observed fewer abnormalities in the male reproductive systems of mice treated with TMEM16a ASO (Figure S4, D-E). The results for G542X mice (Figure 6) were similar to those for F508del mice (Figure 5), showing significance and an increase in life expectancy from 106 to 205 days
(p<0.0001) (Figure 6, B). Regarding weight gain, no significant difference was observed in G542X mice treated with TMEM16a ASO until day 90 compared to WT mice (Figure 6C). In this CF model, we observed significantly higher levels of fecal lipocalin and fecal calprotectin than in WT mice (Figure 6, D and E).

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Although new CF modulator treatments work well for many individuals with CF, they are only suitable for a subset of patients, leaving others with unresolved medical issues.² This study provides data supporting a new therapeutic approach using an antisense oligonucleotide (ASO) that targets the alternative chloride channel TMEM16a, aiming to restore its expression and indirectly compensate for the deficiency of CFTR protein in cystic fibrosis (CF). This new approach is independent of disease-causing CFTR mutations and could benefit all people with cystic fibrosis (pwCF), particularly those who are currently ineligible for curative treatments. The concept of rectifying the ion transport defect regardless of the *CFTR* genotype emerged in the early 1990s when studies showed that triphosphate nucleotides such as adenosine-5'-triphosphate (ATP) and uridine-5'-triphosphate (UTP) activate Cl⁻ efflux in both normal and CF airway epithelia.²³

Before the identification of airway epithelial CaCC in 2008, Denufosol was developed to treat CF lung disease. Despite some promising results in pwCF, the second phase 3 trial failed to demonstrate any benefits due to a lack of specificity and low stability in biological fluids.²⁴ This approach was later abandoned due to limited understanding of the involved channels. Our approach, utilizing ASOs in CF, an innovative therapeutic tool that binds specifically to the targeted mRNA through complementary Watson-Crick base pairing, can prevent such issues, as ASOs do not disrupt calcium (Ca²⁺) mobilization. This study also reveals that activating the TMEM16a channel enhances Cl⁻ activity and improves mucociliary clearance in vitro by modifying ionic movement in the airways, while mucin expression remains unchanged. In our approach, we have treated the mice before the onset of the disease to prevent their death. Based on the experience already acquired with CFTR modulators in pwCF and with antisense
oligonucleotides targeting ENaC in mice, we can assume that treatment in humans, once the disease is already established, could have beneficial effects.²⁵

Our research shows that TMEM16a increases the survival rate of CF mice following intestinal obstruction at an early age. It also relieves other symptoms, including infertility and intestinal blockage. The reduced expression of these channels in the gut contributes to the observed pathology in CF mice. Notably, the improvements in other organs are intriguing, as they have not been documented in pwCF treated with Trikafta. CF mouse models do not permit the observation of respiratory effects because pulmonary involvement is minimal in CF mice due to the presence of TMEM16a channels that are already present in the airways.²⁶,²⁷ It would be interesting to investigate the effects of treatment on other animal models that are more relevant to pulmonary disease, such as ferrets and pigs.²⁸ CFTR KO pigs are significantly affected by intestinal disease.

All newborn piglets experience meconium ileus, which is fatal unless corrective surgery is performed soon after birth to relieve the obstruction. It may be worth treating the mother to see if there is any improvement in the newborns. Phe508del pigs are also impacted, although the intestinal phenotype is somewhat less severe. New CF ferret models have been generated, including a gut-corrected CF KO ferret, which exhibits multi-organ disease and responds to the CFTR potentiator Ivacaftor.²⁹ However, there is currently little data available on TMEM16a expression in the airways of these models. These limitations indicate that using this model is complicated and should only be considered after a more thorough characterization of TMEM16a. Although these models have specific limitations, models representing class I mutations are still unavailable.

A major challenge of this strategy is to maximize the efficacy and minimize the toxicity of ASO drugs. This article presents various results from studies on cells and mice, demonstrating the low
toxicity of this approach, even after extended treatment. This confirms other strategies using ASO in CF models, where ASO is safe and well tolerated.³⁰,³¹ The strategies used for patients are either tailored to specific CFTR mutations or target other ion channels, such as the hyperactivated ENaC channel in pwCF.³² ASO technology has emerged as an appealing therapeutic modality for CF; however, this field has largely remained unexplored in the context of cellular studies.³³ The potency and toxicity of ASOs depend on their chemical modifications, off-target effects, and delivery methods.³² In your experiments, we didn't observe off-target effects on the different CF models analyzed. The transcriptomic analysis results showed indirect connections between this channel and many proteins. This indirect connection may arise from protein and channel interactions, but it could also be attributed to increased chloride activity. Regarding the transcriptomic analysis, TMEM16a ranks among the top ten most highly expressed and significant genes across all tested conditions. We also observed an increase in other genes, which may be an indirect consequence of the expression or activity of TMEM16a, as previously demonstrated for the CFTR protein.³⁴⁻³⁶

Our research shows that TMEM16a ASO has strong specificity, minimal toxicity, and good stability *in vivo*. An important aspect of oligonucleotide use is the route of administration envisaged and its accumulation in specific tissues, such as the liver and kidneys. Studies have shown that pulmonary administration and intravenous injection result in the same accumulation of oligonucleotides in the liver of mice 7 days after administration.³⁷ In our study, administering every 15 days is enough to improve the animals' survival significantly. These data are consistent with the literature, and the results obtained from the persistence of TMEM16a ASO from females to pups may explain the increase in pup survival observed in females treated only with control ASO.³⁸ Under this condition, chronic administration of TMEM16a ASO twice a month has
304 increased the survival of F508del mice from 36 days to 196 days, and from 106 to 205 days for
305 G542X mice. This strategy minimizes adverse side effects by using a minimal dose to restore
306 TMEM16a activity without causing any negative effects from over-induction.16 Through this
307 approach, we have not observed any modifications in the expression or activity of other chloride
308 channels. Concerns have been raised regarding whether the activation of TMEM16a could
309 negatively impact mucus secretion or cause vasoconstriction in the bronchi, as noted in another
310 approach.39 Our *in vivo* models showed no adverse effects, even when the animals were treated for
311 extended periods (over 1 year). The mucus secretion induced by TMEM16a overexpression is
312 more complex, underscoring the limitations of our understanding of cellular lung health
313 physiology.40 A publication by Amaral's team offers a partial answer by showing that TMEM16a
314 does not drive mucus production, which is confirmed by observation in the airways of CF mice.41
315 This strategy is also open for discussion, as many articles indicate that TMEM16a is present in
316 numerous cancers due to its pro-proliferative effects.42,43 Our objective was not to overexpress and
317 drastically increase TMEM16a activity but rather to selectively restore the profile seen in non-CF
318 cells in a targeted way to avoid adverse effects. Furthermore, no tumors were observed in CF mice
319 that were repeatedly treated with TMEM16a over a year.

320 Interestingly, the effects of TMEM16a ASO were not limited to lung improvements but could also
321 affect other CF manifestations when administered subcutaneously, as demonstrated by the effects
322 observed on gastrointestinal obstruction in our CF mouse. Gastrointestinal symptoms were
323 identified as the second priority through a questionnaire completed by over 1,100 pwCF,44 even
324 though some abdominal symptoms appear to be reduced with ETI therapy.44 Another unexpected
325 result was the improvement in fertility of male CF mice by restoring vas deferens abnormalities
326 when the mice were treated very early.20-22 These symptoms in mice are similar to those observed
in CF sufferers, although they differ during the development of the vas deferens.¹ In humans, 98%
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of men with CF are infertile due to obstructive causes, particularly congenital bilateral absence of
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the vas deferens, with abnormalities at birth.⁴⁵ In mice, vas deferens development continues after
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birth, so the positive effects observed with TMEM16a ASO are possible and could explain the
330
results; however, these results are not directly applicable to humans.⁴⁶ The observations noted will
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require targeted additional studies, such as histological analyses during vas deferens development
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and functional assessments of spermatozoa. Furthermore, these findings need further examination
333
to clarify the potential role of TMEM16a in vas deferens formation, since the available data are
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very limited.⁴⁷⁻⁴⁹ Nevertheless, these data indicate that this systemic treatment could benefit other
335
organs, such as alleviating intestinal symptoms in pwCF. Unfortunately, mice do not respond to
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Trikafta, making comparisons with this model impossible. This article demonstrates that
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TMEM16a produces effects similar to Trikafta in cells with class II mutations. Additionally, we
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have evidenced that our strategy can also benefit models with class I mutations, where Trikafta
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falls short. Moreover, our approach utilizing oligonucleotides is highly complementary to the
340
Trikafta strategy, as the differing mechanisms involved allow for enhanced effectiveness. In
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contrast to numerous CF strategies, this study proved effective in class I and II carrier cells and
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mice. Like us, one group proposed a more classical approach utilizing the molecule ETX001,
343
which has recently been developed to target TMEM16a. As observed in our approach, ETX001
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operates independently of Ca²⁺ efflux, ensuring specificity and minimizing secondary effects.⁵⁰
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Similarly, as previously demonstrated, this strategy restored Cl⁻ efflux both *in vitro* and *in vivo* in
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an ovine model.⁵² While the two approaches have similar goals, the TMEM16a ASO acts as a
347
corrector, specifically a facilitator of TMEM16a expression.¹⁶ In contrast, ETX0001 functions
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primarily as a potentiator, requiring TMEM16a expression in this context. Nonetheless, the
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proposed approach, which utilizes ETX001 and our TMEM16a method, could work together to activate the TMEM16a channel. This approach could be very beneficial for patients who are non-responders to modulators but require further analysis.

In conclusion, targeting TMEM16a can address multiple symptoms through a CFTR-independent strategy, applicable to all patients with cystic fibrosis (pwCF) alone or in combination with other CFTR-targeted approaches for eligible patients.

## METHODS

### Antisense oligonucleotides

TMEM16a ASO and Control ASO were synthesized by Qiagen (Les Ulis, France) as sodium salts.

The purification was performed using HPLC and sodium exchange, and the resulting material was provided for each study as lyophilized powder. Physiological serum was used for dilution of TMEM16a ASO and Control ASO. ASO was modified by adding phosphorothioate oligonucleotide linkages and locked nucleic acid (LNA). TMEM16a ASO and control ASO are composed of 15 nucleotides. TMEM16a ASO was designed to specifically bind the 3'UTR of TMEM16a in both human and mouse sequences. The sequence of TMEM16a ASO used is: AATCTTTGGTAGTAA, and the control ASO sequence is: ACGTCTATACGCCA.
367 **Cell culture and transfection**

368 As previously described, CFBE41o⁻, 16HBE14o⁻, CUFI-5 (LGC, France), and CFPAC-1 cell lines
369 were cultured and transfected with ASO (control or TMEM16a).⁵¹,⁵² The 16HBE14o⁻ cell lines
370 with different *CFTR* mutations were generated by the Cystic Fibrosis Foundation.⁵³ Primary
371 human bronchial epithelial cells (hBEC) were isolated from bronchial biopsies and supplied by
372 Epithelix SARL (Geneva, Switzerland).¹⁶ Following the manufacturer's instructions, proliferation
373 was quantified using the CyQUANT NF cell proliferation assay (Thermo-Fischer Scientific,
374 France).

375 **Bacterial strains and cultures**

376 An inoculum of luminescent PAO1 strain of *Pseudomonas aeruginosa* (*P. aeruginosa*) was grown
377 in Luria-Bertani (LB) medium at 37 °C. The bacterial growth of *P. aeruginosa* was monitored over
378 time by measuring luminescence with a plate reader. The MOI (multiplicity of infection) used is
379 0.01, 0.1, 1, and 10.

380 **TMEM16a chloride channel activity assay**

381 Short-circuit current (Isc) was measured under voltage clamp (Physiologic Instruments, San
382 Diego, CA). The differential composition of basal and apical Ringer solutions created the chloride
383 gradient across the epithelium. The basal Ringer solution contained: 145 mM NaCl, 3.3 mM
384 K₂HPO₄, 10 mM HEPES, 10 mM D-Glucose, 1.2 mM MgCl₂, and 1.2 mM CaCl₂. The apical
385 solution contained: 145 mM Na-Gluconate, 3.3 mM K₂HPO₄, 10 mM HEPES, 10 mM D-Glucose,
386 1.2 mM MgCl₂, 1.2 mM CaCl₂. Inhibitors and activators were added after stabilization of baseline
387 Isc: sodium channel blocker Amiloride (100 mM); Forskolin to induce cAMP activation of CFTR
388 (10 µM); CFTR inhibitor CFTR-Inh172 (10 µM) to specifically inhibit CFTR; UTP (100 µM) to
389 challenge the purinergic calcium-dependent Cl⁻ secretion, and Ani9 (10 µM) was utilized as a
specific inhibitor of TMEM16a. The short-circuit currents (Isc) were measured at 1 measurement
per 5-20 seconds. The change of Isc (ΔIsc) upon CFTRInh172 application served as an index of
CFTR function. Chemicals were purchased from Sigma (St. Louis, MO, USA).

In some experiments, TMEM16a channel activity was assessed by iodine quenching of the halide-sensitive YFP-H148Q/I152L protein. ⁵⁴

### Mucociliary clearance assay

As previously described, hBEC were transfected with control ASO or TMEM16a ASO daily for 1
hour over 3 days and/or stimulated with ETI (VX-445, Elexacaftor at 3 µM; VX-661, Tezacaftor,
TEZ at 10 µM; and VX-770, Ivacaftor at 1 µM) in Boston, Massachusetts, USA, for 24 hours.¹⁶

### In vitro pharmaceutical profiling

Pharmaceutical profiling was determined *in vitro*, following the recommendation of Bowes *et al.*
(20). Eurofins Cerep (Celle l'Evescault, France) performed all experimental studies.

### Cytotoxicity evaluation

The LDH (lactate dehydrogenase) cytotoxicity assay was conducted in vitro using the LDH-based
CytoTox 96® Non-Radioactive Cytotoxicity assay, following the manufacturer's guidelines
(Promega, France).

### Calcium mobilization

After treatment with TMEM16a ASO, cells were loaded with Fluo-4 dye, as previously
described.⁵⁵ The cells were incubated with the dye at 37°C for 30 minutes, washed with calcium-
409 free PBS, and then activated with 10 µM ionomycin. The cells were subsequently recorded using
410 a plate reader (BMG LabTech, France).

411 Transcriptomic experiments

412 For cells, miRNA and RNA were extracted using a Nucleospin miRNA kit (Macherey-Nagel,
413 Hoerdt, Germany). The concentration and quality of RNA and miRNA were evaluated using a
414 NanoDrop spectrophotometer, and library quality and size range were assessed using a
415 Bioanalyzer (Agilent Technologies, Leuven, Belgium). The library and transcriptomic analysis
416 were performed at the VIB Nucleomics Core (Leuven, Belgium). Samples were indexed to allow
417 for multiplexing. Libraries (2 nM) were sequenced on an Illumina HiSeq 4000 platform (Illumina,
418 San Diego, CA, USA). Single-end reads of 50 bp length were produced with a minimum of 1 M
419 reads per sample. The alignment files were generated for the reference genome using STAR 2.5.2b.
420 Reads from the alignment that are non-primary mappings or have a mapping quality of < 20 were
421 removed using Samtools 1.15.1. Quality control of raw reads was performed with FastQC v0.11.7.
422 The reads were sorted and indexed according to the chromosomes, and the resulting BAM files
423 were indexed with Samtools. The main sources of sample-specific variation (library size and RNA
424 composition) were corrected using full quantile normalization with the EDASeq package from
Bioconductor. For transcriptomic analysis and to correct for the potential batch effect, CombatSeq
from the sva-devel R package (https://doi.org/10.1093/nargab/lqaa078) was used.

Independent replicates were incorporated, and validation was conducted using quantitative PCR
(qPCR) to confirm mRNA expression. We validated both significant (TMEM16a, SLC26A4,
ERPINB4) and nonsignificant (CXCL8, IL-6, SLC26A14, CFTR, and ENaC) mRNA.

CF mouse model

BALB/cAnNRj-Foxn1 nu/nu were purchased (Janvier-Labs, France). 129-Cftrtm1Eur model mice homozygous for the F508del mutation and their wild-type littermates were obtained from CDTA-CNRS (Orléans, France). Cftr<sup>em3Cwt</sup> G542x model mice homozygous for the G542X mutation and their wild-type littermates were provided by Dr. Craig A. Hodges (Case Western Reserve University, Cleveland, USA). During the TMEM16a ASO treatment, no laxative was provided as previously described to avoid interfering with the treatment. Studies were approved by the Institutional Animal Care and Use Committee (APAFIS #38427-2022091211033928 v8 and APAFIS #43883-2023062011193610 v7).

TMEM16 ASO quantification

The analyses were performed by high-performance liquid chromatography (HPLC) coupled with mass spectrometry (MS). Liquid chromatography was performed on the Ultimate 3000 series Pump System (Thermo Scientific). Detection was carried out on a mass spectrometer, which included an electrospray ionization source (ESI) and an ion trap mass analyzer (Bruker), as well as an ultraviolet-visible spectroscopy detector (VWD3100, Thermo Scientific). Raw data were acquired and processed using Hystar 3.2 and DataAnalysis/QuantAnalysis (Bruker), respectively. The linearity of the calibration curve was tested on TMEM16 ASO with HPLC-MS/MS analysis.
447 Frozen tissue samples (organs) were homogenized with the GentleMACS dissociator device using
448 250 mg of tissue per 1000 µL ATL Buffer and lysed overnight with proteinase K.

449 **Toxicity assessment**

450 C-Ris Pharma (Saint-Malo, France) conducted the *in vivo* toxicity protocol on healthy CD-1 mice.
451 Histology and blood analysis were carried out on mice treated at 500 mg/kg (50 times the effective
452 dose) and sacrificed 14 days after subcutaneous injection. After sacrifice, the organs were
453 collected, embedded in paraffin, and stained with hematoxylin and eosin before being sliced and
454 analyzed.

455 **Non-invasive fluorescence assessment in vivo**

456 Nude mice (BALB/cAnNRj-Foxn1 nu/nu) were treated with fluorescent TMEM16a ASO
457 (Eurogentec, Belgium; ex 640 nm/em 680 nm; 10 mg/kg) through various routes of administration
458 to avoid autofluorescence in the mice's coat. Imaging was performed using the IVIS Spectrum
459 imaging system (PerkinElmer, Inc., France) through two-dimensional (2D) epifluorescence
460 imaging at specified times after probe injection to optimize readouts. Three-dimensional (3D)
461 images were then generated using IVIS software. After 30 days, the organs were extracted and
462 isolated, and imaging was conducted using the IVIS Spectrum imaging system. Fluorescence was
463 recorded, and the images were analyzed and quantified using ImageJ 1.54 software.

464 **Statistical analysis**

465 Statistical analysis was performed as indicated in the respective figure legends, using
466 GraphPad Prism 10.5.3 software (GraphPad Software Inc., San Diego, USA). Extracted contrasts
467 included comparisons between different groups and pairwise comparisons of points in time within
468 each condition. Raw p-values were adjusted for multiple testing using the Benjamini-Hochberg
procedure, and genes with an adjusted p-value. All figures show means and 95% confidence intervals.

471

DATA AVAILABILITY STATEMENT

The datasets generated and/or analyzed during this study are available from the corresponding author upon reasonable request.

475

AUTHOR CONTRIBUTIONS

477

Conceptualization: OT

Methodology: CM, NR, MDW, TF, GD, ML, VG, FS, OT

Investigation: CM, NR, MDW, TF, GB, ML, VG, FS, OT

Visualization: CM, NR, MDW, TF, GB, ML, VG, FS, HC, OT

Funding acquisition: CM, OT

Project administration: CM, OT

Supervision: OT

Writing – original draft: CM, FS, OT

Writing – review & editing: CM, OT

ACKNOWLEDGMENTS

F508del mice were generated by the CF EMCs (Dr. Scholte *et al.*) and the sponsorship of the EUROCARECF (6th framework coordination action program LSHM-CT-2005-018932). G542X mice were generated by the CF Mouse Resource Center at Case Western Reserve University in
the USA (Dr. Craig A. Hodges). The authors thank the Association «Les Motards du Viaduc de Millau» for funding the project.

Grants support this work:

"Blanche pour Vaincre la Mucoviscidose," RF20220503020,

"Vaincre la Mucoviscidose", RF20190502484, RF20200502752, RF20180502589

SATT Lutech, Cystem Flow

Fondation Maladies Rares, FSMR-070805

Anoat Therapeutics, Collaboration agreement N°21118A20

Motards du Viaduc de Millau

Inserm Transfert, MAT-PI-15170-A-03

## DECLARATION OF INTERESTS

CM, FS, HC, and OT are co-founders and board members of Anoat Therapeutics. TMEM16a ASO is patent-protected. CM, FS, HC, and OT are co-inventors of the following patents: EP16736045.2, US15/739,366, EP18 783 539.2, US16/756,917, and EP22305163.2.

## KEYWORDS

Preclinical studies; Cystic fibrosis; CFTR therapy; Anoctamin-1; TMEM16a; Oligonucleotides; Target site blocker

## REFERENCES
513 **Data Availability Statement**All data associated with this study are in the paper or the

514 Supplementary Materials.

515
516

**Figure 1. TMEM16a ASO increases Cl⁻ efflux in F508del cells.**

517

**A.** Relative luciferase activity in HEK cells stably transfected with a luciferase-3'UTR ANO1 and treated with TMEM16a ASO at the indicated concentration. Firefly luciferase activity was normalized to Renilla luciferase activity (n = 4, with 8 replicates). **B.** Representative curve of Cl⁻ efflux assessed by halide-sensitive YFP probe in CFBE41o⁻ cells transfected with control ASO (Ctrl ASO) or TMEM16a ASO for 24h at the specified concentrations in nM. **C.** Quantification of iodine (I⁻) assessed by halide-sensitive YFP probe in CFBE41o⁻ cells transfected with control ASO (Ctrl ASO) or TMEM16a ASO for 24h at the specified concentrations in nM (n=4).

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**D.** Quantification of iodine (I⁻) uptake in (C) (n≥4). **E.** Representative traces of electrophysiological responses in Ussing chambers of non-CF and CF (F508del/F508del) hBECs transfected with Ctrl ASO or TMEM16a ASO for one hour daily over three days at 50 nM. Black arrowheads indicate the addition of amiloride to block sodium channels (100 µM), forskolin to stimulate CFTR (10 µM), or UTP to activate the purinergic calcium-dependent Cl⁻ secretion (100 µM). CFTR was inhibited with CFTR inhibitor 172 (CFTRinh-172) (10 µM). **F.** The normalized total area under the curves of Ussing-chamber assays of CF hBEC cells activated by UTP and pretreated with Ani9 (10 µM) or the vehicle (n=3). Quantification of the normalized total area under the curves of Ussing-chamber assays of CF bronchial primary cells (n=3) before (G) and after (H) Ani9 treatment (10 µM, 1 h). Data are presented as mean values ± SEM with a 95% confidence interval. ANOVA followed by Dunnett's and Bonferroni's post hoc tests. **p**≤0.01; ***p**≤0.001; ****p**≤0.0001,

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**Figure 2. TMEM16a ASO enhances the UTP Cl⁻ response and mucociliary clearance across various cells with different CFTR mutations while not impacting other dysregulated parameters of cystic fibrosis.**

**A.** Representative Ussing experiments of non-CF and CF hBEC transfected with Ctl ASO or TMEM16a ASO for 1 hour daily for 3 days at 50 nM. Black arrowheads indicate the addition of amiloride, forskolin, CFTR inhibitor 172, or UTP during the experiment. **B.** Quantification of the normalized total area under the curves of Ussing-chamber assays of CF hBEC (2184DelA W1282x). Data are presented as mean values ± SD (n=3). **C.** Representative examples of fluorescent bead tracking on control or TMEM16 ASO-treated hBEC (F508del/F508del). Scale bar: 100 µm. **D-F.** Quantification of average bead velocity measured in hBEC with (D) F508del/F508del, (E) 1707-1G>A/F508del, and (F) 2184DelA/W1282x mutations following the specified treatments. (n=5 per condition, with 100 beads tracked per sample). **G.** Representative curve of bioluminescence production and bacterial growth over time for *P. aeruginosa* cultured alone with control or TMEM16a ASO (50 nM, n=5). **H.** Representative curve of bioluminescence production and bacterial growth over time for *P. aeruginosa* cultured at an MOI of 0.1, 1.0, or 10 bacteria/cell, in the presence of CFBE41o- pretreated with either control or TMEM16a ASO for 1 hour daily for 3 days at 50 nM (n=5). **I.** Quantifying infected CFBE41o- cell surfaces treated with control ASO or TMEM16a ASO (24h, 50 nM) using a crystal violet assay. The dotted line indicates the maximum cell area, representing the surface of cells without bacteria (n=5). Data is presented as mean values SD with a 95% confidence interval. ANOVA followed by Dunnett's and Bonferroni's post hoc test. **p**≤0.01; ***p**≤0.001; ****p**≤0.0001.
<caption>**Figure 3. TMEM16a ASO is specific and not toxic.**</caption>

**A.** The results of the binding profile for TMEM16a ASO were expressed as a percentage of inhibition of control-specific binding, calculated as [(100 - measured specific binding/control specific binding) x 100], obtained in the presence of TMEM16a ASO. Binding is deemed positive if it exceeds 100%. **B.** Cytotoxicity was evaluated by quantifying Lactate dehydrogenase (LDH) in CFBE41o- treated with Ctl and TMEM16a ASO (n=7). **C.** The IL-8 secretion from the cells treated with TMEM16a ASO was measured using ELISA and normalized to the secretion from the cells treated with Ctl ASO (n=3). **D.** Mean peak values of ionomycin responses (10 µM) were assessed in 16HBE cell lines with various mutations labeled with fluo-4 after transfection with TMEM16a ASO or Ctl ASO (50 nM) for 24 hours for each cell line (n=5). **E.** Proliferation was assessed in 16HBE cell lines with various mutations following TMEM16a ASO transfection or control ASO transfection (50 nM) for 24 hours (n=3). **F-G.** Volcano plot of transcriptomic analysis of RNA changes between hBEC cells cultured in ALI (n=5 cultures per individual), treated with TMEM16a and Ctl ASO (50 nM, 1h) for 3 days before mRNA extraction and after 24h of incubation. **F.** hBEC cells were obtained from a pwCF with a class II mutation (F508del/F508del), and **G.** was sourced from a pwCF with class I mutations (2184delA/W1282X). Data is presented as mean values SD with a 95% confidence interval. ANOVA followed by Dunnett's and Bonferroni's post hoc test. ANOVA with Dunnett's and Bonferroni's post hoc tests found no significant results.
**Figure 4.** Subcutaneous injection induces optimal ASO biodistribution, which is sustained and does not induce toxicity.

**A.** Diagram illustrating various routes of administration in mice. **B**-**E.** *In vivo* imaging of nude mice following intravenous, subcutaneous, intraperitoneal injections, or intranasal instillation of fluorescent TMEM16a ASO (10 mg/kg) at various time points: **B.** Day 0 (DO), **C.** Day 7 (D7), **D.** Day 21 (D21) and **E.** Day 30 (D30), (n=3/group). **F.** Analysis of fluorescence intensity in organs from mice sacrificed 30 days after treatment (n=5/group). **G.** Quantification of TMEM16a expression in organs 30 days after the injection of the TMEM16a ASO compared to naïve mice (n=5/group). **H.** Quantification of TMEM16a ASO concentration at different times (4h, 24h, 3 days, 1 week, 2 weeks, and 4 weeks) in 25 mg of kidney, liver, lung, intestine, and pancreas (n=3/group). **I.** Quantification of plasma TMEM16a ASO concentration at different times (30min, 1h, 2h, 4h, 24h, 800h) (n=3/group). Data is presented as mean values SD with a 95% confidence interval. ANOVA followed by Dunnett's and Bonferroni's post hoc test. ANOVA with Dunnett's and Bonferroni's post hoc tests.

**Figure 5.** TMEM16a ASO treatment enhances viability and weight gain, while reducing intestinal inflammation in F508del mice.

**A.** TMEM16a ASO and control ASO (10 mg/kg) were injected subcutaneously on days 11 and 18 and then every 15 days thereafter into 129-cftrtm1Eur CF mice (F508del/F508del). **B.** Kaplan-Meier survival curves for overall survival analysis in CF mice treated with either control ASO or
TMEM16a ASO. The data represents the average of more than 100 mice. C. The weight values presented are averages based on as many as 24 mice per group. The values represent the mean and one standard deviation. Ages vary by ± 3 days. D-E. ELISA analyzed fecal lipocalin (D) and calprotectin (E) (n=20/group). F. Kaplan-Meier survival curves for the overall survival analysis in CF pups born to parents treated with TMEM16a ASO or control ASO. Data represents the mean of at least 20 mice per group. The dotted lines indicate median survival when the staircase survival curve intersects 50%. G. Quantification of fluorescence in the pups after birth. The gestating females were treated with fluorescent ASO TMEM16a or vehicle (10 mg/kg) 7 days before giving birth (n=7/group). Survival was evaluated between groups using the Kaplan-Meier survival analysis and log-rank test for (B) and (F), and ANOVA, followed by Dunnett's, Bonferroni's, and Tukey's posthoc tests. (D-E). ns: non-significant; **p≤0.01; ***p≤0.001; ****p≤0.0001.

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Figure 6. Treatment with TMEM16a ASO increases viability and weight gain, and decreases intestinal inflammation in G542X mice

A. TMEM16a ASO and control ASO (10 mg/kg) were subcutaneously injected on days 11 and 18 and subsequently every 15 days into Cftrem3Cwt CF mice (G542X/G542X). B. Kaplan-Meier survival curves for overall survival analysis in CF mice treated with control ASO or TMEM16a ASO show data as the mean of more than 25 mice. C. The histogram of the weight values presented represents averages based on up to 24 mice per group. These values represent the mean and one standard deviation. Ages are ± 3 days. D-E. Fecal lipocalin (D) and calprotectin (E) were analyzed by ELISA (n≥11). Survival was evaluated between groups using the Kaplan-Meier survival analysis and log-rank test (B) and ANOVA, followed by Dunnett's and Bonferroni's post hoc tests. (C-E). ns: non-significant; **p≤0.01; ***p≤0.001; ****p≤0.0001.

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TABLES

<table><caption>**Table 1:** Top 10 mRNA fold changes observed after transcriptomic analysis of CF human primary cells cultured in ALI treated with TMEM16a ASO during 24h (50 nM), including the log of fold change and -LOG10 P value, in a CF individual with F508del/F508del mutations (left) and in a CF individual with 2184delA/1282X mutations (right).</caption><thead><tr><th colspan="3">F508del/F508del</th><th colspan="3">2184delA/1282X</th></tr><tr><th>mRNA</th><th>LogFold Change</th><th>-Log10 P value</th><th>mRNA</th><th>LogFold Change</th><th>-Log10 P value</th></tr></thead><tbody><tr><td>SPDEF</td><td>2,6</td><td>37,3</td><td>SLC26A4</td><td>3,2</td><td>17,8</td></tr><tr><td>CDH6</td><td>2,7</td><td>14,4</td><td>ALOX15</td><td>3,2</td><td>33,8</td></tr><tr><td>TMEM16a</td><td>3,2</td><td>44,1</td><td>C1QTNF1</td><td>3,4</td><td>40,8</td></tr><tr><td>DPP4</td><td>3,3</td><td>37,4</td><td>DPP4</td><td>3,6</td><td>81,2</td></tr><tr><td>SLC26A4</td><td>3,5</td><td>20,9</td><td>TMEM16a</td><td>3,8</td><td>47,6</td></tr><tr><td>SERPINB4</td><td>3,5</td><td>39,9</td><td>SERPINB4</td><td>4,0</td><td>83,3</td></tr><tr><td>CDH26</td><td>3,5</td><td>69,5</td><td>SERPINB2</td><td>4,2</td><td>78,3</td></tr><tr><td>ALOX15</td><td>3,7</td><td>29,6</td><td>CDH26</td><td>4,3</td><td>97,3</td></tr><tr><td>SERPINB2</td><td>3,7</td><td>36,8</td><td>SERPINB10</td><td>5,1</td><td>87,3</td></tr><tr><td>SERPINB10</td><td>4,3</td><td>67,0</td><td>NOS2</td><td>5,3</td><td>41,3</td></tr></tbody></table>

<table><caption>**Table 2:** Vas deferens abnormalities (in %) observed in F508del mice</caption><thead><tr><th></th><th>+/+ mice<br>(n=30)</th><th>Ctl ASO -/- mice<br>(n=24)</th><th>TMEM16a ASO -/- mice<br>(n=32)</th></tr></thead><tbody><tr><td>Lenght (%)</td><td>100%</td><td>-31,5%</td><td>-6,25%</td></tr><tr><td>Atresia (%)</td><td>9%</td><td>79,10%</td><td>18,75%</td></tr><tr><td>Absence (%)</td><td>0%</td><td>16,60%</td><td>0%</td></tr><tr><td>Fertility (%)</td><td>96,67%</td><td>4,16%</td><td>88,23%</td></tr></tbody></table>

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    <td>22.</td>
    <td>McHugh, D.R., Steele, M.S., Valerio, D.M., Miron, A., Mann, R.J., LePage, D.F., Conlon, R.A., Cotton, C.U., Drumm, M.L., and Hodges, C.A. (2018). A G542X cystic fibrosis mouse model for examining nonsense mutation directed therapies. PloS one 13, e0199573. 10.1371/journal.pone.0199573.</td>
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    <td>Knowles, M.R., Clarke, L.L., and Boucher, R.C. (1991). Activation by extracellular nucleotides of chloride secretion in the airway epithelia of patients with cystic fibrosis. N Engl J Med 325, 533-538. 10.1056/NEJM199108223250802.</td>
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    <td>Moss, R.B. (2013). Pitfalls of drug development: lessons learned from trials of denufosol in cystic fibrosis. J Pediatr 162, 676-680. 10.1016/j.jpeds.2012.11.034.</td>
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    <td>McCarron, A., Parsons, D., and Donnelley, M. (2021). Animal and Cell Culture Models for Cystic Fibrosis. The American journal of pathology 191, 228-242. 10.1016/j.ajpath.2020.10.017.</td>
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    <td>Sun, X., Yi, Y., Yan, Z., Rosen, B.H., Liang, B., Winter, M.C., Evans, T.I.A., Rotti, P.G., Yang, Y., Gray, J.S., et al. (2019). In utero and postnatal VX-770 administration rescues multiorgan disease in a ferret model of cystic fibrosis. Sci Transl Med 11. 10.1126/scitranslmed.aau7531.</td>
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    <td>705</td>
    <td>30.</td>
    <td>Caraco, Y., Wanounou, M., Blotnick, S., Friedman, L., Cohen, A., Hart, G., and Kerem, E. (2025). A phase I study assessing the safety and tolerability of SPL84, an inhaled antisense oligonucleotide for treatment of cystic fibrosis patients with the 3849 +10kb C-&gt;T. J Cyst Fibros 24, 66-71. 10.1016/j.jcf.2024.10.004.</td>
  </tr>
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    <td>706</td>
    <td>31.</td>
    <td>Sutharsan, S., Fischer, R., Gleiber, W., Horsley, A., Crosby, J., Guo, S., Xia, S., Yu, R., Newman, K.B., and Elborn, J.S. (2024). Randomised, phase 1/2a trial of ION-827359, an antisense oligonucleotide inhibitor of ENaC. ERJ Open Res 10. 10.1183/23120541.00986-2023.</td>
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    <td>707</td>
    <td>32.</td>
    <td>Kim, Y.J., and Krainer, A.R. (2023). Antisense Oligonucleotide Therapeutics for Cystic Fibrosis: Recent Developments and Perspectives. Mol Cells 46, 10-20. 10.14348/molcells.2023.2172.</td>
  </tr>
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    <td>708</td>
    <td>33.</td>
    <td>Milczewska, J., Syunyaeva, Z., Zabinska-Jaron, A., Sands, D., and Thee, S. (2024). Changing profile of bacterial infection and microbiome in cystic fibrosis: when to use antibiotics in the era of CFTR-modulator therapy. Eur Respir Rev 33. 10.1183/16000617.0068-2024.</td>
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    <td>709</td>
    <td>34.</td>
    <td>Farinha, C.M., and Gentzsch, M. (2021). Revisiting CFTR Interactions: Old Partners and New Players. Int J Mol Sci 22. 10.3390/ijms222413196.</td>
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    <td>710</td>
    <td>35.</td>
    <td>Strub, M.D., and McCray, P.B., Jr. (2020). Transcriptomic and Proteostasis Networks of CFTR and the Development of Small Molecule Modulators for the Treatment of Cystic Fibrosis Lung Disease. Genes (Basel) 11. 10.3390/genes11050546.</td>
  </tr>
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    <td>711</td>
    <td>36.</td>
    <td>Declercq, M., De Zeeuw, P., Conchinha, N.V., Geldhof, V., Ramalho, A.S., García-Caballero, M., Brepoels, K., Ensinck, M., Carlon, M.S., Bird, M.J., et al. (2021). Transcriptomic analysis of CFTR-impaired endothelial cells reveals a pro-inflammatory phenotype. European Respiratory Journal 57, 2000261. 10.1183/13993003.00261-2020.</td>
  </tr>
  <tr>
    <td>712</td>
    <td>37.</td>
    <td>Moschos, S.A., Frick, M., Taylor, B., Turnpenny, P., Graves, H., Spink, K.G., Brady, K., Lamb, D., Collins, D., Rockel, T.D., et al. (2011). Uptake, efficacy, and systemic distribution of naked, inhaled short interfering RNA (siRNA) and locked nucleic acid (LNA) antisense. Mol Ther 19, 2163-2168. 10.1038/mt.2011.206.</td>
  </tr>
  <tr>
    <td>713</td>
    <td>38.</td>
    <td>Straarup, E.M., Fisker, N., Hedtjarn, M., Lindholm, M.W., Rosenbohm, C., Aarup, V., Hansen, H.F., Orum, H., Hansen, J.B., and Koch, T. (2010). Short locked nucleic acid antisense oligonucleotides potently reduce apolipoprotein B mRNA and serum cholesterol in mice and non-human primates. Nucleic Acids Res 38, 7100-7111. 10.1093/nar/gkq457.</td>
  </tr>
  <tr>
    <td>714</td>
    <td>39.</td>
    <td>Danahay, H., Fox, R., Lilley, S., Charlton, H., Adley, K., Christie, L., Ansari, E., Ehre, C., Flen, A., Tuvim, M.J., et al. (2020). Potentiating TMEM16A does not stimulate airway mucus secretion or bronchial and pulmonary arterial smooth muscle contraction. FASEB Bioadv 2, 464-477. 10.1096/fba.2020-00035.</td>
  </tr>
  <tr>
    <td>715</td>
    <td>40.</td>
    <td>Danahay, H.L., Morris, D.G., and Gosling, M. (2020). Reply to Olschewski et al.: TMEM16A Potentiation: Possible Drawbacks. Am J Respir Crit Care Med 202, 905-906. 10.1164/rccm.202004-1214LE.</td>
  </tr>
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    <td>716</td>
    <td>30.</td>
    <td>Caraco, Y., Wanounou, M., Blotnick, S., Friedman, L., Cohen, A., Hart, G., and Kerem, E. (2025). A phase I study assessing the safety and tolerability of SPL84, an inhaled antisense oligonucleotide for treatment of cystic fibrosis patients with the 3849 +10kb C-&gt;T. J Cyst Fibros 24, 66-71. 10.1016/j.jcf.2024.10.004.</td>
  </tr>
  <tr>
    <td>717</td>
    <td>31.</td>
    <td>Sutharsan, S., Fischer, R., Gleiber, W., Horsley, A., Crosby, J., Guo, S., Xia, S., Yu, R., Newman, K.B., and Elborn, J.S. (2024). Randomised, phase 1/2a trial of ION-827359, an antisense oligonucleotide inhibitor of ENaC. ERJ Open Res 10. 10.1183/23120541.00986-2023.</td>
  </tr>
  <tr>
    <td>718</td>
    <td>32.</td>
    <td>Kim, Y.J., and Krainer, A.R. (2023). Antisense Oligonucleotide Therapeutics for Cystic Fibrosis: Recent Developments and Perspectives. Mol Cells 46, 10-20. 10.14348/molcells.2023.2172.</td>
  </tr>
  <tr>
    <td>719</td>
    <td>33.</td>
    <td>Milczewska, J., Syunyaeva, Z., Zabinska-Jaron, A., Sands, D., and Thee, S. (2024). Changing profile of bacterial infection and microbiome in cystic fibrosis: when to use antibiotics in the era of CFTR-modulator therapy. Eur Respir Rev 33. 10.1183/16000617.0068-2024.</td>
  </tr>
  <tr>
    <td>720</td>
    <td>34.</td>
    <td>Farinha, C.M., and Gentzsch, M. (2021). Revisiting CFTR Interactions: Old Partners and New Players. Int J Mol Sci 22. 10.3390/ijms222413196.</td>
  </tr>
  <tr>
    <td>721</td>
    <td>35.</td>
    <td>Strub, M.D., and McCray, P.B., Jr. (2020). Transcriptomic and Proteostasis Networks of CFTR and the Development of Small Molecule Modulators for the Treatment of Cystic Fibrosis Lung Disease. Genes (Basel) 11. 10.3390/genes11050546.</td>
  </tr>
  <tr>
    <td>722</td>
    <td>36.</td>
    <td>Declercq, M., De Zeeuw, P., Conchinha, N.V., Geldhof, V., Ramalho, A.S., García-Caballero, M., Brepoels, K., Ensinck, M., Carlon, M.S., Bird, M.J., et al. (2021). Transcriptomic analysis of CFTR-impaired endothelial cells reveals a pro-inflammatory phenotype. European Respiratory Journal 57, 2000261. 10.1183/13993003.00261-2020.</td>
  </tr>
  <tr>
    <td>723</td>
    <td>37.</td>
    <td>Moschos, S.A., Frick, M., Taylor, B., Turnpenny, P., Graves, H., Spink, K.G., Brady, K., Lamb, D., Collins, D., Rockel, T.D., et al. (2011). Uptake, efficacy, and systemic distribution of naked, inhaled short interfering RNA (siRNA) and locked nucleic acid (LNA) antisense. Mol Ther 19, 2163-2168. 10.1038/mt.2011.206.</td>
  </tr>
  <tr>
    <td>724</td>
    <td>38.</td>
    <td>Straarup, E.M., Fisker, N., Hedtjarn, M., Lindholm, M.W., Rosenbohm, C., Aarup, V., Hansen, H.F., Orum, H., Hansen, J.B., and Koch, T. (2010). Short locked nucleic acid antisense oligonucleotides potently reduce apolipoprotein B mRNA and serum cholesterol in mice and non-human primates. Nucleic Acids Res 38, 7100-7111. 10.1093/nar/gkq457.</td>
  </tr>
  <tr>
    <td>725</td>
    <td>39.</td>
    <td>Danahay, H., Fox, R., Lilley, S., Charlton, H., Adley, K., Christie, L., Ansari, E., Ehre, C., Flen, A., Tuvim, M.J., et al. (2020). Potentiating TMEM16A does not stimulate airway mucus secretion or bronchial and pulmonary arterial smooth muscle contraction. FASEB Bioadv 2, 464-477. 10.1096/fba.2020-00035.</td>
  </tr>
  <tr>
    <td>726</td>
    <td>40.</td>
    <td>Danahay, H.L., Morris, D.G., and Gosling, M. (2020). Reply to Olschewski et al.: TMEM16A Potentiation: Possible Drawbacks. Am J Respir Crit Care Med 202, 905-906. 10.1164/rccm.202004-1214LE.</td>
  </tr>
  <tr>
    <td>727</td>
    <td>38.</td>
    <td>Straarup, E.M., Fisker, N., Hedtjarn, M., Lindholm, M.W., Rosenbohm, C., Aarup, V., Hansen, H.F., Orum, H., Hansen, J.B., and Koch, T. (2010). Short locked nucleic acid antisense oligonucleotides potently reduce apolipoprotein B mRNA and serum cholesterol in mice and non-human primates. Nucleic Acids Res 38, 7100-7111. 10.1093/nar/gkq457.</td>
  </tr>
  <tr>
    <td>728</td>
    <td>39.</td>
    <td>Danahay, H., Fox, R., Lilley, S., Charlton, H., Adley, K., Christie, L., Ansari, E., Ehre, C., Flen, A., Tuvim, M.J., et al. (2020). Potentiating TMEM16A does not stimulate airway mucus secretion or bronchial and pulmonary arterial smooth muscle contraction. FASEB Bioadv 2, 464-477. 10.1096/fba.2020-00035.</td>
  </tr>
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    <td>729</td>
    <td>40.</td>
    <td>Danahay, H.L., Morris, D.G., and Gosling, M. (2020). Reply to Olschewski et al.: TMEM16A Potentiation: Possible Drawbacks. Am J Respir Crit Care Med 202, 905-906. 10.1164/rccm.202004-1214LE.</td>
  </tr>
  <tr>
    <td>730</td>
    <td>38.</td>
    <td>Straarup, E.M., Fisker, N., Hedtjarn, M., Lindholm, M.W., Rosenbohm, C., Aarup, V., Hansen, H.F., Orum, H., Hansen, J.B., and Koch, T. (2010). Short locked nucleic acid antisense oligonucleotides potently reduce apolipoprotein B mRNA and serum cholesterol in mice and non-human primates. Nucleic Acids Res 38, 7100-7111. 10.1093/nar/gkq457.</td>
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    <td>731</td>
    <td>39.</td>
    <td>Danahay, H., Fox, R., Lilley, S., Charlton, H., Adley, K., Christie, L., Ansari, E., Ehre, C., Flen, A., Tuvim, M.J., et al. (2020). Potentiating TMEM16A does not stimulate airway mucus secretion or bronchial and pulmonary arterial smooth muscle contraction. FASEB Bioadv 2, 464-477. 10.1096/fba.2020-00035.</td>
  </tr>
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    <td>732</td>
    <td>40.</td>
    <td>Danahay, H.L., Morris, D.G., and Gosling, M. (2020). Reply to Olschewski et al.: TMEM16A Potentiation: Possible Drawbacks. Am J Respir Crit Care Med 202, 905-906. 10.1164/rccm.202004-1214LE.</td>
  </tr>
  <tr>
    <td>733</td>
    <td>38.</td>
    <td>Straarup, E.M., Fisker, N., Hedtjarn, M., Lindholm, M.W., Rosenbohm, C., Aarup, V., Hansen, H.F., Orum, H., Hansen, J.B., and Koch, T. (2010). Short locked nucleic acid antisense oligonucleotides potently reduce apolipoprotein B mRNA and serum cholesterol in mice and non-human primates. Nucleic Acids Res 38, 7100-7111. 10.1093/nar/gkq457.</td>
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    <td>734</td>
    <td>39.</td>
    <td>Danahay, H., Fox, R., Lilley, S., Charlton, H., Adley, K., Christie, L., Ansari, E., Ehre, C., Flen, A., Tuvim, M.J., et al. (2020). Potentiating TMEM16A does not stimulate airway mucus secretion or bronchial and pulmonary arterial smooth muscle contraction. FASEB Bioadv 2, 464-477. 10.1096/fba.2020-00035.</td>
  </tr>
  <tr>
    <td>735</td>
    <td>40.</td>
    <td>Danahay, H.L., Morris, D.G., and Gosling, M. (2020). Reply to Olschewski et al.: TMEM16A Potentiation: Possible Drawbacks. Am J Respir Crit Care Med 202, 905-906. 10.1164/rccm.202004-1214LE.</td>
  </tr>
  <tr>
    <td>736</td>
    <td>38.</td>
    <td>Straarup, E.M., Fisker, N., Hedtjarn, M., Lindholm, M.W., Rosenbohm, C., Aarup, V., Hansen, H.F., Orum, H., Hansen, J.B., and Koch, T. (2010). Short locked nucleic acid antisense oligonucleotides potently reduce apolipoprotein B mRNA and serum cholesterol in mice and non-human primates. Nucleic Acids Res 38, 7100-7111. 10.1093/nar/gkq457.</td>
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  <tr>
    <td>737</td>
    <td>39.</td>
    <td>Danahay, H., Fox, R., Lilley, S., Charlton, H., Adley, K., Christie, L., Ansari, E., Ehre, C., Flen, A., Tuvim, M.J., et al. (2020). Potentiating TMEM16A does not stimulate airway mucus secretion or bronchial and pulmonary arterial smooth muscle contraction. FASEB Bioadv 2, 464-477. 10.1096/fba.2020-00035.</td>
  </tr>
  <tr>
    <td>738</td>
    <td>40.</td>
    <td>Danahay, H.L., Morris, D.G., and Gosling, M. (2020). Reply to Olschewski et al.: TMEM16A Potentiation: Possible Drawbacks. Am J Respir Crit Care Med 202, 905-906. 10.1164/rccm.202004-1214LE.</td>
  </tr>
  <tr>
    <td>739</td>
    <td>38.</td>
    <td>Straarup, E.M., Fisker, N., Hedtjarn, M., Lindholm, M.W., Rosenbohm, C., Aarup, V., Hansen, H.F., Orum, H., Hansen, J.B., and Koch, T. (2010). Short locked nucleic acid antisense oligonucleotides potently reduce apolipoprotein B mRNA and serum cholesterol in mice and non-human primates. Nucleic Acids Res 38, 7100-7111. 10.1093/nar/gkq457.</td>
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  <tr>
    <td>740</td>
    <td>39.</td>
    <td>Danahay, H., Fox, R., Lilley, S., Charlton, H., Adley, K., Christie, L., Ansari, E., Ehre, C., Flen, A., Tuvim, M.J., et al. (2020). Potentiating TMEM16A does not stimulate airway mucus secretion or bronchial and pulmonary arterial smooth muscle contraction. FASEB Bioadv 2, 464-477. 10.1096/fba.2020-00035.</td>
  </tr>
  <tr>
    <td>741</td>
    <td>40.</td>
    <td>Danahay, H.L., Morris, D.G., and Gosling, M. (2020). Reply to Olschewski et al.: TMEM16A Potentiation: Possible Drawbacks. Am J Respir Crit Care Med 202, 905-906. 10.1164/rccm.202004-1214LE.</td>
  </tr>
  <tr>
    <td>742</td>
    <td>38.</td>
    <td>Straarup, E.M., Fisker, N., Hedtjarn, M., Lindholm, M.W., Rosenbohm, C., Aarup, V., Hansen, H.F., Orum, H., Hansen, J.B., and Koch, T. (2010). Short locked nucleic acid antisense oligonucleotides potently reduce apolipoprotein B mRNA and serum cholesterol in mice and non-human primates. Nucleic Acids Res 38, 7100-7111. 10.1093/nar/gkq457.</td>
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    <td>743</td>
    <td>39.</td>
    <td>Danahay, H., Fox, R., Lilley, S., Charlton, H., Adley, K., Christie, L., Ansari, E., Ehre, C., Flen, A., Tuvim, M.J., et al. (2020). Potentiating TMEM16A does not stimulate airway mucus secretion or bronchial and pulmonary arterial smooth muscle contraction. FASEB Bioadv 2, 464-477. 10.1096/fba.2020-00035.</td>
  </tr>
  <tr>
    <td>744</td>
    <td>40.</td>
    <td>Danahay, H.L., Morris, D.G., and Gosling, M. (2020). Reply to Olschewski et al.: TMEM16A Potentiation: Possible Drawbacks. Am J Respir Crit Care Med 202, 905-906. 10.1164/rccm.202004-1214LE.</td>
  </tr>
  <tr>
    <td>745</td>
    <td>38.</td>
    <td>Straarup, E.M., Fisker, N., Hedtjarn, M., Lindholm, M.W., Rosenbohm, C., Aarup, V., Hansen, H.F., Orum, H., Hansen, J.B., and Koch, T. (2010). Short locked nucleic acid antisense oligonucleotides potently reduce apolipoprotein B mRNA and serum cholesterol in mice and non-human primates. Nucleic Acids Res 38, 7100-7111. 10.1093/nar/gkq457.</td>
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    <td>746</td>
    <td>39.</td>
    <td>Danahay, H., Fox, R., Lilley, S., Charlton, H., Adley, K., Christie, L., Ansari, E., Ehre, C., Flen, A., Tuvim, M.J., et al. (2020). Potentiating TMEM16A does not stimulate airway mucus secretion or bronchial and pulmonary arterial smooth muscle contraction. FASEB Bioadv 2, 464-477. 10.1096/fba.2020-00035.</td>
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    <td>747</td>
    <td>40.</td>
    <td>Danahay, H.L., Morris, D.G., and Gosling, M. (2020). Reply to Olschewski et al.: TMEM16A Potentiation: Possible Drawbacks. Am J Respir Crit Care Med 202, 905-906. 10.1164/rccm.202004-1214LE.</td>
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    <td>748</td>
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    <td>Straarup, E.M., Fisker, N., Hedtjarn, M., Lindholm, M.W., Rosenbohm, C., Aarup, V., Hansen, H.F., Orum, H., Hansen, J.B., and Koch, T. (2010). Short locked nucleic acid antisense oligonucleotides potently reduce apolipoprotein B mRNA and serum cholesterol in mice and non-human primates. Nucleic Acids Res 38, 7100-7111. 10.1093/nar/gkq457.</td>
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790
A

<figure><img src="image_2.png" /></figure>

C

TMEM16a ASO concentration (nM)

<figure><img src="image_3.png" /></figure>

E

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G

Class II
CF primary cells

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B

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D

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F

NCF primary cells

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H

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Fig. 1
<figure><img src="image_10.png" /><figcaption>Fig. 2</figcaption></figure>
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<figure><img src="image_17.png" /><figcaption>Fig. 3</figcaption></figure>
<figure><img src="image_18.png" /><figcaption>A</figcaption></figure>

<figure><img src="image_19.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_20.png" /><figcaption>D0</figcaption></figure>

<figure><img src="image_21.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_22.png" /><figcaption>D7</figcaption></figure>

<figure><img src="image_23.png" /><figcaption>D21</figcaption></figure>

<figure><img src="image_24.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_25.png" /><figcaption>D30</figcaption></figure>

<figure><img src="image_26.png" /><figcaption>G</figcaption></figure>

<figure><img src="image_27.png" /><figcaption>H</figcaption></figure>

<figure><img src="image_28.png" /><figcaption>I</figcaption></figure>

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Fig. 4
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<figure><img src="image_36.png" /><figcaption>Fig. 5</figcaption></figure>
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<figure><img src="image_41.png" /><figcaption>Fig. 6</figcaption></figure>
SUPPLEMENTAL INFORMATION

<figure><img src="image_42.png" /><figcaption>B</figcaption></figure>

F508del/F508del Ctl
50mM 48h

<figure><img src="image_43.png" /><figcaption>C</figcaption></figure>

2184delA/W1282X
50 mM 48h

<figure><img src="image_44.png" /><figcaption>Figure S1</figcaption></figure>
Fig. S1. TMEM16a ASO does not induce inflammation or toxicity.

A. After 24 hours of treating cells with either control ASO or TMEM16a ASO (50 nM), the media were collected, and cytokines were analyzed using multiplex analysis (Bio-Rad, France). The induction of cytokines was recorded in the control results for each cell line (n=5). No significant differences were observed in any of the cytokines tested. B. List of the various proteins analyzed using the in vitro profiling method. **B-C.** Volcano plot of transcriptomic analysis of hBEC cells cultured in ALI (n=5 cultures per patient). hBEC cells were obtained from a pwCF with class II mutations (F508del/F508del and 2184delA/W1282X). The cells were treated with TMEM16a or Ctrl ASO (50 nM, 1h) for 3 days before mRNA extraction after a 48-hour incubation. **C-D** shows the top 10 mRNA fold changes observed after transcriptomic analysis, including the log of fold change and the -LOG10 P value.
<figure><img src="image_45.png" /><figcaption>Supplemental Fig. 2</figcaption></figure>

Fig. S2. TMEM16a ASO does not induce histological effects on mice after one year of TMEM16a ASO treatment.
Representative histology of different tissues at 200x (A, scale bar 250µm) and 400x (B, scale bar 100 µm) magnification of the129-cftrtm1Eur CF mice. Following euthanasia after 1 year of treatment with TMEM16a ASO (10 mg/kg every 15 days). Mice were euthanized and mouse tissues (liver, kidney, ovary, testis, pancreas, lung, and intestine) were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned (5µm), and subsequently stained with hematoxylin and eosin staining for histological analysis. C Semi-quantitative histological analysis of the H&E staining in the different tissues of CF mice treated with TMEM16a ASO for one year compared to untreated WT mice (n=9 per group). D MPO activity in different tissues. Values are expressed as mean values SD with a 95% confidence interval in fold of increase compared to tissues of WT mice. Statistical analysis was performed with ANOVA followed by Dunnett's and Bonferroni's post hoc tests. **p≤0.01; ***p≤0.001; ****p≤0.0001,
A

<figure><img src="image_46.png" /></figure>

B

<figure><img src="image_47.png" /></figure>

C

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D

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E

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F

<figure><img src="image_51.png" /></figure>

G

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Figure S3

Fig. S3. TMEM16a does not induce short or long-term toxicity.
A. Serum alanine aminotransferase (ALAT), B. Aspartate aminotransferase (ASAT), C. Urea, and D. amylase levels were determined in the blood of control and treated male and female mice (F508del/F508del) after injecting TMEM16a ASO subcutaneously at 0, 10, 20, 100, 300, and 500 mg/kg at 14 days post injection (n=3/group). E. ALAT, ASAT, F. urea, and creatinine levels were determined 200 days after subcutaneous injection of TMEM16a ASO or control ASO (10 mg/kg) for long-term toxicity observations (n=5/group). Data are represented as mean values ± SD, and Student's t-test was used to determine statistical significance. No significant difference was found. G. Kaplan-Meier survival curves of overall survival analysis in CF mice treated with only two injections of TMEM16a ASO at J11 and J18 (10 mg/kg) after birth, compared to mice treated with ASO control. Data are the mean of at least 15 mice per group.
The dotted lines represent median survival when the staircase survival curve crosses 50%.
<figure><img src="image_53.png" /><figcaption>Supplemental Fig. 4</figcaption></figure>

<caption>Fig. S4. TMEM16a ASO enhances fertility in CF male mice</caption>

Schematic representation of the reproduction of mice. In the first generation, heterozygote (-/+)
mice were crossed to generate CF (-/-), WT (+/+), or -/+ mice. In the second generation, CF
mice were crossed. Untreated TMEM16a ASO -/- male mice are not fertile (2nd generation, left panel). For treated mice, TMEM16a ASO was administered repeatedly by subcutaneous injections (10 mg/kg) to parents (males and females) of the 2nd generation (right panel). Pups were born from CF-treated mice (3rd generation). **B.** Picture of an F508del/F508del litter born from a TMEM16a ASO-treated male and female CF mice. **C.** Representative genotyping gel after Ssp1 restriction enzyme digestion. This gel represents the normal breeding results (1st and 2nd generation) versus those of CF mice that are treated and can reproduce. **D-E.** Representative pictures of reproductive tissues of CF male mice after 4 weeks of control ASO (D) or TMEM16a ASO treatment (E).