Maelle, Nawal Marie Marion

Pauline Sylvia Tobias

Albane Gabrielle Sylvain

Inès, Eloi, Camille Manon ✿ Claire

Nathalie Lhousseine

Manon Loïc

Johan Claire

Development of an RNA therapy : a new approach to treat all cystic fibrosis patients with antisense oligonucleotide

Christie Mitri

vii Altogether all these results demonstrated that this strategy could be applied to all patients with CF, regardless of their mutations (i.e., mutation-agnostic treatment).

This novel strategy can be combined with CFTR modulators or TMEM16A potentiators to obtain additive effects and target multiple fronts, such as pulmonary symptoms and other manifestations observed in patients with CF.

List of Figures

Introduction

Chapter 1: Cystic Fibrosis Cystic fibrosis (CF) is a common life-limiting disease. It is due to autosomal recessive mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that encodes for the cyclic adenosine monophosphate (cAMP)-dependent chloride (Cl -) channel CFTR. This chapter reviews the history and epidemiology of CF, describes the pathophysiology, genetics, and different manifestations, as well as summarizes current and future innovative treatments.

Early Descriptions and Knowledge

"Cystic fibrosis of the pancreas" was first described by pathologist Dorothy Andersen in 1938 based on autopsies of infants [START_REF] Andersen | Cystic fibrosis of the pancreas and its relation to celiac disease: a clinical and pathologic study[END_REF]. At the time, children died early of meconium ileus and malnutrition subsequent to pancreatic insufficiency and not from severe lung disease [START_REF] Farber | Pancreatic Function and Disease in Early Life. I. Pancreatic Enzyme Activity and the Celiac Syndrome[END_REF]. In 1945, the generalized impairment in mucus production clogging the glandular ducts throughout the body gave the alternative designation of "mucoviscidosis" by pediatric pathologist Sydney Farber.

During the 1948 heat wave, a critical discovery was made in New York by pediatrician Paul Di Sant'Agnese, who noticed that many children with CF developed severe hyponatremic dehydration [START_REF] Sant'agnese | Abnormal electrolyte composition of sweat in cystic fibrosis of the pancreas[END_REF]. His findings led many years later to the discovery of salt loss through sweat, which was the foundation of the later development of diagnostic testing via sweat sodium (Na + ) and Cl -testing. This "sweat test" helped identify milder patients, of which some even were pancreatic sufficient.

These sweat abnormalities implied that the fundamental defect could not be directly related to mucus.

In the early 1980s, Knowles et al. compared the transepithelial electrical potential difference of airways between patients with CF and controls and identified increased Na + reabsorption in the airways as a regular feature of CF [START_REF] Knowles | Increased Bioelectric Potential Difference across Respiratory Epithelia in Cystic Fibrosis[END_REF][START_REF] Knowles | Abnormal ion permeation through cystic fibrosis respiratory epithelium[END_REF]. At about the same time, while studying the problem of the sweat ducts, Paul Quinton identified in 1983 that Cl -transport is the fundamental defect in CF [START_REF] Quinton | Chloride impermeability in cystic fibrosis[END_REF]. He established that sweat/salt remained on the skin surface in patients with CF because of the inability to reabsorb Cl -ions into the blood. These discoveries significantly advanced the understanding of CF basic defects; however, the defective protein was still not identified. a maximum bound of 186,620 and a minimum bound of 144, 606) (J. [START_REF] Guo | Worldwide rates of diagnosis and effective treatment for cystic fibrosis[END_REF].

Most of the undiagnosed patient burden was in India; other countries with significant undiagnosed patient populations were extrapolated from prevalence estimates and include Morocco, the United Arab Emirates, Oman, Japan, Turkey, Argentina, and Egypt (J. [START_REF] Guo | Worldwide rates of diagnosis and effective treatment for cystic fibrosis[END_REF]. [START_REF] Shteinberg | Cystic fibrosis[END_REF].

Attributable to advances in research and improved disease management, the life expectancy of patients with CF continues to increase. CF went from being a childhood disease to now being an adult disorder. The average life expectancy of a patient is currently between 40 and 50 years, whereas it used to be only five years in the 1960s.

Life expectancy will likely further improve for children born with CF in the coming years.

Cystic Fibrosis Transmembrane conductance Regulator

CFTR Gene

CF is a disease resulting from recessive mutations in the CFTR gene, which contains 27 exons spanning 250 kb of chromosome 7at q31.2 and encodes a 6.5 kb mRNA (B. J. [START_REF] Wainwright | Localization of cystic fibrosis locus to human chromosome 7cen-q22[END_REF] (Figure 2). In 1989, the CFTR gene was identified by positional cloning, along with the most common mutation, F508del, which accounts for 70% of the mutant CFTR gene worldwide (B. [START_REF] Kerem | Identification of the Cystic Fibrosis Gene: Genetic Analysis[END_REF]Riordan et al., 1989;[START_REF] Rommens | Identification of the cystic fibrosis gene: chromosome walking and jumping[END_REF]. Today, more than 2,110 mutations have been identified and grouped into different categories based on the CFTR mutation molecular defects, ranging from defects in biosynthesis to defects in the regulation of CFTR activities (cftr2.org, genet.sickkids.on.ca). [START_REF] Prasad | Use of Potentiators and Correctors to Rescue the Various Effects of Mutations in Cystic Fibrosis[END_REF].

According to the traditional classification system, CFTR mutations are categorized into six classes [START_REF] Boyle | A new era in the treatment of cystic fibrosis: correction of the underlying CFTR defect[END_REF][START_REF] De Boeck | Progress in therapies for cystic fibrosis[END_REF][START_REF] Marson | Classification of CFTR mutation classes[END_REF].

Classes I to III are associated with little to no CFTR function and, therefore, with a more severe phenotype. Classes IV to VI have residual CFTR function and are less severe (Figure 3).



Class I mutations are responsible for defects in the early steps of biosynthesis and mainly include premature termination codons or stop codons, often causing mRNA degradation. In class I, some mutations (class Ia) do not allow mRNA synthesis; they are sometimes called class VII. [START_REF] De Boeck | Progress in therapies for cystic fibrosis[END_REF]. Class I is the most challenging CFTR defect to address since no CFTR protein is produced.

Nonsense and frameshift mutations account for 8.39% and 16.21% of the total mutations (cftr2.org), respectively.



Class II mutations disrupt protein maturation and trafficking to the plasma membrane (PM). Thus, the protein is either absent or present in reduced quantity at the apical membrane. The F508del mutation represents around 70% of all associated alleles; thus, given the recessive inheritance of the disease, approximately 90% of patients with CF carry at least one class II mutation.



Class III mutations are most often located in the adenosine-5'-triphosphate (ATP) binding domains and disrupt the regulation of the CFTR channel requiring ATP hydrolysis at nucleotide-binding domains and cAMP-dependent phosphorylation at the cytoplasmic domain for activation. Hence, the abnormal protein can reach the PM but is not activated by ATP or cAMP.



Class IV mutations are missense mutations located in the transmembrane channel and affect the conductance of the CFTR channel. Some transmembrane domain segments participate in ionic pore formation. Missense mutations in these regions produce a correctly positioned protein with cAMP-dependent Cl -activity.

Nevertheless, these channels' characteristics differ from those of the endogenous CFTR channel, with decreased ion flux and altered selectivity, making the protein partially functional.

 Class V mutations correspond to molecular anomalies that affect transcription (quantitative defect). They alter the mRNA stability; thus, the protein is functional, but the density of CFTR protein expressed at the membrane is low.



Class VI mutations include some infrequent mutations resulting in a mature functional protein but with an altered stability

CFTR Protein

CFTR Structure

The CFTR gene encodes a 1480 amino acid transmembrane CFTR protein (165 kDa) (Figure 2) belonging to the ATP-binding cassette transporter superfamily, which actively transports substrates across cell membranes, with ATP hydrolysis providing energy for this transport [START_REF] Anderson | Nucleoside triphosphates are required to open the CFTR chloride channel[END_REF][START_REF] Berger | Identification and regulation of the cystic fibrosis transmembrane conductance regulatorgenerated chloride channel[END_REF]. CFTR functions as a cAMP-dependent chloride channel. In CFTR, ATP binding opens the channel, and its subsequent hydrolysis causes channel closure.

CFTR contains two repetitive motifs, each consisting of a hydrophobic transmembrane domain (TMD) containing six α-helices and a sizeable hydrophilic nucleotide-binding domain that can bind ATP (Figure 4). A cytoplasmic domain (R-domain), encoded by exon 13, links these two motifs and contains numerous charged residues and the majority of potential phosphorylation sites [START_REF] Collins | Cystic fibrosis: molecular biology and therapeutic implications[END_REF][START_REF] Ma | Function of the R domain in the cystic fibrosis transmembrane conductance regulator chloride channel[END_REF])

CFTR Localization

CFTR is expressed at the apical membrane of epithelial cells, including sweat glands, salivary glands, airway, biliary and pancreatic ducts, liver, intestine, and vas deferens, and its density varies depending on the different tissues (Figure 5) (Ideozu et al., 2019). Within the same tissue, CFTR is expressed only by specific cells. In the lungs, it was long considered that CFTR was mainly expressed in ciliated cells (Appendix 1).

However, multiple other epithelial cell types of the airway and submucosal epithelium express the protein, including club cells, secretory cells, basal cells, submucosal gland cells, alveolar cells, and ionocytes (Figure 6, Appendix 1) [START_REF] Engelhardt | Expression of the Cystic Fibrosis Gene in Adult Human Lung[END_REF]3.2.3. CFTR Function and Dysfunction CFTR protein is primarily an anion transporter channel allowing the transport of Cl - and bicarbonate (HCO3 -) ions across the apical PM of epithelial cells [START_REF] Quinton | Chloride impermeability in cystic fibrosis[END_REF].

Cl -secretion in physiological conditions contributes to water flux that hydrates the airway surface liquid (ASL), modulating mucin entanglement [START_REF] Morrison | Treatment of cystic fibrosis airway cells with CFTR modulators reverses aberrant mucus properties via hydration[END_REF].

HCO3 -transport regulates the ASL pH and is vital for the proper tethering and detachment of mucins [START_REF] Kunzelmann | Bicarbonate in cystic fibrosis[END_REF]Quinton, 2008;Shah et al., 2016).

Recent publications have also highlighted that a loss of CFTR-mediated HCO3 - secretion and pH acidification impairs airway host defense by increasing mucus viscosity and reducing bacterial killing (Shah et al., 2016). Studies in CF pig models confirmed that in ASL, pH strongly influences bacterial killing, and therefore, a change in pH might induce impaired innate immunity by reducing the function of the antimicrobial peptide [START_REF] Hoegger | Impaired mucus detachment disrupts mucociliary transport in a piglet model of cystic fibrosis[END_REF][START_REF] Keiser | Defective Innate Immunity and Hyperinflammation in Newborn Cystic Fibrosis Transmembrane Conductance Regulator-Knockout Ferret Lungs[END_REF]A. A. Pezzulo et al., 2012). Besides being a Cl -and HCO3 -transporter, CFTR is also permeable to halide and polyatomic anions with different relative permeability: thiocyanate (SCN -) > nitrate (NO3 -) > bromide (Br -) > Cl -> iodide (I -) > formate (HCOO -) > fluoride (F -) [START_REF] Linsdell | Molecular Determinants of Anion Selectivity in the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Pore[END_REF][START_REF] Mccarty | Identification of a region of strong discrimination in the pore of CFTR. American journal of physiology[END_REF].

Moreover, this low conductance channel is a regulator of separate populations of ion channels, particularly sodium channels such as the amiloride-sensitive epithelial sodium channel (ENaC), which has a vital role in the hydration and viscosity of mucus.

The dysfunction of CFTR, and therefore the loss of CFTR inhibitory effects on ENaC, leads to increased Na + and water resorption (Figure 7) [START_REF] Knowles | Mucus clearance as a primary innate defense mechanism for mammalian airways[END_REF]Quinton, 2008). Along with Cl -loss, it causes the dehydration of ASL essential for ciliary function, thus leading to impaired mucociliary clearance (MCC) and progressive cycles of chronic infection and inflammation, leading to lung function decline (Figure 8). CFTR also has a role in water conductance via aquaporin channels [START_REF] Schreiber | The Cystic Fibrosis Transmembrane Conductance Regulator Activates Aquaporin 3 in Airway Epithelial Cells[END_REF] and potassium ion (K + ) transport at the basolateral surface of the airway epithelium via K + channel (Figure 7), of which renal outer medullary K + channel (ROMK). Finally, CFTR is required for the activation of the outward rectifying chloride channel (ORCC) by protein kinase A or ATP mainly, but not exclusively, at the basolateral membrane, such that outward rectifying chloride channel is unresponsive to any stimuli in CF [START_REF] Gabriel | CFTR and outward rectifying chloride channels are distinct proteins with a regulatory relationship[END_REF][START_REF] Jovov | Interaction between Cystic Fibrosis Transmembrane Conductance Regulator and Outwardly Rectified Chloride Channels[END_REF][START_REF] Schwiebert | CFTR regulates outwardly rectifying chloride channels through an autocrine mechanism involving ATP[END_REF][START_REF] Szkotak | The role of the basolateral outwardly rectifying chloride channel in human airway epithelial anion secretion[END_REF]. CFTR is also able to regulate calcium-activated chloride channels (CaCCs), however, some studies suggest that CFTR expression and activation at the PM require TMEM16A, the primary CaCC [START_REF] Benedetto | Plasma membrane-localized TMEM16 proteins are indispensable for expression of CFTR[END_REF]Benedetto et al., 2017;[START_REF] Ousingsawat | CFTR and TMEM16A are Separate but Functionally Related Cl-Channels[END_REF] (See Chapter 2, paragraph 6). secretion and regulates Na + absorption by inhibiting ENaC on the apical surface. CFTR also positively regulates the Cl -channels (ANO1/TMEM16A and SLC26A9) and the anion exchanger SLC26A4, increasing Cl -and HCO 3 -secretion and therefore increasing ASL hydration and pH. Na + and water follow the electrochemical gradient through the paracellular pathway. On the basolateral membrane, Na-K-2Cl cotransporter-1 (NKCC1) accumulates Cl - intracellularly, supported by electrochemical gradients established by the basolateral Na + /K + ATPase. K + recycling across the basolateral membrane is governed by the K + channel and serves to adjust membrane potential. From [START_REF] Saint-Criq | Role of CFTR in epithelial physiology[END_REF].

Current studies have established other functions for CFTR than ion transport regulation (Figure 8). Results have suggested a significant role of CFTR as a surface receptor for the internalization of Pseudomonas aeruginosa (P. aeruginosa) via endocytosis and consequent bacteria removal from the airway (Pier, 2000). Also, the loss of functional CFTR in immune cells results in the dysregulation of critical functions [START_REF] Bonfield | Absence of the cystic fibrosis transmembrane regulator (<i>Cftr</i>) from myeloid-derived cells slows resolution of inflammation and infection[END_REF][START_REF] Brennan | Innate immune activation and cystic fibrosis[END_REF][START_REF] Bruscia | Cystic Fibrosis Lung Immunity: The Role of the Macrophage[END_REF][START_REF] Bruscia | Macrophages directly contribute to the exaggerated inflammatory response in cystic fibrosis transmembrane conductance regulator-/-mice[END_REF][START_REF] Bubien | Cell cycle dependence of chloride permeability in normal and cystic fibrosis lymphocytes[END_REF][START_REF] Mcdonald | Human lymphocytes transcribe the cystic fibrosis transmembrane conductance regulator gene and exhibit CF-defective cAMP-regulated chloride current[END_REF]. In addition, CFTR is permeable to anionic antioxidant tripeptide glutathione, implicating CFTR's role in controlling oxidative stress in the airways (Figure 8) [START_REF] Kogan | CFTR directly mediates nucleotide-regulated glutathione flux[END_REF][START_REF] Linsdell | Glutathione permeability of CFTR[END_REF].

Dysfunctional CFTR induces multiple important metabolic changes in lipids, the main component of membranes that also regulates inflammation (Figure 8). For example, eicosanoid synthesis through the arachidonic acid pathway plays a vital role in inflammation (See 4.1.1). The CFTR protein is not just a Cl -channel. It is also involved in HCO 3 -, glutathione (GSH), and SCN -transport, regulation of ENaC, immune cells, and metabolism of lipids. In CF, the loss of functional CFTR results in bronchial obstruction (in yellow), inflammation (in orange), and infection (in green), leading to lung injury. From [START_REF] Hanssens | CFTR Protein: Not Just a Chloride Channel?[END_REF].

Clinical Manifestations

Absent or dysfunctional CFTR protein causes damage in organs lined with epithelial cells and mucus, including the lungs, the pancreas, the gastrointestinal tract, the liver, and the reproductive tract. Moreover, it leads to complications, including CF-related diabetes, metabolic bone disease, gastrointestinal malignancy, and comorbidities (Table 1, Figure 9). (Elborn, 2016).

Respiratory

The following paragraph is an adapted extract from the review "Novel Anti-Inflammatory Approaches for Cystic Fibrosis Lung Disease: Identification of Molecular Targets and Design of Innovative Therapies," written by Mitri et al. and published in Frontiers in July 2020 (Mitri et al., 2020) (Appendix 6).

While most patients have multiple organ alterations, the leading causes of morbidity and mortality in more than 90% of patients remain chronic progressive pulmonary disease and respiratory failure (Elborn, 2016). In patients with CF, the lack of CFTR chloride channel activity leads to dehydrated, thick secretions that cause mucus 4.1.1. Inflammation Although inflammation is an innate and protective defense mechanism resulting from pathogen invasion, this natural process plays a significant role in CF lung pathogenesis. Chronic and elevated inflammation in a patient with CF causes lung tissue damage that can eventually cause respiratory failure (Cantin et al., 2015). It is unclear whether the inflammation is a direct consequence of the CFTR mutation or a result of infection and mucus accumulation since pulmonary inflammation and infection are often present in patients with CF even before symptom onset.

Recent results show that bronchial epithelial cells play a significant role in the progression of the disease. In addition to being a physical barrier, epithelial cells secrete many inflammatory factors such as cytokines, eicosanoids, enzymes, and adhesion molecules (Roesch et al., 2018). In CF, infiltrating inflammatory cells across the epithelium into the lumen can be deleterious to epithelia and requires precise regulation. CF airway inflammation is characterized by large numbers of neutrophils and macrophages, and other inflammatory cells, accompanied by excessive interleukin-8 (IL-8) secretion by airway epithelial cells. In the airways of patients with CF, additional cytokines (TNF-α, IL-1β, IL-6, IL-8, IL-33, GM-CSF, and G-CSF) are increased, along with pro-inflammatory metabolites of arachidonic acid metabolism.

Different groups have highlighted the central role of neutrophils in CF airway inflammation, and many believe that bronchiectasis results from the proteolytic and oxidative damage induced by these cells (Nichols & Chmiel, 2015;Perrem & Ratjen, 2019). Several mechanisms are suggested to explain how CF basal inflammation promotes subsequent bacterial infection. One possible explanation is that serine protease, released by activated neutrophils, degrades innate immune components, including antimicrobial peptides, participates in secondary infection, and initiates a vicious cycle. In 2016, researchers discovered leukocyte adhesion deficiency IV, a defect in monocyte integrin activation in patients with CF (Sorio et al., 2016). The study showed that CFTR mutations could lead to a monocyte-specific adhesion deficiency (~80%) and impairment in transmigration into the alveolar space, which could explain the intense infiltration of neutrophils since monocytes play a crucial part in inflammation and its resolution. Thus, failing to recruit monocytes in the lungs of patients with CF may explain the excessive production of cytokines, inadequate inflammation resolution, and impaired pathogen capture (Sorio et al., 2016).

Furthermore, different groups showed more than 15 years ago that there is a dysregulation of lipid metabolism in CF with an imbalance between pro-inflammatory metabolites of arachidonic acid metabolism and pro-resolving mediators from the eicosanoid pathway (Freedman et al., 2004;Karp et al., 2004;Roesch et al., 2018;Serhan, 2017). Ceramide is an airway component composed of sphingosine and a fatty acid that may alter the CF inflammatory response. Ceramide is present in the cell membrane, and when in contact with a specific stimulus, like a bacteria, it participates in transmembrane signaling processes to help regulate cellular responses to infection by activating the inflammatory processes. This could be an interesting alternative to treating CF inflammatory dysregulation by inhibiting ceramide synthesis (Mingione et al., 2020). However, there is currently no consensus regarding the regulation of ceramide in CF cells, even though more recent data have demonstrated the implication of these waxy lipid molecules in the progression of CF lung disease (Horati et al., 2020).

The development of new anti-inflammatory strategies in CF remains limited due to the paucity of research in this area compared to the more general topic of infection.

Bacterial Infection

In healthy people, MCC is the primary innate lung defense mechanism protecting against pathogens in the airways. In CF, ASL dehydration causes mucin concentration and decreases cilia beating, which in turn causes slow impaired MCC. In progressive lung diseases like CF, typical pathogens (P. aeruginosa, Streptococcus aureus, Burkholderia cepacia, Achromobacter xylosoxidans) colonize the airways (Palser et al., 2019). Respiratory bacterial colonization in CF occurs during childhood. More than 50% of children diagnosed at birth have positive P. aeruginosa cultures by five years of age (Palser et al., 2019). If P. aeruginosa is neither spontaneously cleared nor eradicated with antibiotic therapy, the CF lung environment facilitates the colonization.

Moreover, P. aeruginosa colonization leads to a poor prognosis and accelerated disease evolution.

The presence of pathogens triggers inflammatory processes in the airways contributing to the destruction of the cell barrier. Since inflammation is an innate defense mechanism designed to eradicate pathogens, suppressing this important process for an extended period could be counterproductive. For this reason, antibiotics are more frequently recommended than anti-inflammatory drugs in CF lung disease treatment, and their mechanism of action can indirectly diminish airway inflammation (Oermann et al., 1999;Saint-Criq et al., 2012). Anti-inflammatory drugs that can alter natural lung defenses are only prescribed during exacerbations. As a result of long-term antibiotic treatment, the decreased bacterial and inflammatory burden is associated with lung function improvements and reduction of pulmonary exacerbations (Waters, 2018). The constant development and patient-to-patient adapted usage of new antimicrobial compounds are vital for improving patient survival and quality of life (Waters & Smyth, 2015).

In healthy conditions, the airways defend against inhaled particles by forming a physical barrier between the outside and the inside. In case of infection, the lung is capable of secreting cytokines that will allow the recruitment of inflammatory cells, but it is also capable of secreting antibacterial molecules. Thus, many natural antimicrobial peptides produced in the airways are part of the innate immune response that protects the lungs (R. E. W. Hancock et al., 2016). In CF, unfavorable environments for antimicrobial peptides actions, such as abnormal salt concentration or acidification and inactivation by proteases in ASL, have been shown to inactivate bactericidal functions, which may explain increased airway infections (Bals et al., 2001;Lecaille et al., 2016;Simonin et al., 2019). Normalizing ASL pH might enhance innate airway defense in CF newborns during the onset of infections.

Mucus Alteration

ASL is a thin liquid-rich milieu consisting of two main layers: (1) an apical polymeric mucus layer overlying (2) a periciliary layer (PCL) that surrounds the cilia and facilitates cilia beating (Atanasova & Reznikov, 2019) (Figure 10). In healthy people, ASL covers the airways preventing airway desiccation and governing MCC.

Historically, studies suggested that different secretory cells (serous and mucous cells of the submucosal glands, club cells, and goblet cells) contribute to ASL production (Tarran et al., 2001). The recent finding of the airway "ionocyte" could result in a revised understanding of fluid secretion in the lungs (Plasschaert et al., 2018). Normal mucus comprises 97% water and 3% proteins, lipids, and salt. The mucus gel layer acts as a physical barrier to prevent most pathogens from accessing the cells (Button et al., 2012). Mucin 5B (MUC5B) and mucin 5AC (MUC5AC), the predominant mucins produced by airway epithelial cells, are the principal components of airway mucus and serve different functions. MUC5B ensures normal mucus clearance, and MUC5AC has pathologic roles in mechanisms of allergen-induced airway hyperresponsiveness, mucous metaplasia, and airway mucus plugging [START_REF] Evans | The polymeric mucin Muc5ac is required for allergic airway hyperreactivity[END_REF][START_REF] Hancock | Muc5b overexpression causes mucociliary dysfunction and enhances lung fibrosis in mice[END_REF][START_REF] Livraghi-Butrico | Contribution of mucus concentration and secreted mucins Muc5ac and Muc5b to the pathogenesis of muco-obstructive lung disease[END_REF]X. Wang et al., 2017). Hydration and mucin concentration dramatically affect the viscoelastic mucus properties, which, in turn, determine how effectively it is cleared from the distal airways toward the trachea by MCC (Fahy & Dickey, 2010).

As mentioned earlier, a defective CFTR is associated with (1) low HCO3 - concentration, (2) acidic pH ASL, and (3) inadequate ASL hydration, altogether altering the mucus physical properties in CF airways. In addition to altered physical properties, CF mucus composition is modified, which participates in the pathophysiology by altering host proteins (Henderson et al., 2014). Increased mucin concentration and an abnormal mucus composition are implicated in the formation of endobronchial mucus plaques and plugs. The bronchioles become the primary site of airway mucus obstruction [START_REF] Cabrini | Role of Cystic Fibrosis Bronchial Epithelium in Neutrophil Chemotaxis[END_REF][START_REF] Cabrini | Overview of CF lung pathophysiology[END_REF][START_REF] Yu | StatPearls. Treasure Island[END_REF], and subsequent chronic infection and persistent inflammation lead to airway structural integrity damage, succeeded by bronchiectasis development (Chalmers et al., 2017).

Gastrointestinal Tract

Early in the history of the disease, infants with CF died shortly after birth from severe pancreatic insufficiency and gastrointestinal complications, as these manifestations are more commonly encountered in the first year of life, and treatments were rapidly developed. In brief, CFTR mutations cause impaired Cl -and HCO3 [START_REF] Burton | Luminal Gastrointestinal Manifestations of Cystic Fibrosis[END_REF][START_REF] Sathe | Meconium ileus in Cystic Fibrosis[END_REF]. Studies have shown that CF mice and piglet models also display poor growth and develop bowel obstructions that are typically lethal [START_REF] Rosen | Animal and model systems for studying cystic fibrosis[END_REF][START_REF] Semaniakou | Animal Models in the Pathophysiology of Cystic Fibrosis[END_REF][START_REF] Wilke | Mouse models of cystic fibrosis: Phenotypic analysis and research applications[END_REF]. Approximately 20% of untreated children have rectal prolapse, secondary to increased abdominal pressures from cough, constipation, malabsorption, malnutrition, and bulky stool.

Distal intestinal obstruction syndrome is a common condition in CF that occurs at all ages and is characterized by complete or partial obstruction in the terminal ileum and caecum due to thick mucus. The pathophysiology of distal intestinal obstruction syndrome is still unclear. Risk factors like intestinal dysmotility, inflammation, fat malabsorption, and defective ion and water secretion into the gut lumen may contribute to its development.

Constipation is one of the most common gastrointestinal manifestations among patients with CF, affecting nearly one-half of pediatric and most adult patients [START_REF] Burton | Luminal Gastrointestinal Manifestations of Cystic Fibrosis[END_REF]. The most common symptoms include abdominal pain, flatulence, and decreased appetite. Constipation begins distally in the colon and extends proximally as the condition worsens, as opposed to distal intestinal obstruction syndrome, in which the opposite is true, although both conditions often occur together.

Motility

CFTR expression is commonly found on the apical surface of enterocytes throughout the gastrointestinal tract. With CFTR dysfunction, gastrointestinal motility problems are widespread in CF and are probably due to viscous mucus in the luminal tract contributing to dysmotility [START_REF] Strong | Localization of cystic fibrosis transmembrane conductance regulator mRNA in the human gastrointestinal tract by in situ hybridization[END_REF]. In addition, frequent antibiotic use, intestinal inflammation, and small bowel bacterial overgrowth can all impact gut motility [START_REF] De Lisle | Mast Cells and Gastrointestinal Dysmotility in the Cystic Fibrosis Mouse[END_REF][START_REF] De Lisle | Enteric circular muscle dysfunction in the cystic fibrosis mouse small intestine[END_REF] as well as poor glycemic control [START_REF] Kuo | Gastric Emptying, Incretin Hormone Secretion, and Postprandial Glycemia in Cystic Fibrosis-Effects of Pancreatic Enzyme Supplementation[END_REF], fat-rich and/or carbohydrate-rich meals, previous surgery, and hydration [START_REF] Camilleri | Clinical Guideline: Management of Gastroparesis[END_REF].

Gastroesophageal Reflux Disease

Pathological gastroesophageal reflux has been reported in more than 50% of children and 86% of adult patients. An association between reflux episodes and worsening pulmonary outcomes in patients with CF has been described [START_REF] Gustafsson | Gastrooesophageal reflux and severity of pulmonary disease in cystic fibrosis[END_REF]. [START_REF] Coffey | Gut Microbiota in Children With Cystic Fibrosis: A Taxonomic and Functional Dysbiosis[END_REF][START_REF] Eng | Infants with cystic fibrosis have altered fecal functional capacities with potential clinical and metabolic consequences[END_REF][START_REF] Kristensen | Development of the gut microbiota in early life: The impact of cystic fibrosis and antibiotic treatment[END_REF][START_REF] Nielsen | Disrupted progression of the intestinal microbiota with age in children with cystic fibrosis[END_REF].

Gastrointestinal Malignancy

Intestinal and colon cancers are increasing in the adult CF population. Male patients and patients with a history of distal intestinal obstruction syndrome have a higher risk of developing malignancies [START_REF] Hadjiliadis | Cystic Fibrosis Colorectal Cancer Screening Consensus Recommendations[END_REF][START_REF] Neglia | The Risk of Cancer among Patients with Cystic Fibrosis[END_REF][START_REF] Yamada | Risk of gastrointestinal cancers in patients with cystic fibrosis: a systematic review and meta-analysis[END_REF]. Malignancies are also found in the pancreas and biliary tree. The risk for colorectal cancer in adults with CF is five to ten times greater than in the general population. The incidence increases further (25 to 30 times) in patients who have undergone organ transplantation.

Pancreatic Disease

Pancreatic insufficiency is characterized by significantly decreased pancreatic ductal flow and absent digestive enzymes. Over 95% of severe patients (class I-III) are pancreatic insufficient or progress to pancreatic insufficiency [START_REF] Ahmed | Molecular consequences of cystic fibrosis transmembrane regulator (CFTR) gene mutations in the exocrine pancreas[END_REF].

Impaired Cl -and HCO3 -transport results in pancreatic secretions with a lower pH, increased viscosity, and higher protein concentration, leading to ductal obstruction.

The result of alterations in fluid volume, viscosity, and flow is that proenzymes build up within the obstructed ducts, leading to early activation of pancreatic enzymes that trigger acute or recurrent inflammation and lead to progressive pancreatic destruction.

The pancreatic disease starts in utero [START_REF] Gibson-Corley | Pancreatic pathophysiology in cystic fibrosis[END_REF]. Approximately 70% of patients with CF have pancreatic insufficiency from birth. An additional 20% develop this condition during the first three years of life. The most severe signs are steatorrhea, failure to thrive and poor weight gain due to fat and protein malabsorption, and fat-soluble vitamins (A, D, E, and K) deficiency. Patients that are pancreatic sufficient are at risk for developing pancreatitis.

Moreover, patients with a damaged pancreas, and thus, damaged islet cells, often develop dysfunction of the endocrine pancreas, leading to glucose intolerance and CF-related diabetes [START_REF] Gibson-Corley | Pancreatic pathophysiology in cystic fibrosis[END_REF]. The latter is an age-dependent increasing problem as 20 to 30% of adolescents and at least 50% of adults with CF develop diabetes. It is a combined problem of insulin resistance and insulin deficiency.

CF-related diabetes is associated with a decline in pulmonary function and nutritional status; thus, early diabetes detection may prevent this decline. Diabetes should be well controlled, as patients develop microvascular complications after 5-10 years of diabetes.

Hepatobiliary Disease

While the leading cause of death in CF continues to be pulmonary disease complications followed by lung transplantation complications, CF-associated liver disease has been identified as the third most common and most crucial non-pulmonary cause of death [START_REF] Pals | Cirrhosis associated with decreased survival and a 10-year lower median age at death of cystic fibrosis patients in the Netherlands[END_REF][START_REF] Paranjape | Cystic fibrosis in the era of precision medicine[END_REF]. The most common hepatic finding in CF is hepatic steatosis which can be related to malnutrition and altered phospholipids' metabolism. Biliary cirrhosis and cholestasis caused by impaired bile flow are also clinical manifestations.

Nutrition and Growth Disorder

Poor nutrition is a serious concern for patients with CF and is often due to malabsorption related to defective pancreatic enzymes. Inadequate nutritional intake and increased energy losses can increase undernutrition, leading to impaired cognitive function, worse lung function, and poor survival [START_REF] Corey | A comparison of survival, growth, and pulmonary function in patients with cystic fibrosis in Boston and Toronto[END_REF][START_REF] Stallings | Evidence-based practice recommendations for nutrition-related management of children and adults with cystic fibrosis and pancreatic insufficiency: results of a systematic review[END_REF].

Bone and Teeth Problems

Up to 30% of patients with CF have low bone density and increased fracture rates, while this proportion approaches 75% in adults with CF [START_REF] Aris | Guide to Bone Health and Disease in Cystic Fibrosis[END_REF]. This is primarily due to the malabsorption of fat-soluble vitamins such as vitamin D and K, poor nutritional status, low physical activity, glucocorticoid use, delayed puberty, and hypogonadism (Isabelle Sermet-Gaudelus et al., 2011). Severe enamel defects in permanent dentition are also observed in CF patients. As patients with CF are already at risk of infection, dental caries and periodontal diseases should be monitored closely to prevent autoinfection as they may be a reservoir of microorganisms, including P.

aeruginosa [START_REF] Pawlaczyk-Kamienska | Dental and periodontal manifestations in patients with cystic fibrosis -A systematic review[END_REF].

Fertility Problems

More than 98% of men with CF are infertile due to congenital bilateral absence of the vas deferens resulting in obstructive azoospermia. Meanwhile, females with CF are less fertile than healthy women due to malnutrition and abnormal tenacious cervical mucus production. Nonetheless, with proper treatment, females with CF may become pregnant [START_REF] Ahmad | Cystic fibrosis and fertility[END_REF][START_REF] Naz Khan | CF and male health: Sexual and reproductive health, hypogonadism, and fertility[END_REF].

Therapies

Conventional Symptomatic Therapies

Management of CF is a multidisciplinary therapy tailored to the individuals and their comorbidities to help control the symptoms, prevent complications, and make the condition easier to tolerate (cff.org/managing-cf, mayoclinic.org, vaincrelamuco.org).

For pulmonary problems, medications are often inhaled and include the following:

 Antibiotics, to prevent and treat lung infections,  Mucolytics, to make the mucus in the lungs thinner and easier to cough up,  Bronchodilators, to widen the airways and reduce inflammation, Exercise has many beneficial effects and has become strongly recommended.


Complementary practices such as yoga, massage, acupuncture, meditation, and chiropractic often provide significant relief for anxiety and depression while improving overall well-being.

Cystic fibrosis is a complex disease with numerous medical, psychiatric, and social consequences. For patients and their families, it means a lifetime of adjustments.

Back in 1938, the life expectancy for patients with CF was less than a year. Over the last six decades, significant improvements have been made in patient care allowing patients with CF to live beyond 50 years (Figure 11) [START_REF] Elborn | Personalised medicine for cystic fibrosis: treating the basic defect[END_REF]. With the improved conventional therapy treating CF symptoms, disease progression now generally occurs at a slower pace. Further slowing might become evident in the next decade with the widespread adoption of highly effective CFTR modulator therapies.

Basic Defect Therapies

The CF community has recently witnessed remarkable scientific advances with the therapeutic development of CFTR modulators treating its fundamental defect. As of today, 4 CFTR modulator drugs have obtained the Food and Drug Administration (FDA) and the European Medicines Agency (EMA) marketing approval (Table 2). To accurately determine the effects of a CFTR modulator treatment on clinical outcomes, several endpoints were measured, including change in percent predicted FEV1

(Forced Expiratory Volume in 1 second) and change in sweat chloride concentration, which is often used to aid in the diagnosis of CF by measuring CFTR activity. In healthy people, sweat chloride levels are typically less than 30 mmol/L, while levels exceed 60 mmol/L in patients with CF [START_REF] Tice | Modulator Treatments for Cystic Fibrosis: Effectiveness and Value; Final Evidence Report and Meeting Summary[END_REF].

Ivacaftor

Approved in July 2012, Ivacaftor (trade name Kalydeco, developed as VX-770) was the first CFTR potentiator developed by Vertex Pharmaceuticals (Boston, MA). As a potentiator, Ivacaftor binds to the defective CFTR at the cell membrane, increases the channel gating, and restores its activity. To date, the binding site for Ivacaftor remains unclear, with studies proposing that it interacts directly with CFTR by binding to a site most likely located between the TMDs [START_REF] Yeh | Identifying the molecular target sites for CFTR potentiators GLPG1837 and VX-770[END_REF]. This molecule significantly increased FEV1 from patients eligible to take this drug by 10.6 percentage points and decreased sweat chloride concentration by -48.1 mmol/L compared to placebo after 48 weeks [START_REF] Ramsey | A CFTR Potentiator in Patients with Cystic Fibrosis and the<i>G551D</i>Mutation[END_REF]. Ivacaftor was initially discovered to target the G551D et al., 2016;[START_REF] De Boeck | Efficacy and safety of ivacaftor in patients with cystic fibrosis and a non-G551D gating mutation[END_REF][START_REF] Ramsey | A CFTR Potentiator in Patients with Cystic Fibrosis and the<i>G551D</i>Mutation[END_REF]. However, most patients are heterozygous for gating mutations and 3000 patients can currently benefit from it worldwide. Today, Ivacaftor can be prescribed to infants aged four months and older. Wainwright et al., 2015); however, this molecule brought undeniable hope to the CF community and was seen as a breakthrough drug for CF. Limitations of this combination include respiratory adverse events and several drug-drug interactions, which led Vertex to develop better-tolerated correctors with fewer drug-drug interactions, such as Tezacaftor (VX-661).

Tezacaftor-Ivacaftor

The combination Tezacaftor-Ivacaftor licensed as Symdeko (or Symkevi Europe) in 2018 has similar efficacy to Orkambi without the side effects [START_REF] Schwarz | Tezacaftor/ivacaftor in people with cystic fibrosis who stopped lumacaftor/ivacaftor due to respiratory adverse events[END_REF]. It is marketed for patients homozygote for F508del or patients with at least one copy of the F508del mutation and one of the 14 other residual function mutations and was approved for ages six years and older in 2019. Symdeko provides a modest but sustained 3 to 4 percentage points improvement in predicted FEV1 for homozygote patients and a 10,1 mmol/L decrease in sweat chloride concentration (Rowe et al., 2017;[START_REF] Taylor-Cousar | Tezacaftor-Ivacaftor in Patients with Cystic Fibrosis Homozygous for Phe508del[END_REF][START_REF] Tice | Modulator Treatments for Cystic Fibrosis: Effectiveness and Value; Final Evidence Report and Meeting Summary[END_REF].

Elexacaftor-Tezacaftor-Ivacaftor

Trikafta, a combination of Tezacaftor, Ivacaftor, and Elexacaftor, is the last of the four modulator therapies developed by Vertex and is considered a game-changer for patients with CF. It was first approved by the FDA in 2019 and one year later by the EMA in 2020 (licensed and marketed as Kaftrio). The triple modulator combination can be prescribed to patients 6 years and older with at least one copy of the F508del mutation or a mutation in the CFTR gene that is responsive to Trikafta based on in vitro data, which makes up almost 85% of patients with CF. The estimated absolute increase for predicted FEV1 compared to placebo for Trikafta (14.0 percentage points) (Middleton et al., 2019) is markedly more significant than the estimates for Orkambi

Current Limitation

Although current modulator therapies target 85% of patients with CF, the exact mechanisms of action for these drugs are still poorly understood. Furthermore, one significant downside of CFTR modulators is their price: treatment costs up to €200,000 per patient per year, which can challenge the availability of fair and equitable treatment between patients in different countries. Besides their extremely high costs, there are additional hurdles to overcome.

Post-approval safety and efficacy studies showed that the frequency of adverse events in response to CFTR modulator treatment is higher than what was observed in clinical trials. In different cohorts from the United States, Canada, Italy, United Kingdom, Scotland, France, Germany, Switzerland, Australia, Ireland, Greece, and Spain, the prevalence of combined adverse events related to respiratory (Chest tightness/dyspnea, bronchospasm, increased cough/sputum, hemoptysis), digestive (diarrhea, abdominal pain), menstrual abnormality, fatigue, and headaches, were greater than two percent [START_REF] Dagenais | Real-World Safety of CFTR Modulators in the Treatment of Cystic Fibrosis: A Systematic Review[END_REF]. Hepatic events, nausea, cutaneous rash, tachycardia, depression, and anxiety were also observed [START_REF] Dagenais | Real-World Safety of CFTR Modulators in the Treatment of Cystic Fibrosis: A Systematic Review[END_REF].

In some cases, pronounced adverse events led to the discontinuation or interruption of the treatment. Moreover, a recent study has found that Trikafta treatment for patients post-lung transplants resulted in limited improvements and were not well tolerated, with risks for adverse pulmonary and mental health effects [START_REF] Doligalski | Poor tolerability of cystic fibrosis transmembrane conductance regulator modulator therapy in lung transplant recipients[END_REF]. In the study, out of 13 transplanted patients, 40% discontinued treatment before the end of the first year. In addition, efficacy observed in clinical trials may be more modest in the "real world".

A significant challenge to currently available CFTR modulators is that they require oral administration with fatty food while avoiding food or drugs that may interact with.

Moreover, patients ingesting roughly 20 tablets daily make the treatment a heavy burden as they require increasing time and effort. Also, when novel therapies become accessible, patients must add on their medications instead of replacing some, escalating the burden of cost and treatments.

Another drawback is that every single CFTR modulator mentioned above is mutation class-specific and thus restricted to people with specific mutations. Patients with class I mutations or carrying rare/unique mutations account for 10 to 15% of the CF population and cannot benefit from such treatments, leaving an unmet medical need.

Also, under the current license, some patients are ineligible for this effective therapy because of their age, genotype, or transplantation status. Moreover, although CFTR modulators are very effective for 85% of the patients, taking a drug combination for life is not ideal as some patients may become intolerant to the treatment or irresponsive.

Therefore, alternative strategies continue to be explored. was abandoned in a Phase II proof-of-concept study as it failed to meet the prespecified efficacy criteria.

Alternative Therapies in Development

Many pharmaceutical companies are developing numerous drugs to restore CF function, enhance mucociliary clearance, treat inflammation and/or infection, or treat gastrointestinal and nutritional problems (Figure 12). Although most of the drugs target the CFTR gene or protein, other exciting approaches targeting other channels are also arising. This paragraph will briefly review some of the alternative therapies that are currently in development based on the US Cystic Fibrosis Foundation database (https://apps.cff.org/trials/pipeline, last accessed August 2022).

ELX-02

This drug, developed by Eloxx Pharmaceuticals, targets patients with at least one copy of the G542X CFTR mutation (class I). ELX-02 is a read-through agent of mRNA transcripts bearing nonsense mutations. Through interaction with the ribosome, the aminoglycoside analog induces read-through of nonsense mutations leading to fulllength functional protein production [START_REF] Crawford | Targeting G542X CFTR nonsense alleles with ELX-02 restores CFTR function in human-derived intestinal organoids[END_REF][START_REF] Kerem | ELX-02: an investigational read-through agent for the treatment of nonsense mutation-related genetic disease[END_REF]. This compound is currently in a phase II study to test the safety and tolerability in adults with at least one G542X mutation.

4D-710

4D Molecular Therapeutics is working on an adeno-associated virus vector designed to deliver a wild-type (WT) CFTR gene to cells in the lungs. A phase I study in adults with CF is underway. Although the development of these drugs was discontinued after phase II studies (Figure 13), it highlights the interest in targeting channels other than CFTR.

TMEM16A Channel Modulators

TMEM16A (also called Anoctamin-1 or ANO1), an alternative chloride channel, is the basis of our work and will be extensively described in the next chapter, "TMEM16A".

Briefly, different studies suggest the activation of TMEM16A as an alternative approach to compensate for CFTR deficiency. Hence, compounds such as Denufosol (Inspire Pharmaceuticals) and ETX001 (or ETD002, Roche, Genentech) were developed to potentiate TMEM16A activity (See chapter 2, paragraph 7).

Chapter 2: TMEM16A

Identification

CaCCs were described for the first time in 1981 in Rana pipiens eggs (Cross, 1981) and then in Xenopus laevis oocytes (Miledi, 1982). Today, CaCCs have been identified in many cell types. In neurons [START_REF] André | Axotomy-induced expression of calciumactivated chloride current in subpopulations of mouse dorsal root ganglion neurons[END_REF]Frings et al., 2000) and cardiac muscle cells (D. [START_REF] Guo | Calcium-activated chloride current contributes to action potential alternations in left ventricular hypertrophy rabbit[END_REF]Leblanc et al., 2005), CaCCs contribute to electrical activity. In olfactory receptors [START_REF] Kurahashi | Olfactory transduction. Tale of an unusual chloride current[END_REF] and photoreceptors [START_REF] Macleish | Ion channel compartments in photoreceptors: evidence from salamander rods with intact and ablated terminals[END_REF], CaCCs are involved in stimuli transduction. In the epithelia, CaCCs have also been shown to play essential roles in transepithelial fluid and mucus secretion [START_REF] Danahay | Interleukin-13 induces a hypersecretory ion transport phenotype in human bronchial epithelial cells[END_REF][START_REF] Galietta | The TMEM16 Protein Family: A New Class of Chloride Channels[END_REF]Kidd & Thorn, 2000). Although Cl -is the predominant extracellular and intracellular anion, and thus, Cl -fluxes are CaCCs' primary conducted currents, CaCCs can also conduct different anions, with a preference for larger anions (Zhiqiang [START_REF] Qu | Anion Permeation in Ca2+-Activated Cl-Channels[END_REF]. The relative permeabilities measured under bi-ionic conditions by changes in reversal potential were as follows: CaCC.

C(CN)3 > SCN > N(CN)2 > ClO4 > I > N3 > Br > Cl >
CaCCs molecular identity remained unidentified for decades until recently. Several channels, such as calcium-activated chloride channel regulator (CLCA), bestrophins (BEST), and chloride voltage-gated channel 3 (CLC-3), were proposed as potential candidates for CaCCs but were later dismissed due to the properties of these channels.

In 2008, three independent research groups identified TMEM16A as a CaCC [START_REF] Caputo | TMEM16A, a membrane protein associated with calcium-dependent chloride channel activity[END_REF]Schroeder et al., 2008;Y. D. Yang et al., 2008) (Caputo et al., 2008a;Schroeder et al., 2008;Y. D. Yang et al., 2008).

TMEM16A belongs to the anoctamin family (ANO for anion channel and OCTA for their eight predicted transmembrane domains or TMEM16). This family consists of 10 proteins: ANO1 through 10, or TMEM16 A to K, excluding I, and is separated into two groups (TMEM16A-B and the rest). TMEM16A and TMEM16B work as Cl -channels, whereas the rest serve as Ca 2+ -activated lipid scramblases with non-selective ion channel activity. Despite being a Cl -channel TMEM16B has different biophysical characteristics than TMEM16A [START_REF] Scudieri | TMEM16A-TMEM16B chimaeras to investigate the structure-function relationship of calcium-activated chloride channels[END_REF], and its expression is limited to the vomeronasal epithelium (Dibattista et al., 2012;Kunzelmann et al., 2011). The TMEM16A gene is located on chromosome 11q13 and contains 26 exons, resulting in proteins ranging in size from 840 to 1008 amino acids (114 kDa).

The Overall Structure of the TMEM16A Channel

Since its discovery 14 years ago, several studies have been published describing the topological structure of TMEM16A. In 2011, a chemical cross-linking study performed by Sheridan et al. revealed that TMEM16A exists as a dimer (Sheridan et al., 2011).

The dimeric structure of TMEM16A was also confirmed by Fallah et al. using biochemical techniques such as native PAGE and chemical cross-linking (Fallah et al., 2011). Furthermore, Brunner et al. published the X-ray structure of Ca 2+ -activated TMEM16A, showing that both subunits are symmetrical and have identical conformation (Brunner et al., 2014). Recently, two research groups proposed, based on high-resolution single-particle cryo-electron microscopy, a standard structure of TMEM16A consistent with earlier biochemical studies showing homodimers in the structure of anoctamins (Dang et al., 2017;Paulino et al., 2017a). Each subunit contains N-and C-terminal domains on the cytoplasmic side of the membrane, a transmembrane unit consisting of 10 membrane-spanning α-helices, one independent ion-conducting pore, and two Ca 2+ binding sites (Figure 14). The Ca 2+ binding sites are located near the membrane cytosolic end of the pore. The pore, which includes TMD3-7, adopts an hourglass shape with a wide intracellular vestibule and a smaller extracellular vestibule connected by a restricted neck region.

Ten pore-lining residues on TMD3-7 were identified as crucial for the anion selectivity of TMEM16A channels. Five conservative acidic residues (E654, E702, E705, E734, and D738) group together on TM6-8 to form two Ca 2+ ion-binding sites and, thus, regulate the opening of the channel (Greenwood et al., 2001;Ledoux et al., 2003) (Figure 14). Each monomeric subunit is regulated independently (Jeng et al., 2016;[START_REF] Lim | Independent activation of ion conduction pores in the double-barreled calcium-activated chloride channel TMEM16A[END_REF]. The N-terminal domain also participates in an intra-subunit interaction with the cytoplasmic C terminus. In Dang et al. and Paulino et al. proposed cryo-electron microscopy structure, the channel is supposed to be closed because TMEM16A reduces the Ca 2+ in a time-dependent manner, pushing the channel into an inactive state (Dang et al., 2017;Paulino et al., 2017b). Despite several attempts by researchers to decipher the structure of TMEM16A in the open state, the fully open pore structure of TMEM16A has not been elucidated (A. Lam & Dutzler, 2021).

Regulation and Activation of TMEM16A

TMEM16A has typical characteristics of CaCCs, including Ca 2+ and voltagedependent activation (Kuruma & Hartzell, 2000;Liu et al., 2021;Terashima et al., 2013). When Ca 2+ is absent, no TMEM16A current is measured (Y. D. Yang et al., 2008). At low Ca 2+ concentrations, TMEM16A activates outwardly rectifying currentvoltage, and at high Ca 2+ concentrations, the outwardly rectifying current-voltage becomes linear (Caputo et al., 2008a;Y. D. Yang et al., 2008). Like most natively expressed CaCCs, TMEM16A activation is sensitive to cytosolic Ca 2+ at very low concentrations, and the current is voltage-dependent (Hartzell et al., 2005;Kleene & Gesteland, 1991;[START_REF] Qu | Characterization of Ca2+-activated Clcurrents in mouse kidney inner medullary collecting duct cells[END_REF]. The synergistic regulatory mechanism is due to the Ca 2+ binding site localized within the transmembrane segment of the channel, which makes CaCCs sensitive to membrane potential (Arreola et al., 1996;Le & Yang, 2020;Terashima et al., 2013). According to a few studies, the required Ca 2+ concentration for TMEM16A activation ranges from 0.4-0.6 µM at a positive membrane potential, and this value increases as the membrane potential deviates to the negative side (Figure 15) (Ferrera et al., 2009;Kato et al., 2021;Y. D. Yang et al., 2008). When activated by Ca 2+ , TMEM16A exhibits an instantaneous current reflecting the channel opening at the holding potential, followed by a time-dependent current reflecting the channel opening by depolarization (Figure 15). When Ca 2+ concentration is high, the channel activation period is shortened (Xiao et al., 2011). Moreover, studies have shown that the TMEM16A channel exhibits permeability to anions larger than Cl -such as SCN -, I -, Br -, and HCO3 - [START_REF] Ji | Recent advances in TMEM16A: Structure, function, and disease[END_REF]. The HCO3 -and Cl - permeability is regulated by Ca 2+ concentration such as TMEM16A is more permeable to Cl -at submaximal cytosolic Ca 2+ levels (1 µM), but when Ca 2+ concentration increases to 3 µM, TMEM16A becomes more permeable to HCO3 - (Jung et al., 2013a).

Subsequently, intracellular Ca 2+ release delays the release of inositol tetrakisphosphate (IP4); thus, a similar model may work as an inhibitory feedback mechanism of TMEM16A (Xie et al., 1998). Some fatty acids, such as stearic, arachidonic, oleic, and docosahexaenoic, were also found to have inhibitory effects on TMEM16A activity in a concentration-and voltage-dependent manner, probably due to altered lipid channel-protein interaction (Antollini & Barrantes, 2016).

Expression

TMEM16A is widely expressed in epithelial and non-epithelial cells, and its expression differs significantly among different cells. In humans, TMEM16A is expressed throughout the respiratory tract, from the large bronchi to the alveoli (Kunzelmann et al., 2012), with somewhat larger expression in goblet cells. TMEM16A is less expressed in ciliated epithelial cells [START_REF] Bai | The diverse roles of TMEM16A Ca(2+)-activated Cl(-) channels in inflammation[END_REF]. This channel is also ubiquitously expressed in all exocrine secretory glands (salivary gland, mammary gland, pancreas, kidneys, and gut) (Figure 16, Appendix 2). Moreover, TMEM16A is present in the interstitial cells of Cajal that generate the pacemaker activity of gastrointestinal smooth muscles (Huang et al., 2009;Pedemonte & Galietta, 2014) and in smooth muscles of the respiratory and reproductive systems (Kunzelmann et al., 2016) and sensory neurons. It is also expressed in proliferating cells of different types (Pinto et al., 2020).

TMEM16A cellular localization varies between cell types. For example, TMEM16A is mainly expressed at the apical PM of acinar cells of the salivary gland, exocrine pancreas, and airway epithelial cells. In the colon, TMEM16A was found primarily on the basolateral side of the colonic epithelial cells, although it is also localized in the apical membrane of goblet cells and enterocytes (Benedetto et al., 2019a;[START_REF] He | Activation of the basolateral membrane Cl-conductance essential for electrogenic K+ secretion suppresses electrogenic Cl-secretion[END_REF]Kunzelmann et al., 2019a;[START_REF] Schreiber | Anoctamins support calcium-dependent chloride secretion by facilitating calcium signaling in adult mouse intestine[END_REF].

Huang et al. showed that TMEM16A is expressed explicitly in mucus-producing cells and poorly expressed in ciliated cells. TMEM16A was upregulated in asthma-like inflammatory conditions in the airways and is associated with airway goblet cell metaplasia (Huang et al., 2012b). The induction of mucus cell hyperplasia with IL-4, a potent inducer of mucus secretion and mucus cell metaplasia in the airways, increases the number of cells co-expressing TMEM16A and MUC5AC (Huang et al., 2012b).

Scudieri et al. suggested that TMEM16A expression is required under mucus hypersecretion conditions to ensure adequate water and electrolyte secretion (Scudieri et al., 2012a). TMEM16A is also involved in HCO3 -ion secretion, particularly under conditions of high Ca 2+ mobilization [START_REF] Jung | Dynamic modulation of ANO1/TMEM16A HCO3(-) permeability by Ca2+/calmodulin[END_REF]. It has been shown that HCO3 -is particularly important for the mucus biophysical properties as it influences polycation chelation and ASL pH, both important for mucin network organization [START_REF] Okuda | Mucins and CFTR: Their Close Relationship[END_REF]Quinton, 2008).

In a mice model, Rock et al. showed that TMEM16A plays a critical role in tracheal cartilage development (Rock et al., 2008). Homozygous mice for the TMEM16A null allele died within one month of birth and exhibited severe tracheomalacia with gaps in the tracheal cartilage rings along the entire length of the trachea, as well as a mucus build-up leading to airway obstruction, which contributed to neonatal mortality (Rock et al., 2008;Rock et al., 2009). Impaired MCC has also been demonstrated in TMEM16A knocked-out mice (Ousingsawat et al., 2009). In this study, the authors observed a movement of the polystyrene microspheres on freshly isolated mice tracheas when TMEM16A was present and activated. However, in mice lacking TMEM16A, no movement was observed, suggesting that TMEM16A could participate in MCC regulation in the airways. Hence, activation of TMEM16A can induce Cl -and HCO3 -secretion, a process needed to ameliorate MCC and restore antimicrobial activity.

Moreover, due to its wide distribution in various tissues, TMEM16A is also involved in several critical physiological processes such as the transmission of nociceptive signals, cell proliferation and migration, airway and exocrine gland secretion, regulation of smooth muscle contraction, and rhythmic movements of the gastrointestinal system (Pedemonte & Galietta, 2014).

TMEM16A also plays a cell type-dependent role in different disease conditions, as described next. Studies on TMEM16A knocked-out mice showed the importance of TMEM16A in maintaining coordinated Ca 2+ transients and slow waves within the interstitial cells of Cajal, which generate electrical pacemaker activity in gastrointestinal smooth muscles to regulate gastrointestinal motility [START_REF] Choi | Effects of Ca2+-Activated Cl-Channel ANO1inhibitors on Pacemaker Activity in Interstitial Cells of Cajal[END_REF]Cobine et al., 2017;Hwang et al., 2009). In the nervous system, TMEM16A hyperactivity increases the excitability of neurons under inflammatory or neuropathic conditions, thereby aggravating inflammation or tissue injury-induced pain (B. [START_REF] Lee | Anoctamin 1 Contributes to Inflammatory and Nerve-Injury Induced Hypersensitivity[END_REF]. TMEM16A is also involved in different cancers such as gastrointestinal stromal tumors, breast cancer, head and neck squamous cell carcinoma, colon cancer, pancreatic ductal adenocarcinoma, and oesophageal cancer (Figure 17), and inhibition of TMEM16A can inhibit proliferation, migration, and invasion of cancer cell (Britschgi et al., 2013;[START_REF] Kim | Anoctamin-1 affects the migration and invasion of anaplastic thyroid carcinoma cells[END_REF]Z. Qu et al., 2014;Song et al., 2018;H. Wang et al., 2017;Wanitchakool et al., 2014). In contrast, a decrease in TMEM16A activity leads to Cl -transport and gland dysfunction at the epithelia (such as salivary or lacrimal gland) and is associated with diseases. In fact, TMEM16A inhibition by niclosamide significantly reduced goblet cell metaplasia and mucus production in asthmatic mice and inhibited MUC5AC expression in Calu-3 human submucosal cells, suggesting that TMEM16A inhibition might be beneficial in inflammatory airway diseases (Centeio et al., 2021). Another study showed that TMEM16A inhibitors reduced asthmatic mucus secretion and airway hyperactivity in normal human bronchial epithelial (NHBE) and airway smooth muscles, respectively (Huang et al., 2012b). Nevertheless, a transcriptome metaanalysis revealed that CF and asthma pathways are highly divergent (Clarke et al., 2015). Thus, what is seen in asthma is not always applicable in CF. In 2008, Benedetto et al. reported defects in mucus secretion and accumulation in airway club cells and intestinal goblet cells when TMEM16A was knocked out, respectively, in FoxJ1expressing ciliated epithelial cells or intestinal goblet cells, highlighting the crucial role of TMEM16A in mucus secretion (Benedetto et al., 2019a). Authors suggest that ATPinduced mucus secretion by airway club cells is inhibited when TMEM16A is knocked out in ciliated cells, possibly due to the compromised release of prosecretory cytokines. Moreover, another study showed that TMEM16A plays an important role in regulating inflammation, as the authors showed that TMEM16A overexpression decreases proinflammatory IL-8 secretion in CF hBECs (Veit et al., 2012).

On the other hand, using a human respiratory basal cell line (BCi-NS1.1), Simões et al. showed that MUC5AC production does not require TMEM16A, and their simultaneous upregulation is only circumstantial under cell proliferation (Simoes et al., 2019). The authors also showed a decrease in ASL when inhibiting TMEM16A by inhibitor Ani9. Furthermore, while replying to Olschewski et al.'s concerns on increasing TMEM16A activity, Danahay et al. declared that positive modulation of TMEM16A did not induce bronchospasm in the conscious sheep model nor did it affect vascular smooth muscle contraction (unpublished observations) (Danahay et al., 2020c;Olschewski et al., 2020). Overall, TMEM16A possible role in mucus production remains obscure and evokes controversial opinions over the beneficial or deleterious results of stimulating the channel in CF.

Furthermore, TMEM16A is suggested to be essential for proper activation and expression of CFTR at the PM, indicating a close relationship between the two chloride channels [START_REF] Ousingsawat | CFTR and TMEM16A are Separate but Functionally Related Cl-Channels[END_REF]Ousingsawat et al., 2009). In 2017, Benedetto et al. demonstrated that in conditional TMEM16A knock-out mice, absence of TMEM16A function eliminated cAMP-activated Cl -currents in intestinal and respiratory epithelia.

The authors also showed that TMEM16A activated CFTR by enhancing Ca 2+ store release from the endoplasmic reticulum and activating Ca 2+ -regulated cAMP (Benedetto et al., 2017). Two years later, the same group showed that TMEM16A, or its paralogue TMEM16F, must be co-expressed in the same cell for membrane exocytosis, as well as CFTR expression and activation at the PM [START_REF] Benedetto | Plasma membrane-localized TMEM16 proteins are indispensable for expression of CFTR[END_REF].

Several observations propose that increasing TMEM16A activity can compensate for CFTR deficiency (Figure 19):

(1) TMEM16A absence decreases airway secretion (Simoes et al., 2019),

(2) TMEM16A is ubiquitously expressed in all the tissue affected by CF, including airway epithelial cells where TMEM16A is a secondary chloride channel, (3) TMEM16A provides a Cl -pathway important for fluid secretion and mucus hydration, and which is CFTR-independent (which is important for rare CFTR mutations), (4) TMEM16A is involved in HCO3 -secretion, which is highly important for mucus biophysical properties and antimicrobial protection.

Based on those observations, researchers have developed different approaches targeting TMEM16A to compensate for CFTR dysfunction and address the unmet needs of CF patients. In healthy airways, TMEM16A is colocalized with CFTR within the apical membrane of epithelial cells, contributing to ion and water homeostasis. In CF airways, dysfunctional CFTR leads to compromised ions efflux. Na + entry is upregulated, leading to a dehydrated ASL and impaired MCC favoring mucostasis, causing chronic inflammation and infection. In CF, the expression of TMEM16A is also diminished. Modulating TMEM16A, as an alternative CF therapy, could compensate for defective CFTR and, thus, enhance fluid secretion by epithelial cells, regulating ASL height and pH. Adapted from (Mitri et al., 2021).

Therapeutical Compounds Targeting TMEM16A

In order to understand TMEM16A functions better and develop specific pharmacological approaches, many small molecules have been used to modulate TMEM16A activity, but few have been shown to be efficient and, most importantly, specific to TMEM16A (Table 3). (Deterding et al., 2007;Deterding et al., 2005;Goss et al., 2008;Kellerman et al., 2008) concentration which is below the threshold for TMEM16A activity. In order for ETX001

to act as a potentiator and enhance the TMEM16A activity, ETX001 requires a minimal TMEM16A activation threshold. In the study, a pre-treatment with IL-13 (10 ng/ml; 48 h) elevated intracellular Ca 2+ concentration, effectively allowing ETX001 to increase TMEM16A Cl -currents. Currently, a phase I study to test the safety of ETX001 in healthy participants is in progress (Danahay et al., 2020b).

TMEM16A Regulation by microRNAs

Like other proteins, ion channel expression is regulated by a large number of small non-coding RNAs (∼22 nucleotides) known as microRNA (miRNA). MiRNAs were first described in Caenorhabditis elegans by Ambros and colleagues in 1993 (R. C. [START_REF] Lee | The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14[END_REF]. These short RNA sequences are transcribed from DNA in the cell nucleus.

The DNA is first transcribed by the enzyme RNA polymerase II into primary RNA, recognized, and cleaved by RNase III; a precursor miRNA is then generated within the nucleus. After leaving the nucleus, the precursor miRNA is then cleaved by Dicer, resulting in the mature miRNA [START_REF] Filipowicz | Post-transcriptional gene silencing by siRNAs and miRNAs[END_REF]. The miRNA and RNA-induced silencing complex (RISC) then form a miRISC complex that binds to the target mRNA's 3'-untranslated region (3'UTR), resulting in translational repression, cleavage, or degradation of the target mRNA and, in consequence, alters the protein function (Figure 20) (J. K. [START_REF] Lam | siRNA Versus miRNA as Therapeutics for Gene Silencing[END_REF][START_REF] Liang | Translation efficiency of mRNAs is increased by antisense oligonucleotides targeting upstream open reading frames[END_REF][START_REF] O'brien | Overview of MicroRNA Biogenesis, Mechanisms of Actions, and Circulation[END_REF]. Most miRNAs repress gene expression, but some induce gene upregulation [START_REF] O'brien | Overview of MicroRNA Biogenesis, Mechanisms of Actions, and Circulation[END_REF]. If miRNAs are 100% complementary or partially complementary to their mRNA target, they can either lead to mRNA degradation or result in blocked protein formation, respectively [START_REF] Jing | Involvement of MicroRNA in AU-Rich Element-Mediated mRNA Instability[END_REF][START_REF] Lim | Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs[END_REF]. Each miRNA can regulate hundreds of target genes.

Multiple studies have demonstrated the involvement of miRNAs in the regulation of TMEM16A. For example, when miRNA-381 expression is downregulated, TMEM16A

is overexpressed in gastric cancer [START_REF] Cao | MicroRNA-381 inhibits the metastasis of gastric cancer by targeting TMEM16A expression[END_REF]. The same applies in colorectal cancer when miRNA-144, miRNA132, and miRNA-9 are downregulated [START_REF] Jiang | MicroRNA-144 suppresses aggressive phenotypes of tumor cells by targeting ANO1 in colorectal cancer[END_REF][START_REF] Mokutani | Down-Regulation of microRNA-132 is Associated with Poor Prognosis of Colorectal Cancer[END_REF][START_REF] Park | Down-regulation of miR-9 promotes epithelial mesenchymal transition via regulating anoctamin-1 (ANO1) in CRC cells[END_REF]. Regulation of TMEM16A by miRNA-9

was also confirmed in idiopathic pulmonary fibrosis [START_REF] Dai | Downregulation of microRNA-9 reduces inflammatory response and fibroblast proliferation in mice with idiopathic pulmonary fibrosis through the ANO1-mediated TGF-beta-Smad3 pathway[END_REF], sepsis (Zhen et al., 2019), breast cancer [START_REF] Selcuklu | MicroRNA-9 inhibition of cell proliferation and identification of novel miR-9 targets by transcriptome profiling in breast cancer cells[END_REF], and CF (Sonneville et al., 2017). An upregulation of miRNA-9 decreases the expression of TMEM16A responsible for inhibiting TGF-b signaling in bacterial lipopolysaccharides (LPS)-induced sepsis. On the contrary, miRNA-9 knockdown improves TMEM16A-mediated TGF-b signaling inhibition, suggesting that miRNA-9 might be a potential target for sepsis treatment (Zhen et al., 2019). In idiopathic pulmonary fibrosis, TMEM16A downregulation by miRNA-9 induced increased proliferation and inflammation and inhibited fibroblast apoptosis in an idiopathic pulmonary fibrosis mice model, while miRNA-9 downregulation reduced inflammatory response and fibroblast proliferation [START_REF] Dai | Downregulation of microRNA-9 reduces inflammatory response and fibroblast proliferation in mice with idiopathic pulmonary fibrosis through the ANO1-mediated TGF-beta-Smad3 pathway[END_REF]. In 2013, Ruffin et al. demonstrated that TMEM16A expression and activity were significantly decreased in CF hBECs, and, TMEM16A inhibition or overexpression were associated respectively with a decrease or increase in cell proliferation and migration (Ruffin et al., 2013). The same group predicted later, by computational approach, a variety of miRNAs that could target the 3′UTR of TMEM16A mRNA, and then demonstrated by luciferase reporter assay that miRNA-9 directly binds to the 3'UTR of TMEM16A mRNA and induces the downregulation of TMEM16A expression and activity in CF hBECs (Sonneville et al., 2017) (Figure 21). Since the therapeutic goal is to improve Cl -activity in CF, increasing TMEM16A activity seemed an exciting therapeutic approach. As miRNAs have multiple mRNA targets, miRNA inhibitors cannot be utilized to prevent specific downregulation. Hence, our team designed an antisense oligonucleotide to inhibit miRNA-9 binding to TMEM16A 3'UTR with the goal to restore channel expression and activity in CF cells (Sonneville et al., 2017) (See chapter 3, paragraph 5). The next chapter will discuss in more detail the technology used for this approach. [START_REF] Stephenson | Inhibition of Rous sarcoma viral RNA translation by a specific oligodeoxyribonucleotide[END_REF].

However, the ASO therapeutic potential began to attract interest only in the late 1980s, after the use of ASOs to suppress the human T-cell lymphotropic virus type III [START_REF] Zamecnik | Inhibition of replication and expression of human T-cell lymphotropic virus type III in cultured cells by exogenous synthetic oligonucleotides complementary to viral RNA[END_REF], inhibit HIV [START_REF] Renneisen | Inhibition of expression of human immunodeficiency virus-1 in vitro by antibodytargeted liposomes containing antisense RNA to the env region[END_REF], and the malaria parasite Plasmodium falciparum replication [START_REF] Barker | Inhibition of Plasmodium falciparum malaria using antisense oligodeoxynucleotides[END_REF]. Since then, several chemistry developments have led to the widespread of ASOs as therapeutic tools to target RNA.

In 2021, ASOs annual market sales were estimated to be approximately $1 billion and are expected to increase to $1.62 billion by 2025 [START_REF] Crooke | Antisense drug discovery and development technology considered in a pharmacological context[END_REF]. The current pipeline includes more than 100 ASOs in development and more than ten approved ASOs for a broad range of genetic disorders [START_REF] Dhuri | Antisense Oligonucleotides: An Emerging Area in Drug Discovery and Development[END_REF][START_REF] Scharner | Clinical Applications of Single-Stranded Oligonucleotides: Current Landscape of Approved and In-Development Therapeutics[END_REF]) (Figure 22, Appendix 5).

Chemistries: Antisense Oligonucleotide Synthesis

The development of RNA-based therapies has required overcoming several major hurdles, including (1) rapid degradation of exogenous RNA by endonucleases and exonucleases;

(2) delivery of negatively charged RNA across the hydrophobic PM;

and (3) cell toxicity [START_REF] Damase | The Limitless Future of RNA Therapeutics[END_REF][START_REF] Mansoor | Advances in Antisense Oligonucleotide Development for Target Identification, Validation, and as Novel Therapeutics[END_REF]. Chemical modifications were made to provide drug properties to ASOs, with the primary goal of providing nucleotide stability without affecting hybridization and affinity with the RNA target [START_REF] Bennett | RNA targeting therapeutics: molecular mechanisms of antisense oligonucleotides as a therapeutic platform[END_REF]. The chemistry used also depended on the desired mechanism of action (See 3. Mechanisms of Action). The different modifications classify ASOs into three groups: the first, second, an third generations [START_REF] Khvorova | The chemical evolution of oligonucleotide therapies of clinical utility[END_REF].

The first generation applied modification in the ribonucleotide structure (Figure 23) is the phosphorothioate (PS) backbone (Table 4) which consists of a sulfur atom replacing a non-bridging oxygen atom [START_REF] Eckstein | Phosphorothioate oligodeoxynucleotides: what is their origin and what is unique about them?[END_REF]. Other modifications with methyl and amine groups exist, although PS is the most widely used in first-generation ASOs. This modification was initially designed to provide nuclease stability (half-life of 9 to 10h in human serum vs. 1h for unmodified oligodeoxynucleotides). However, it also impacted cellular uptake as PS-ASOs are negatively charged and do not need carriers [START_REF] Crooke | Progress in antisense technology: the end of the beginning[END_REF]. Once bound to their target RNA, PS-ASOs recruit RNase H, a ubiquitously expressed endonuclease that cleaves the ASO target mRNA. Although PS improved ASOs stability, the ASO plasma half-life was still very short due to nuclease degradation in vivo and repeated administration at high doses induced cell toxicity [START_REF] Levin | A review of the issues in the pharmacokinetics and toxicology of phosphorothioate antisense oligonucleotides[END_REF] due to their binding to certain proteins, causing immune stimulation and complement cascade activation. To further improve nuclease resistance and binding affinity, the second generation of ASOs included sugar modifications like 2'-O-methyl (2'-OMe) (Table 4). However, 2'-OMe-PS-ASOs initially showed less activity than PS-ASOs. Since that time, many analogs were synthesized to increase RNase H activity and 2'-O-methoxyethyl (2'-MOE) emerged as one of the most useful [START_REF] Johannes | Current Challenges in Delivery and Cytosolic Translocation of Therapeutic RNAs[END_REF] and has been widely employed in clinical drug trials (Inotersen, Nusinersen, Mipomersen, Malisen) (Appendix 5). These second-generation ASOs are less toxic than PS-ASOs and have a slightly enhanced affinity towards their complementary RNAs; however, they are inefficient in inducing RNase H cleavage and are limited to a steric hindrance and splicing modulation (See paragraph 2) (Jens [START_REF] Kurreck | Antisense technologies. Improvement through novel chemical modifications[END_REF]. To restore RNAse H activity, hybrid "gapmer" ASOs that contain sugar-modified nucleotides on both ends and an unmodified or PS-DNA cleavage-sensitive sequence in the central gap region were created (Jens Kurreck, 2003;[START_REF] Monia | Evaluation of 2'-modified oligonucleotides containing 2'-deoxy gaps as antisense inhibitors of gene expression[END_REF].

The third-generation of ASOs was developed to improve the nuclease stability, target affinity, and pharmacokinetic profile. Heterogeneous modifications were developed, such as phosphorodiamidate morpholino oligomer (PMO), in which a morpholino ring replaces the sugar unit, and peptide nucleic acids (PNA) [START_REF] Larsen | Antisense properties of peptide nucleic acid[END_REF], where the sugar-phosphate backbone is replaced by aminoethyl glycine units linked by amide bonds (Table 4). Phosphorodiamidate morpholino oligomers and peptide nucleic acids are charge-neutral, which reduces the likelihood of non-specific interactions, but at the same time, accelerates their clearance from the body and renders their solubility and uptake difficult [START_REF] Chen | Chemical modification of gene silencing oligonucleotides for drug discovery and development[END_REF]. Therefore, third-generation ASOs also need to be administered at very high doses for in vivo applications and do not degrade the target RNA as they do not recruit RNase H; however, they are used mainly in steric blocking/splicing modulation approaches. A significant improvement in the ASOs design was the development of locked nucleic acid (LNA) (Table 4), one of the most used third-generation ASO. This bicyclic sugar analog links the 2' oxygen and 4' carbon of ribose, thus, reducing the conformational flexibility of nucleotides and significantly increasing their binding affinity and stability (Jens [START_REF] Kurreck | Antisense technologies. Improvement through novel chemical modifications[END_REF][START_REF] Mansoor | Advances in Antisense Oligonucleotide Development for Target Identification, Validation, and as Novel Therapeutics[END_REF]. LNAs can also be incorporated with DNA to form a gapmer, improving their nuclease resistance and affinity as well as the ability to recruit RNase H (J. [START_REF] Kurreck | Design of antisense oligonucleotides stabilized by locked nucleic acids[END_REF].

Given this significant variability, the choice of ASO chemistry is now based on the desired therapeutical approach.

Mechanisms of Action

Before protein synthesis, mRNA goes through a range of necessary processing phases such as 5'-capping, polyadenylation, intron-exon splicing, nuclear export, cytoplasmic stabilization, and ribosomal binding. These steps are highly regulated and are important for mRNA synthesis and function. By interfering with any of these phases, the ASOs can generate different outcomes depending on their mechanisms of action. ASOs are divided into two most common categories: ( 1) RNase Hdependent ASOs that mediate cleavage and degradation of target RNA [START_REF] Wu | Determination of the Role of the Human RNase H1 in the Pharmacology of DNA-like Antisense Drugs[END_REF], and ( 2) RNase H-independent ASOs that modulate splicing events or block their RNA target activity [START_REF] Heidenreich | RNase H-independent antisense activity of oligonucleotide N3'->P5' phosphoramidates[END_REF] (Figure 24).

RNase H-dependent ASO

To knockdown the target protein, the RNase H-activating ASOs or gapmers bind to a small mRNA region and activate the RNase enzyme, which degrades and reduces the mRNA levels in the nucleus and cytoplasm; hence, downregulating gene expression (Figure 24.1). The cleavage leaves the ASO intact and free to bind to another mRNA copy. Thus, on the condition that ASOs reach their target, once inside the cell, the recycling makes their effect long-lasting [START_REF] Mansoor | Advances in Antisense Oligonucleotide Development for Target Identification, Validation, and as Novel Therapeutics[END_REF]. Fomivirsen was the first approved first-generation ASO and is the only RNase H-activating ASO with no gapmer structure. Nevertheless, Fomivirsen was withdrawn in 2006 from the US market (in 2002 from the European market) for toxicity reasons.

As to gapmers, they are used to degrade genes in an allele-independent or specific manner [START_REF] Helm | Towards Personalized Allele-Specific Antisense Oligonucleotide Therapies for Toxic Gain-of-Function Neurodegenerative Diseases[END_REF]. For example, Mipomersen and Volanesorsen are drugs used to reduce the overexpressed genes or pathways responsible for the disease in

RNase H-independent ASO

In this category, gapmers are unnecessary as the major use of this class is not to seek the recruitment of RNase H but to alter or correct splicing patterns [START_REF] Hua | Enhancement of SMN2 Exon 7 Inclusion by Antisense Oligonucleotides Targeting the Exon[END_REF], modulate translation [START_REF] Baker | 2′-O-(2-Methoxy)ethyl-modified Anti-intercellular Adhesion Molecule 1 (ICAM-1) Oligonucleotides Selectively Increase the ICAM-1 mRNA Level and Inhibit Formation of the ICAM-1 Translation Initiation Complex in Human Umbilical Vein Endothelial Cells[END_REF][START_REF] Liang | Translation efficiency of mRNAs is increased by antisense oligonucleotides targeting upstream open reading frames[END_REF], or block mRNA activity.

In splicing modulation, different approaches are possible: exon exclusion or exon inclusion (Figure 24.2). For example, in the case of Duchenne muscular dystrophy, Eteplirsen specifically recognizes exon 51 of the Duchenne muscular dystrophy gene.

The ASO binding to the pre-mRNA transcript corrects the disrupted reading frame and influences the splicing machinery to skip exon 51, thus producing a functional protein (K. R. [START_REF] Lim | Eteplirsen in the treatment of Duchenne muscular dystrophy[END_REF]. Conversely, Nusinersen is used in spinal muscular atrophy to facilitate the integration of exon 7 into the mRNA, prevent the splicesome from accessing the transcript site, and thereby enhance full-length spinal muscular atrophy protein levels [START_REF] Wurster | Nusinersen for spinal muscular atrophy[END_REF].

Other approaches are based on a steric-blocking ASO with LNA modifications, known as a target site blocker (TSB) (Figure 25). TSB prevents the activity of miRNA, a small

Pharmacokinetic Parameters

Absorption, distribution, metabolism, and excretion (ADME) are the critical biological processes for determining a drug pharmacokinetic parameters [START_REF] Migliorati | Absorption, Distribution, Metabolism, and Excretion of US Food and Drug Administration-Approved Antisense Oligonucleotide Drugs[END_REF][START_REF] Shadid | Antisense oligonucleotides: absorption, distribution, metabolism, and excretion[END_REF]. The processes of drug uptake by the body, such as biotransformation, distribution, metabolite interactions in different tissues, and elimination from the body, are all influenced by the route of administration and have direct impacts on efficacy and adverse drug reactions.

Absorption

Absorption is the unmetabolized drug movement from the administration site to the local or systemic circulation. The time to reach maximum drug concentration in the plasma reflects drug absorption into the body. Drug administration routes and firstpass metabolism can affect drug absorption and impact drug bioavailability, which is the percentage of the bioactive form of the drug out of the total amount of administered drug.

ASOs can be given by the typical administration routes, including oral [START_REF] Hammond | Delivery of oligonucleotide-based therapeutics: challenges and opportunities[END_REF]. So far, oral delivery is not yet commercially feasible because it requires

ASOs to be enteric coated to avoid acid-based depurination and precipitation in the stomach and must include a penetration enhancer to enhance absorption; hence, additional modifications are necessary to ensure bioavailability [START_REF] Crooke | Antisense drug discovery and development technology considered in a pharmacological context[END_REF].

Intravenous infusion (IV) has been widely used to maximize bioavailability, as most peak plasma concentrations can be reached within 1 to 2 hours. This route avoids first-pass metabolism in the liver and affords quick access to the target tissue through systemic circulation. IV leads to rapid distribution to organs that are highly vascularized (liver, kidney, and spleen) but slower distribution to tissues such as the heart, muscles, and lungs, thereby necessitating more frequent dosing [START_REF] Migliorati | Absorption, Distribution, Metabolism, and Excretion of US Food and Drug Administration-Approved Antisense Oligonucleotide Drugs[END_REF][START_REF] Shadid | Antisense oligonucleotides: absorption, distribution, metabolism, and excretion[END_REF], which also means lengthy hospital visits for the patients. ASO half-life with IV administration is usually 3 to 4 hours.

A longer half-life of ASO drugs allows less frequent dosing requirements. Hence, for a longer ASO half-life of around 30 days, subcutaneous (SC) injections are used. This way of administration is easy to use and allows gradual absorption from the site of injection of the ASO into the systemic or lymphatic system and a wide range of tissues afterward. Peak plasma concentrations for SC are reached after 2 to 4 hours.

Local delivery has been successfully used so far to bypass the blood-brain barrier and/or circumvent limitations in systemic drug distribution. ASOs have been delivered

locally via inhalation to the lungs [START_REF] Drevinek | Antisense oligonucleotide eluforsen is safe and improves respiratory symptoms in F508DEL cystic fibrosis[END_REF]I. Sermet-Gaudelus et al., 2019) or site-specific injection to the eye [START_REF] Temsamani | Antisense approach for the treatment of cytomegalovirus infection[END_REF] or cerebrospinal fluid [START_REF] Rinaldi | Antisense oligonucleotides: the next frontier for treatment of neurological disorders[END_REF]. Local delivery into the eye via intravitreal injection requires a minimal amount of drug to allow direct distribution to the retina. Similarly, ASO localized administration into the cerebrospinal fluid via lumbar puncture resulted in a better distribution throughout the central nervous system than IV administration.

Distribution

Distribution refers to the movement of a drug between the intravascular and extravascular compartments of the body. Route of delivery, acid-base characteristics, lipophilicity, size, free-drug concentration, and tissue blood perfusion are all factors that affect ASO distribution.

The activity of ASOs depends on reaching the correct compartment of the targeted cell. The highest ASO concentration is found in the liver and kidneys following systemic administration, making these organs less challenging to target. However, other tissues like skeletal muscles and heart are less prone to ASO uptake. Achieving sufficient uptake might require different strategies to transport ASOs to the nucleosome and protect it from nucleases. ASOs with negatively charged backbones display high binding capacity (>85%) to plasma proteins, while uncharged ASOs have low protein binding, limiting their cellular uptake [START_REF] Hammond | Delivery of oligonucleotide-based therapeutics: challenges and opportunities[END_REF][START_REF] Roberts | Advances in oligonucleotide drug delivery[END_REF]. One of the strategies is increasing protein binding by conjugating ASOs to fatty acids or cholesterol, thereby decreasing the elimination rate and increasing distribution to tissue. Palmitate, for example, increases the ASO activity in skeletal and cardiac muscles. Different vectors have also been suggested, such as (1) liposomes like the lipid nanoparticles that can carry anionic RNA, although subjects are pretreated with steroids to avoid inflammatory response; (2) peptides conjugation like arginine-rich cell-penetrating peptides that reached clinical development, however, they can only be used with charge-neutral ASOs to avoid aggregation; (3) N-acetylgalactosamine (GalNAc) carbohydrate conjugation for hepatocyte delivery improvement [START_REF] Damase | The Limitless Future of RNA Therapeutics[END_REF][START_REF] Hammond | Delivery of oligonucleotide-based therapeutics: challenges and opportunities[END_REF][START_REF] Roberts | Advances in oligonucleotide drug delivery[END_REF].

Limited information is available on how ASOs are taken into cells after distribution.

ASOs can pass through the PM with no vectorization. They enter the cytoplasm via diffusion or are taken up by clathrin-coated endocytic vesicles from the PM; 1 to 2% escape endocytic vesicles and, depending on whether their target is in the cytoplasm or the nucleus, ASOs may need to further translocate to the nucleus at sufficient concentrations to produce the desired biological response [START_REF] Bennett | Therapeutic Antisense Oligonucleotides Are Coming of Age[END_REF][START_REF] Crooke | Cellular uptake and trafficking of antisense oligonucleotides[END_REF]. ASOs have long half-lives (1 to 4 weeks) inside the cells and prolonged biological activity. ASOs are primarily excreted in urine (the primary route for ASOs clearance) or feces as unchanged drugs or are metabolized as cleaved short fragments [START_REF] Migliorati | Absorption, Distribution, Metabolism, and Excretion of US Food and Drug Administration-Approved Antisense Oligonucleotide Drugs[END_REF].

Preclinical Safety Assessment of Therapeutic Oligonucleotides

Developing new chemistries and delivery technologies has undoubtedly led to more potent clinical ASOs candidates. To enter clinical trials and get safety approval, ASOs have to undergo preclinical safety assessments consisting of three groups: (1) sequence and hybridization-dependent effects, (2) sequence and hybridizationindependent effects, and (3) sequence-dependent but hybridization-independent effects (Figure 26) [START_REF] Andersson | Preclinical Safety Assessment of Therapeutic Oligonucleotides[END_REF][START_REF] Frazier | Antisense oligonucleotide therapies: the promise and the challenges from a toxicologic pathologist's perspective[END_REF].

Sequence and Hybridization-Dependent Effects

This category includes assessing on-and off-target effects. On-target safety, also known as exaggerated pharmacology, refers to adverse consequences or too strong intended effects induced by the ASO excessive or prolonged activity in the target organ. The on-target safety risk study is mainly theoretical because target sequences may not be conserved across species. Off-target safety corresponds to the ASO activity on unintended targets. The guidelines of The Oligonucleotide Safety Working Group (OSWG) for off-target assessment recommend (1) identifying candidates for off-target hybridization by in silico evaluation, (2) in vitro confirmation of the in silico hits, and ( 3) in vivo evaluation [START_REF] Marlowe | Recommendations of the Oligonucleotide Safety Working Group's Formulated Oligonucleotide Subcommittee for the Safety Assessment of Formulated Oligonucleotide-Based Therapeutics[END_REF].

However, these safety studies must take into account (1) species differences in sequences, ( 2) slow tissue elimination, hence extended effect but also on-or off-target toxicity duration, and ( 3) the significant difference in ASO uptake in different cells and tissues depending on different administration route, different chemistries, and conjugates.

Sequence and Hybridization-Independent Effects

Maximal plasma concentration of PS backbone ASOs displays high plasma protein binding affinities, causing prolongation of coagulation time and alternative complement activation, which can lead to significant drops in blood pressure and vascular inflammation. Both side effects can be regulated by dose reduction or extended infusion, although with modern ASOs, the plasma concentration rarely exceeds the activation thresholds [START_REF] Hammond | Delivery of oligonucleotide-based therapeutics: challenges and opportunities[END_REF].

Sequence-Dependent but Hybridization-Independent Effects

Other hybridization-independent toxicities can be highly dependent on the ASO sequence and the chemical modifications, inducing inflammation as well as liver and kidney toxicities.

Immunostimulation is caused by ASOs activating the innate immune system through binding to pattern-recognition receptors such as toll-like receptors. PS modifications, for example, are known to induce an ASO immune stimulation. The pro-inflammatory reaction can be illustrated in different ways, such as reactions at the site of infusion following SC or IV administration and flu-like symptoms [START_REF] Andersson | Preclinical Safety Assessment of Therapeutic Oligonucleotides[END_REF][START_REF] Gait | Introduction and History of the Chemistry of Nucleic Acids Therapeutics[END_REF].

Thrombocytopenia is also a rare event observed in rodents and non-human primates treated with ASOs, and was also observed recently in clinical trials. In most cases, platelet count reduction is mild to moderate and reversible. However, in non-human primates studies, severe decline after repeated dosing has been observed, but platelet count normalized after drug cessation [START_REF] Hammond | Delivery of oligonucleotide-based therapeutics: challenges and opportunities[END_REF].

Highly exposed organs like the liver and the kidneys are also prone to high toxicity levels translated by considerable necrosis in regions of the liver, renal lesions in proximal tubules, and a significant increase in liver enzyme levels and renal injury biomarkers, such as B2-microglobulin. Different mechanisms have been proposed, including the accumulation of RNase -cleaved metabolites.

Examples of Antisense Oligonucleotides Based Therapeutics for Cystic Fibrosis

After the recent successful ASOs developments for Duchenne muscular dystrophy, spinal muscular atrophy, and macular degeneration diseases, some have started to develop ASO treatments for various common respiratory diseases, including asthma, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, and CF. This nucleotides as a cryptic exon and generating a fully functioning CFTR protein [START_REF] Oren | Antisense oligonucleotide-based drug development for Cystic Fibrosis patients carrying the 3849+10 kb C-to-T splicing mutation[END_REF]. CF is an autosomal recessive genetic multiorgan disease caused by an absent or dysfunctional CFTR channel that mainly mediates Cl -and HCO3 -anion transport across the apical membrane of epithelial cells. CF affects the epithelial cells of multiple organs, including the lungs, pancreas, liver, intestine, vas deferens, and sweat glands (Cutting, 2015). For example, CFTR dysfunction in the lungs destabilizes the epithelial TMEM16A is one of the most promising alternative channels to compensate for the lack of epithelial CFTR-mediated anion secretion in CF [START_REF] Galietta | TMEM16A (ANO1) as a therapeutic target in cystic fibrosis[END_REF]Liu et al., 2021;Mitri et al., 2021). TMEM16A is ubiquitously expressed in all exocrine secretory glands in the epithelia, including in airway epithelial cells.


Sonneville et al. demonstrated that the downregulation of TMEM16A expression and activity in CF hBECs was correlated with a high expression of miRNA-9, which inhibits TMEM16A translation by binding to the 3′UTR of TMEM16A mRNA (Ruffin et al., 2013). To amplify TMEM16A at the apical surface, the team has developed a new specific strategy with oligonucleotide antisense (ASO) that binds to the TMEM16A 3′UTR and prevents miRNA-9 binding, therefore, re-establishing TMEM16A

expression and activity at the cell surface (Sonneville et al., 2017).

This project aims to investigate in vitro and in vivo effects of ASO TMEM16A on CF dysregulated parameters and prepare for future clinical studies.

Results

86

This article was submitted to the journal of Nature Communications.

The three models used to perform this study were cell lines, human primary cell lines, and the 129-Cftr tm1 Eur CF mouse model (Appendix 3, Appendix 4). Following the cloning of the CFTR gene in 1989 2 , early hope for a therapy to treat patients with CF was founded firmly in the realm of gene therapy, but clinical trials were never successful 3,4 . Vertex Pharmaceuticals' therapies have revolutionized the clinical approach for the last ten years by providing strategies to address and activate CFTR at the apical membrane 5,6 . Unfortunately, these approaches are mutationdependent and still exclude more than 15% of patients with nonsense and rare mutations non-responsive to the drugs 7 . Hence the need for alternative therapies that are CFTR-independent.

Key Points

In 2008, TMEM16a (also called anoctamin-1 or ANO1) was identified by three different groups as the gene encoding the calcium-activated chloride channel (CaCC) 8-10 .

TMEM16A, like CFTR, is located at the epithelia, participates in the homeostasis of the airway surface fluid, and has been proposed as an alternative strategy to compensate for CFTR deficiency 11 . However, in 2013, we demonstrated that this protein is downregulated in CF bronchia 12 . Thus, an approach to increasing TMEM16A could bypass CFTR deficiency and be applied to all patients with CF, particularly those who cannot benefit from current curative treatments. In 2017, we first demonstrated the inhibitory role of microRNA-9 (miR-9) in the regulation of TMEM16A 13 , which other groups have since confirmed in different pathologies 14-17 .

Then, we developed a strategy based on antisense oligonucleotide (ASO) to inhibit the binding of miR-9 on the 3'UTR of TMEM16A by competitive interaction and consequently increase its expression 13 .

To limit adverse effects, the design of the ASO is specific to TMEM16A 3'UTR and acts as a corrector to increase the expression at the apical membrane of the epithelia.

Here, we report that this strategy could apply to all patients with CF to correct Cl -efflux and mucociliary clearance without inducing inflammation or toxicity. We also demonstrate that this strategy increased the life expectancy of CF mice without laxatives and enhanced CF male mice' fertility. Herein, we support the concept that an increase in TMEM16A Cl -activity could represent a therapeutic potential for drug development for all patients with CF.

Results

TMEM16A ASO potentiates chloride efflux in CF cells

TMEM16A ASO robustly potentiated TMEM16A Cl -channel in a concentration manner. We performed a dose-response study on the CFBE41o-cell line with a YFP halide sensor to determine the optimal TMEM16A ASO concentration (Figure 1A-B).

The assay combines the YFP halide sensor with iodide (a chloride surrogate); upon We measured by Ussing chamber the transepithelial Cl -current, driven by cAMP for CFTR or Ca 2+ mediated signaling pathways for TMEM16A activity. After adding to the apical compartment of primary human bronchial epithelial cells (hBEC) amiloride to block ENaC-mediated Na + absorption and forskolin to activate CFTR efflux by an agonist of cAMP-dependent protein kinase, we observed a strong CFTR-dependent Cl -efflux in non-CF hBEC. This activity was abolished when we added the specific CFTR inhibitor Inh-172 (Figure 1C). In CF hBEC, no cAMP-dependent Cl -activity was observed as expected (Figure 1 D-E). After activating the CaCC channel, UTPinduced Ca 2+ -mediated Cl -efflux was significantly increased in both ASO TMEM16A treated CF and non-CF cells compared to control (Figure 1 C-E). In order to verify the specificity of TMEM16A ASO and be sure about the CaCC's molecular identity, we pretreated the cells with Ani9, the most potent and selective inhibitor of TMEM16A 18 .

The TMEM16A-mediated Cl -efflux was strongly inhibited (Figure 1F), which confirms the ASO's targeted channel specificity.

The effects of TMEM16A ASO on Cl -efflux were analyzed by halide-sensitive YFP probe in different F508del cells from lungs (tracheal gland serous cells KM4, bronchial epithelial CUFI and IB3-1, primary human glandular cells HBG) or pancreas (CFPAC).

The effects are significant in all cells tested, with differences in the activation level

(Figure 1 G-H) as the significant induction of Cl -efflux was between 20 to 25% in KM4, CUFI, and IB3-1, and close to 50% in the most responsive cells HBG and CFPAC.

TMEM16A ASO increases mucociliary clearance of CF cells with different mutations

TMEM16A ASO induced strong and significant activation of mucociliary clearance evaluated by the movement of fluorescent beads in all the different tested mutations (Figure 2 A-D). The effects of Orkambi® were only positively significant on class II F508del/F508del cells, although significantly lower than those treated with TMEM16A ASO (Figure 2B). Interestingly, when combining TMEM16A ASO and Orkambi®, we observed additive effects on F508del/F508del cells compared to cells treated with ASO TMEM16A or Orkambi® alone. The impact of Orkambi® on Cl -efflux with or without TMEM16A ASO was negative on mucociliary clearance with 1717-1G>A/F508del cells (Figure 2C). Interestingly, for cells with class I mutation on which Orkambi® does not have any effect, we observed a significant increase of Cl -efflux in cells treated with TMEM16A ASO (4.5±0.3 vs. 9.6±0.3) (Figure 2D). No significant change is observed with the combination of both molecules.

Along with mucociliary clearance, infection and inflammation of bronchial epithelial cells are the most critical dysregulated parameters in CF. For the eventual implementation of our therapeutic strategy, we wanted to ensure that ASO TMEM16A did not worsen those parameters. TMEM16A ASO did not have any bacteriostatic or bactericidal effect on Pseudomonas aeruginosa (P. aeruginosa) proliferation when alone (Figure 2E) or in the presence of cells (Figure 2F). Moreover, cells treated with TMEM16A ASO did not tend to get infected more than the control (Figure 2G). Finally, inflammation is considered a critical dysregulated parameter in CF, and as inflammatory cytokines like interleukin-8 (IL-8) are excessively secreted, it is critical to avoid further inflammation. We have analyzed the impact of TMEM16A ASO on IL-8 secretion, which is one of the main chemokines responsible for recruitment, activation, and accumulation of neutrophils in the lungs (Figure 2H), and also on various cytokines by multiplex (Supplemental data Figure 1A). No significant induction of cytokines by TMEM16A ASO was observed.

TMEM16A ASO is specific and not toxic

We have performed an in vitro pharmaceutical profiling described by Bowes et al. 19 and used by major pharmaceutical companies (AstraZeneca, GlaxoSmithKline, Novartis, and Pfizer) to identify undesirable off-target interactions that could hinder or halt candidate drugs' development. The tested targets panel identified no significant unspecific binding or cross-reaction of TMEM16A ASO with other channels/proteins (Figure 3A). We also evaluated TMEM16A ASO toxicity on CFBE41o-cells by measuring LDH levels and found no difference (Figure 3B). TMEM16A's overexpression is associated with cell proliferation in different cancers 21 ; therefore, we looked at the proliferation rate in cells with varying mutation classes.

After TMEM16A ASO treatment and TMEM16A activation, we have not observed any significant modification in cell proliferation compared to cells treated with control ASO (Figure 3 E-F).

Subcutaneous administration allows a good distribution and is not toxic in vivo

Fluorescent TMEM16A ASO was administered in nude mice (10 mg/kg) by intravenous, subcutaneous, intraperitoneal injection or intranasal installation to determine the best administration ways. Fluorescence in mice was recorded every week for one month (Figures 4 A-D). In these pseudocolor images in which white represents the greatest intensity of photon emission, minimal light emission is detected outside the injection site at D0 (Figure 4A). With the intraperitoneal injection, the molecule dispersed to a small extent, while intravenous, subcutaneous, and intranasal administration showed a good distribution in all tissues after seven days (Figure 4B), and it continued to improve after 21 days (Figure 4C). After day 21, we observed a decrease in the fluorescence level for all the conditions, as we can observe on day 30 (Figure 4D). We chose the subcutaneous injection, a standard ASO administration method, for the following experiments.

Toxicity was assessed in vivo by monitoring general signs (morbidity and mortality, clinical signs, body weight, food, and water consumption) three times a week for 14 days. Mice were injected once, subcutaneously, with six different doses from 0 to 500 mg/kg, and were sacrificed 14 days after the injection. With 50 times the active dose (500 mg/kg), we observed no significant difference in the mice's behavior, macroscopic autopsy, or histology analysis (Figure 4E). In blood samples, the different parameters reflecting toxicity in the liver (ALT and AST), kidney (urea), or pancreas (amylase) were similar in all tested doses (Supplemental data Figure 1 B-E).

Hematology analysis (hematocrit, hemoglobin concentration, mean corpuscular hemoglobin, mean corpuscular volume, mean corpuscular hemoglobin concentration, platelet count), blood chemistry analysis (including coagulation parameters), and urinalysis showed similar results (data not shown). For CF mice treated with long-term repeated injections (every 15 days), the transaminases, urea, and creatinine concentrations were similar to control mice (Supplemental data Figure 1 F-G).

TMEM16A ASO increases survival and fertility in CF mice

CF mice usually die of intestine occlusion during the weaning or after 22 . We treated the CF mice before weaning, at days 11 and 18 after birth, by subcutaneously injecting the control ASO or TMEM16A ASO (10 mg/kg) (Figure 5A). With ASO TMEM16A treatment, we observed that the median survival went from 36 days in control mice to 101 days in mice treated twice with TMEM16A ASO (p<0.001) (Figure 5B). Thus, the treatment seemed to protect the CF mice temporarily when mice were treated only with two injections. We treated the mice repeatedly every 15 days after weaning (Figure 5A). With this chronicle treatment, we observed a significant increase in the mice's survival, up to 196 days (p<0.0001) (Figure 5B). These data are consistent with the chronically treated mice's weight gain being higher than control mice or mice treated with two injections (data not shown).

The literature shows that homozygous male mice (F508del/F508del) are sterile 23,24 .

It might be explained by the vas deferens atresia, making them non-functional and resulting in infertility (Supplemental data Figure S2A; 2 nd generation left). In our study, homozygous male mice (F508del/F508del) chronically treated from day 11 after birth (Supplemental data Figure S2A, 2 nd generation right) could reproduce, and the pups were viable (Supplemental data Figure S2A, 3 rd generation, B-C). These CF 3 rd generation mice born from TMEM16A ASO treated 2 nd generation breeding survive the weaning stage, whether treated with ASO TMEM16A or the control, and have a longer life expectancy than CF mice born from untreated parents (Ctl mice median survival: 106 vs. 36) (Figure 5C). This suggests a placental passage from the treated gestating female to the fetus during the gestation period and persistence of the TMEM16A ASO in time in the pups. By injecting fluorescent TMEM16A ASO into the gestating female mice before parturition, we tracked the fluorescence in the newborn pups. We found a significantly higher level of fluorescence (Figure 5D), validating the ASO transmission through the placenta from the gestating female to the pups.

Discussion

Even though current CF treatments look promising, they cannot be applied to all patients, leaving some patients with unmet medical needs and a high disease burden 5 . Furthermore, adverse events were reported, such as rash, blood pressure changes, diarrhea, severe liver damage, and transiently increased respiratory symptoms upon initiation 6 . This study presents data that validate a novel therapeutic strategy with an ASO targeting the alternative chloride channel TMEM16A to reestablish its expression and activity and compensate for CFTR deficiency in CF. This approach is independent of the disease-causing CFTR mutations and could apply to all patients with CF, particularly those who cannot benefit from current curative treatments.

In this article, we have also demonstrated the strong specificity, the absence of toxicity, and the good persistence in vivo of TMEM16A ASO. We have also established that activating the TMEM16A Cl -channel increases Cl -activity and mucociliary clearance in vitro and improves the survival of CF mice that die of intestinal obstruction shortly after being weaned, along with other symptoms such as fertility problems and intestinal obstruction. We have also observed that TMEM16A ASO is transmissible from the treated mother to the pups by a maternal-fetal transmission as previously described 25 .

Previously, we have demonstrated that TMEM16A is specifically regulated by miR-9

and could be an exciting target for CF pathology by increasing alternative Cl -efflux 13 .

Further to our original publication, different groups have confirmed TMEM16A

regulation by miR-9 in other pathologies such as breast and colorectal cancer, sepsis, or idiopathic pulmonary fibrosis 14,15,17 .

The idea to correct the ion transport defect independently of the CFTR genotype was proposed in the early 1990s when studies showed that triphosphate nucleotides such as adenosine-5'-triphosphate (ATP) and uridine- With four RNA-targeting drugs approved for commercial use and scored in clinical development, researchers and clinicians have proved that the technology is efficient and supports approaching "undruggable" targets 28 . The pharmaceutical properties of ASO have been documented for certain diseases. Still, much data is missing, and it often remains empirical. However, it is well known that ASO has interesting characteristics for the industry because they are highly water-soluble (>50 mM), stable for years under refrigeration, and could be administered in simple saline solutions 29 .

Different groups have described that the pharmacokinetics of ASO in humans is predictable from data measured in animals 30 . Single-stranded ASOs are amphipathic and bind to proteins in the serum, the cell surface, and intracellularly. These interactions facilitate cell uptake and distribution such that ASOs of this type can be given by nearly all routes of administration in saline solutions and distributed to most body tissues 28 .

In our study, we have identified the most effective way of administration by injecting fluorescent TMEM16A ASO into mice. The idea was to select classical routes of administration (intravenous, intraperitoneal) and more specific routes (intranasal, subcutaneous) that could easily be used to treat patients. In our study, intranasal administration could be of interest because ASO activity could be significantly more effective in the airway than intraperitoneal or intravenous administration 31 .

Conversely, to avoid interaction with other drugs administrated by aerosol, subcutaneous seems to be a good alternative and does not require vectorization or modification in the formulation of the ASO facilitating industrial development 30 . This route of administration is generally well tolerated and easily applicable to patients receiving multiple treatments.

Additionally, our results show that the molecule's half-life in animals is quite long since we can still detect the ASO fluorescence 30 days after administration. More, an administration every 15 days is sufficient to increase the survival of the animals significantly. These data are consistent with the literature. For example, a 20-MOE ASO administered subcutaneously weekly at 300 mg per week has been shown to reduce Factor XI levels by 80% and reduce deep vein thromboses in patients undergoing total knee replacement by 7-fold enoxaparin 32 . In this condition, a twicemonthly chronic administration of TMEMA16a ASO has been demonstrated to increase the survival of mice from 36 to 294 days. This strategy minimizes adverse side effects with a minimal dose to restore and not over-induced TMEM16A activity 13 .

Concerns about whether activating TMEM16A would adversely affect mucus secretion or induce vasoconstriction in the bronchi were expressed. Vasoconstriction can only be considered if TMEM16A activates intracellular Ca 2+ mobilization, which is not the case in our approach 33 . The mucus secretion that may be induced by TMEM16A activation is more complex and show the limitations of our knowledge of cellular lung physiology 34 . A recent publication by Amaral's team provides a partial answer as they have demonstrated that TMEM16A does not drive mucus production 35 . This strategy is also open to discussion as many papers show that TMEM16A is present in many cancers because it has a pro-proliferative capacity 21,36 . Hence, our goal was not to overexpress and drastically increase TMEM16A activity but only to restore the profile observed in non-CF cells in a specific way to limit adverse effects. Moreover, no tumors were observed for CF mice repeatedly treated with TMEM16A for over a year.

Interestingly, TMEM16A ASO effects were not restricted to lung improvements but could impact other CF manifestations. Indeed, although lung pathology is preponderant and plays a significant role in the morbidity of patients, ancillary involvement of the other organs is not negligible, and few therapies seem to have any effect. These symptoms remain debilitating and impact the patients' personal development. The relief of gastrointestinal obstruction in our CF mice model shows that our molecule can act on the intestinal tract and that this could limit patients' abdominal pain. This is a crucial issue, as it was the second priority that emerged from a questionnaire completed by more than 1100 patients with CF 37 . Surprisingly, the priority was not to prevent disease progression but to simplify treatment, which our approach provides. Finally, one unexpected result was the fertility improvement in male CF mice by restoring vas deferent abnormalities, also described in CF male patients 1 . Of course, these results will have to be pursued to understand better the role that TMEM16A may play in vas deferens formation as little data is available 33,38,39 .

Recently, a more classical approach using a chemical molecule ETX001 was also developed to target TMEM16A. Like ours, ETX001 acts independently of Ca 2+ efflux to remain as specific as possible and limit secondary effects 40 . In the same way, as we had shown previously, this strategy restored Cl -efflux in vitro and in vivo in a sheep model. Even though the purposes are similar, the TMEM16A ASO acts as a corrector, more precisely as a facilitator of TMEM16A expression, while ETX0001 acts more as a potentiator where TMEM16A needs to be expressed and have a minimal level of activation. Nonetheless, we think that the two approaches could be complementary.

In conclusion, targeting TMEM16A can treat multiple symptoms in a CFTRindependent strategy that can be applied to all patients with CF alone or in combination with other CFTR-targeted strategies for eligible patients.

Methods

Cell culture and transfection

As previously described, CFBE41o-and 16HBE14o-cell lines were cultured and transfected with ASO (control or TMEM16A) 41,42 . 16HBE14o-with different CFTR mutations were generated by Cystic Fibrosis Foundation 43 . Primary human bronchial epithelial cells (hBEC) isolated from bronchial biopsies and provided by Epithelix SARL (Geneva, Switzerland) were cultured in an air-liquid interface and transfected with ASO 13 . Cell surfaces were evaluated by the crystal violet method. Proliferation was quantified using CyQUANT NF cell proliferation assay following manufacturer instruction (Thermo-Fischer, France) and recorded with a plate reader.

Bacterial strains and cultures

An inoculum of luminescent Pseudomonas aeruginosa (P. aeruginosa) was grown in In some experiments, TMEM16A channel activity was assessed by iodine quenching of halide-sensitive YFP-H148Q/I152L protein 45 . Fluorescence was recorded and analyzed using a plate reader as previously described 13 .

Mucociliary clearance assay

hBEC were transfected with control ASO or TMEM16A ASO 1h every day for three Axiovert 200 microscope (Zeiss), and the average speed (µm/ms) was determined as previously described 13 .

In vitro pharmaceutical profiling

In vitro pharmaceutical profiling was determined following the recommendation published by Bowes et al. 19 . The experimental studies were performed by Eurofins

Cerep (Celle l'Evescault, France).

Cytotoxicity evaluation LDH (lactate dehydrogenase) cytotoxicity assay was determined in vitro using the LDH-based CytoTox96®Non-RadioactiveCytotoxicity Assay according to the manufacturer's instructions (Promega, France).

Calcium mobilization

After the treatment with the TMEM16A ASO, cells were plated at 30K cells/well in MEM supplemented with 10% SVF into a 96 wells plate 48 hours prior to the assay. After the treatment, cells were loaded with Fluo-4 dye as previously described 46 . Cells were incubated with dye at 37 °C for 30 minutes, washed with calcium-free PBS, activated with ionomycin (10 µM), and recorded with a plate reader (BMG LabTech).

CF mice model

129-Cftr tm1Eur CF model mice homozygous for the F508del mutation and their wild-type littermates were obtained from CDTA-CNRS (Orléans, France). All adult animals had free access to drinking water without laxatives and were fed ad libitum on a standard diet. Studies were approved by the Institutional Animal Care and Use Committee.

Noninvasive fluorescence assessment in vivo

Nude mice (BALB/cAnNRj-Foxn1 nu/nu) were treated with fluorescent TMEM16A ASO (Eurogentec, Belgium; ex 640 nm/em 680 nm; 10mg/kg) by different routes of administration (10 mg/kg) as indicated in the legend of figure 4. Fluorescence was measured at 1h, 4h, 1d, 7d, 15d, 21d, and 30 days. Prior to imaging, mice were anesthetized using isoflurane inhalation. Immediately afterward, the mice were placed in a light-sealed chamber connected to an image intensifier coupled to a CCD camera fitted with a 25-mm macro lens. Imaging was performed on the IVIS Spectrum imaging system (PerkinElmer, Inc., France) by two-dimensional (2D) epifluorescence imaging at indicated times after probe injection to optimize readouts. 3D images were generated by Ivis software.

Toxicity assessment

In vivo toxicity protocol was performed by C-Ris Pharma (Saint-Malo, France) on healthy CD-1 mice. Histology and blood analysis was performed on mice treated at 500 mg/kg (50 times the effective dose) and sacrificed 14 days post subcutaneous injection. Hematology parameters were analyzed on whole blood using the ProCyte Dx hematology analyzer (IDEXX, France). Biochemical assays were performed using the KBIO04-DUO biochemistry analyzer (Kitvia, France). After sacrifice, the organs were collected, embedded in paraffin, and stained with hematoxylin and eosin before cutting and analyzing.

Statistical analysis

Statistical analysis was performed as indicated in the respective figure legends, using GraphPad Prism 7.05 software (GraphPad Software Inc, San Diego, USA).

Body weight

The body weight of CF mice treated with either ASO control or TMEM16A ASO was measured every 15 days. TMEM16A ASO-treated mice tend to gain weight better than control mice (Figure 30). In order to better understand how TMEM16A resolves intestinal obstruction, we started to investigate intestinal inflammation that partly reflects the obstruction. Mice feces were collected and extracted to measure fecal lipocalin, which marks neutrophil infiltration in the gut. Only lipocalin from WT and CF mice feces were dosed at this time to describe the gastrointestinal status of the mice model (Figure 33). As observed, lipocalin was five times higher in the feces of CF mice compared to control mice (±466.7 vs. ±88.27), highlighting an inflammatory bowel in CF mice (Figure 33). regulated chloride channel expressed in epithelial cells. Highly effective modulator therapies are now available for most patients with CF. However, some individuals have CFTR variants that do not produce CFTR protein and, therefore, cannot benefit from pharmacologic rescue approaches. Thus, it is critical to emphasize the importance of a CFTR-independent strategy regarding the 15% remaining patients, notably class I patients and nonresponders, who should not be forgotten. TMEM16A constitutes a promising alternative for treating those patients [START_REF] Amaral | Activating alternative chloride channels to treat CF: Friends or Foes?: Report on the Meeting of the Basic Science Working Group in Dubrovnik, Croatia[END_REF]Danahay et al., 2020b;[START_REF] Galietta | TMEM16A (ANO1) as a therapeutic target in cystic fibrosis[END_REF][START_REF] Pinto | Systems Approaches to Unravel Molecular Function: High-content siRNA Screen Identifies TMEM16A Traffic Regulators as Potential Drug Targets for Cystic Fibrosis[END_REF].

In 2017, Sonneville et al. showed that TMEM16A expression was dysregulated in CF due to miRNA-9 inhibition by directly targeting its 3′UTR. They then synthesized a target site blocker, an ASO designed to compete specifically with miRNA-9 for the 3'UTR of the TMEM16A mRNA. By binding to the 3'UTR of TMEM16A mRNA, TMEM16A ASO prevents miRNA-9 from gaining access, thus, enhancing TMEM16A expression and function by enabling protein translation (Sonneville et al., 2017).

In this work, we looked further into the TMEM16A ASO effects on CF dysregulated parameters and acquired preclinical data to determine if this approach was reasonably safe for initial use in humans.

 Chloride efflux, mucociliary clearance, and mucus production Dysfunction of the CFTR channel impairs transepithelial fluid and electrolyte permeation, resulting in an abnormal MCC associated with infection and inflammation, ultimately leading to pulmonary failure and pancreatic insufficiency. Hence, activating TMEM16A could compensate for CFTR function impairment by restoring the ionic balance and water content, as shown previously (Ousingsawat et al., 2009).

After choosing the optimal TMEM16A ASO concentration, TMEM16A channel activity was assessed in hBECs by iodine quenching of halide-sensitive YFP protein and confirmed by the Ussing chamber measurements. We found that TMEM16A ASO increased the Cl -efflux at the apical membrane, which correlates with increased TMEM16A proteins in CF primary cells in Sonneville et al. publication (Sonneville et al., 2017). Furthermore, MCC in CF primary cells bearing class I and/or II mutations was restored, even more so than those treated with Orkambi. Our findings are supported by Danahay et al., showing that TMEM16A potentiation using ETX001 enhances airway epithelial anion and fluid secretion in CF hBEC and promotes mucus clearance in an in vivo MCC sheep model. However, unlike TMEM16A ASO, ETX001 acts as a potentiator and requires a minimal TMEM16A activation acquired by IL-13

or UTP pre-treatments in the study (Danahay et al., 2020b).

Conceivably, we can attribute improved MCC to better mucus hydration and changes in either ionic composition, mucin composition, or ASL pH, affecting the rheological properties of gel-forming mucins. HCO3 -is critical to normal mucus formation and pH regulation [START_REF] Ambort | Calcium and pH-dependent packing and release of the gel-forming MUC2 mucin[END_REF][START_REF] Curnutt | Chemical and Microstructural Characterization of pH and [Ca2+] Dependent Sol-Gel Transitions in Mucin Biopolymer[END_REF]. Altered HCO3 Although TMEM16A has been demonstrated to play an essential role in MCC, its role in mucus production has generated conflicting evidence. Some studies proposed that TMEM16A activation can lead to excessive mucus secretion and goblet cell metaplasia under IL-4/IL-13 [START_REF] Cabrita | TMEM16A Mediates Mucus Production in Human Airway Epithelial Cells[END_REF]Centeio et al., 2021;[START_REF] Benedetto | Plasma membrane-localized TMEM16 proteins are indispensable for expression of CFTR[END_REF][START_REF] Scudieri | Association of TMEM16A chloride channel overexpression with airway goblet cell metaplasia[END_REF], while others demonstrated that TMEM16A does not induce cell metaplasia and mucus induction occurs independently of TMEM16A expression (Danahay et al., 2020a;Simoes et al., 2019). To date, no effects of TMEM16A activation were observed on excessive mucus production with ETX001 (Danahay et al., 2020b).

Further studies (MCC, chloride efflux, etc.) will also be needed in order to compare the effect of ASO TMEM16A to the highly effective new modulator Trikafta. Moreover, combining the two treatments might be interesting since studies have shown that TMEM16A is vital for CFTR activation and its expression at the PM [START_REF] Benedetto | Plasma membrane-localized TMEM16 proteins are indispensable for expression of CFTR[END_REF]Benedetto et al., 2017;[START_REF] Ousingsawat | CFTR and TMEM16A are Separate but Functionally Related Cl-Channels[END_REF].

 Bacterial overgrowth

Chronic lung infections are associated with the deterioration of pulmonary functions and the general health of patients with CF [START_REF] Mesinele | Risk factors for Pseudomonas aeruginosa airway infection and lung function decline in children with cystic fibrosis[END_REF]. Antimicrobial functions among numerous elements within the airway depend on pH regulation to carry out their normal functions (A. Pezzulo et al., 2012). Pezzulo et al. found that as pH becomes more acidic in CF, it inhibits antimicrobial activity and thereby impairs bacteria killing (A. Pezzulo et al., 2012). We were curious to see if TMEM16A would have any direct or indirect bactericidal or bacteriostatic activity by restoring anion efflux and MCC. After putting the bacteria directly with the TMEM16A ASO or pre-treating the hBECs prior to infection, no difference was observed compared to the control. It seems that ASO TMEM16A does not affect bacterial overgrowth either directly or indirectly. It would still be interesting to look at the expression and activity of antimicrobial peptides in the future, along with measuring pH variations, in TMEM16A ASO-treated CF primary cells under infectious and inflammatory conditions.

 Cellular proliferation

TMEM16A overexpression is associated with different cancer types, such as breast or colorectal cancer. Hence, our goal is not to overexpress or drastically increase TMEM16A but only to restore its normal expression. Experiments on cell proliferation in 16HBE noted no difference in the proliferation rate of pulmonary cells. Further investigations with different cell types, such as mammary glandular and colorectal cells, are anticipated. Noteworthy, no tumor was detected in mice (WT or CF) treated with ASO TMEM16A in long term (18 months) testing.

 Inflammation and toxicity

Chronic inflammation in people with CF causes lung damage, ultimately leading to respiratory failure. It is critical to avoid further stimulation of inflammation to an already inflamed environment. In our case, inflammation could be induced theoretically by (1) TMEM16A activation or (2) the ASO structure itself. On the one hand, TMEM16A

activates diverse inflammatory signaling pathways, including the inositol 1,4,5trisphosphate receptor (IP3R)-mediated Ca 2+ signaling pathway, the NF-κB signaling pathway, and the ERK signaling pathway [START_REF] Bai | The diverse roles of TMEM16A Ca(2+)-activated Cl(-) channels in inflammation[END_REF]. Thus, we treated CFBE and 16HBE cells with TMEM16A ASO to examine the inflammatory response to increasing TMEM16A expression and activity. No induced cytokine secretion was observed in the treated cells. However, future experiments should include primary cells that would better reflect the cell environment in CF in presence or absence of bacterial infection to better mimic the conditions observed in patients with CF.

TMEM16A and miRNA-9 expression should also be quantified in presence of bacteria since it has been demonstrated that multiple inflammatory mediators, including cytokines (IL-4, IL-13, IL-6), histamine, bradykinin, and ATP/UTP, as well as bacterial and viral infections, promote TMEM16A expression and activity [START_REF] Bai | The diverse roles of TMEM16A Ca(2+)-activated Cl(-) channels in inflammation[END_REF].

On the other hand, as mentioned previously in the third chapter, ASOs can induce adverse effects depending on their hybridization, sequence, and/or chemical modifications (Figure 3). To be able to go into clinical studies, it was essential for us to demonstrate that TMEM16A ASO did not induce toxicity. This was first confirmed in vitro by quantifying the lactate dehydrogenase on treated CFBE cells. We also injected WT mice with 50 times the active dose, monitored general signs, and did a macroscopic autopsy, histology analysis, and blood analysis to check for signs of toxicity, notably in the liver and kidneys often associated with toxic effects. No particular change was detected. We also observed the CF mice repeatedly treated with the usual dose (10mg/Kg) for the survival study, and no abnormal behavior or disease was noticed. Next, we will assess (1) Hence, further investigations will be needed to assess the potential adverse effects.

 Specificity

The specificity of Watson-Crick hybridization between an ASO and mRNA suggests that its use will result in a sequence-specific protein downregulation. Nevertheless, binding specificity is essential in drug design for a molecule to validate pharmaceutical regulations. Indeed, an effective drug molecule must bind with high specificity to its intended target. Hence, early screening can help avoid off-target interactions and thus reduce failure at a later stage [START_REF] Bowes | Reducing safety-related drug attrition: the use of in vitro pharmacological profiling[END_REF]. For that purpose, the in vitro pharmaceutical profiling method was carried out. In vitro pharmacological profiling involves the screening of compounds against a broad range of targets (receptors, ion channels, enzymes, and transporters) that are distinct from the intended therapeutic target (or targets) in order to identify specific molecular interactions that may cause adverse drug reaction [START_REF] Bowes | Reducing safety-related drug attrition: the use of in vitro pharmacological profiling[END_REF]. In the case of TMEM16A ASO, no offtarget activity was identified. However, we must remain cautious about other missed targets or targets that may not be particularly relevant to off-target safety [START_REF] Van Vleet | Screening Strategies and Methods for Better Off-Target Liability Prediction and Identification of Small-Molecule Pharmaceuticals[END_REF]. For later off-target interaction studies, computational identification should be considered. This approach uses two complementary paradigms: protein structurebased (target centric), which uses knowledge of the protein 3D structure and its binding pockets, and ligand-based (ligand centric), which relies on biological response variations with diverse ligand chemical structures [START_REF] Van Vleet | Screening Strategies and Methods for Better Off-Target Liability Prediction and Identification of Small-Molecule Pharmaceuticals[END_REF].

Advancements on emerging screening strategies were achieved with the "organ-ona-chip" platforms where toxicity studies are being developed to study toxicity-disease interactions or effects on one organ resulting in toxicities in another organ [START_REF] Esch | Organs-on-chips at the frontiers of drug discovery[END_REF][START_REF] Rezaei Kolahchi | Microfluidic-Based Multi-Organ Platforms for Drug Discovery[END_REF]. Another common use of this method includes tumor cells into common metastatic sites to assess malignancy or dormancy. This approach can be applied in the case of our study to evaluate the TMEM16A effects on cancer development on a breast or colorectal tissue, for example [START_REF] Rezaei Kolahchi | Microfluidic-Based Multi-Organ Platforms for Drug Discovery[END_REF]. Finally, it can also be used to study some levels of immune function, on a lung level, for instance.

Based on the denufosol experience, scientists have learned that any pharmacological modulation of alternative chloride channels, notably TMEM16A, should not increase intracellular Ca 2+ to avoid potential Ca 2+ -dependent side effects. To this end, we performed a Ca 2+ mobilization study on 16HBE cells. As expected, this study demonstrated that TMEM16A ASO does not interfere with Ca 2+ mobilization since TMEM16A ASO targets specifically TMEM16A mRNA via complementary Watson-Crick base pairing. Further experiments on Ca 2+ mobilization in primary cells should be completed.

 Biodistribution and persistence

To establish the optimal molecule administration route, we administered a fluorescent TMEM16A ASO to nude mice. Four routes were tested: intravenous, subcutaneous, intraperitoneal, and intranasal. In our study, subcutaneous injection and intranasal instillation had the best results, with persistence that lasted over 30 days; however, we chose the subcutaneous injection for multiple reasons. First, substances administered subcutaneously are often absorbed in a slower rate by different routes, providing a sustained effect [START_REF] Turner | Administration of substances to laboratory animals: routes of administration and factors to consider[END_REF]. In a study comparing the pharmacokinetics of intranasal versus subcutaneous insulin injections in the mouse, researchers trying to target the brain without the periphery found that the subcutaneous route achieves significantly higher peripheral exposure than the the intranasal route [START_REF] Nedelcovych | Pharmacokinetics of Intranasal versus Subcutaneous Insulin in the Mouse[END_REF]. Thus, subcutaneous route should increase the diffusion rate in the body, which is, in our case, desirable for multi-organ effects as CF is a multi-organ disease. Also, in the case of patients with CF, subcutaneous injection allows getting around the thick and sticky mucus that might prevent the oligonucleotide from accessing the cells.

Moreover, treatment compliance is often poor for patients with CF who already have a high treatment burden (estimates suggest the regimen takes two to three hours per day for routine CF care, with more than 50 medications)(G. [START_REF] Davies | Characterising burden of treatment in cystic fibrosis to identify priority areas for clinical trials[END_REF].

Therefore, patients would adhere better to subcutaneous than intranasal treatment because it is faster and less complicated. Also, since ASO by subcutaneous injection persist for days, we can imagine using it once weekly, like Mipomersen, Inotersen, and Volanesonersen, to simplify dosing regimens and improve patient compliance.

Finally, it seemed there was less accumulation in the kidney and liver when the subcutaneous route was used compared to intranasal, which is also beneficial in terms of toxicity. We have currently started to quantify the distribution of fluorescent TMEM16A ASO in each organ individually to determine if the organ concentrations are in the therapeutical range, especially in the lungs. Future pharmacokinetic 131 analyses will better clarify the absorption, distribution, metabolism, and excretion of the ASO in the body.

 ASO TMEM16A Effects on CF Physiological Parameters in Vivo

Studies have shown that TMEM16A expression and activity in mice airways are more significant than in humans and that the Ca2+-dependent Cl -efflux compensates for low CFTR activity in mouse airways and maintains a proper MCC (G. [START_REF] Davies | Characterising burden of treatment in cystic fibrosis to identify priority areas for clinical trials[END_REF], which could explain why the CF mouse model does not develop pulmonary manifestations. Although the mouse CF model does not entirely reflect the human pulmonary manifestations, these mice develop the classic muco-obstructive pathology observed in the human CF intestine, which was initially the primary cause of death in patients with CF in the 1950s. Also, many phenotypic abnormalities of CF mice can mimic CF human pathologies, such as male infertility, reduced growth, osteopenia, abnormal enamel, and many others [START_REF] Wilke | Mouse models of cystic fibrosis: Phenotypic analysis and research applications[END_REF]. In this study, we investigated the effects of TMEM16A on some of these CF phenotypes.

The colon is involved in mucus and HCO3 -secretion that relies on luminal CFTR [START_REF] Kunzelmann | Electrolyte transport in the mammalian colon: mechanisms and implications for disease[END_REF]. So mice with CFTR deficiency die of intestinal obstruction around weaning due to intestinal obstruction [START_REF] Wilke | Mouse models of cystic fibrosis: Phenotypic analysis and research applications[END_REF]. Kunzelmann et al. and Benedetto et al. showed that TMEM16A was expressed in murine intestinal epithelium in the distal colon but not in the small intestine [START_REF] Benedetto | Plasma membrane-localized TMEM16 proteins are indispensable for expression of CFTR[END_REF]Kunzelmann et al., 2019a). Moreover, the role of TMEM16A in gut motility has been demonstrated in mice homozygous for the null TMEM16A allele (Cobine et al., 2017;Hwang et al., 2009). In this study, CF-treated mice exhibited a significant increase in median survival, probably due to a TMEM16A compensating for CFTR lack of activity.

Further ion permeability studies using short-circuit current should be carried out in the intestinal segments to validate our hypothesis along with TMEM16A protein expression and histology analysis.

Furthermore, we are currently measuring the expression of lipocalin and calprotectin, biomarkers of neutrophil infiltration found in feces, and indicating the occurrence of intestinal inflammation due to obstruction, among other reasons. These biomarkers are used to screen patients with suspected inflammatory bowel disease. Recently, fecal calprotectin was measured in young patients with CF and was suggested to be an indicator of rectal inflammation [START_REF] Roda | Faecal calprotectin and rectal histological inflammatory markers in cystic fibrosis: a single-centre study[END_REF]. Hence, fecal calprotectin should inform us in a none invasive way about the evolution of the inflammation, which can reflect the obstruction. Relieving gastrointestinal symptoms in patients with CF is set among the "top 10" priorities by the CF community (Smith et al., 2020). Activating TMEM16A might have a therapeutic effect in patients with CF who suffer from intestinal obstruction syndrome, which can also improve their nutritional status and body weight, as observed in treated CF mice, for better clinical outcomes (Ranganathan et al., 2011).

In a recent case report, seven male patients between 17 and 39 years of age, treated with Trikafta ® , reported testicular pain (Rotolo et al., 2020). It is suggested that this may be related to CFTR function restoration in the male reproductive tract, where TMEM16A is also expressed (https://www.proteinatlas.org; accessed on August 2022). In the same way, we speculate that TMEM16A activation can also treat CF infertility. Male CF mice, like male CF patients, are generally infertile partly due to the frequent plugging or absence of the vas deferens. This study showed an improvement in CF male fertility; TMEM16A expression and histology analyses will be carried out to better explain these promising results.

With 36% of adults with CF being over 40 in 2030 versus 21% in 2017 [START_REF] Keogh | The changing demography of the cystic fibrosis population: forecasting future numbers of adults in the UK[END_REF][START_REF] Kunzi | Cystic Fibrosis Lung Disease in the Aging Population[END_REF], the aging CF population needs are changing as new symptoms are being described. For example, an additional phenotype developing in CF patients is osteoporosis manifested by low bone mineral density and increased fracture rates (Isabelle Sermet-Gaudelus et al., 2011) due to defective osteoblast maturation [START_REF] Velard | Cystic fibrosis and bone disease: defective osteoblast maturation with the F508del mutation in cystic fibrosis transmembrane conductance regulator[END_REF]. A similar phenotype was reported in CFTR mutant and KO mice [START_REF] Dif | Severe osteopenia in CFTR-null mice[END_REF][START_REF] Haston | Persistent osteopenia in adult cystic fibrosis transmembrane conductance regulatordeficient mice[END_REF][START_REF] Pashuck | Murine Model for Cystic Fibrosis Bone Disease Demonstrates Osteopenia and Sex-Related Differences in Bone Formation[END_REF][START_REF] Wilke | Mouse models of cystic fibrosis: Phenotypic analysis and research applications[END_REF]. Since an enhanced F508del-CFTR channel activity ameliorates bone pathology in CF mice [START_REF] Henaff | Enhanced F508del-CFTR Channel Activity Ameliorates Bone Pathology in Murine Cystic Fibrosis[END_REF], we believe that TMEM16A potentiation could have the same effect, especially since a recent study by Sun et al. identified

TMEM16A as an important regulator of osteoclast function by interacting by coupling

Cl -channel activation with RANKL activating signaling pathway involved in bone resorption [START_REF] Partridge | Ca(2+)-activated chloride channel ANO1: A new regulator of osteoclast function[END_REF][START_REF] Sun | Anoctamin 1 controls bone resorption by coupling Cl(-) channel activation with RANKL-RANK signaling transduction[END_REF]. This phenotype is being explored in collaboration with Dr. Frederic Velard for further insights.

Similarly, CF mice are easily recognized by their typical white incisor teeth due to abnormal enamel development [START_REF] Arquitt | Cystic fibrosis transmembrane regulator gene (CFTR) is associated with abnormal enamel formation[END_REF]Wright et al., 1996a;Wright et al., 1996b). One of the explanations is that altered pH regulation is necessary for producing an environment conducive to enamel growth and mineralization [START_REF] Sui | Altered pH regulation during enamel development in the cystic fibrosis mouse incisor[END_REF]. It is important to note that mice wear down and renew their four incisors every 35 to 45 days. In our study, we have noticed that the teeth of CF treated mice were more resemblant to WT than CF. Following this observation, we developed a collaboration with Dr. Francoise Tilotta to validate these findings. Indeed, preliminary results show a restoration in the tooth structure. Further studies will be needed to know if TMEM16A promotes enamel formation, reduces its degradation, or regulates the pH providing an adequate mineralization environment.

 Further validation models

Our mutation-agnostic approach was developed to treat all patients with CF, especially those still lacking treatment, like patients with nonsense mutations and nonresponders. So far, TMEM16A ASO has been mainly validated in models (cell lines, primary cells, CF mice) with the most common F508del class II mutation.

Therefore, the assessment of in vivo effectiveness of TMEM16A ASO on nonsense mutation is critical. For that reason, we recently imported a CF mouse model carrying the G542X mutation, the most common CFTR nonsense mutation. This model was generated by Hodges et al. in 2018 using CRISPR/Cas9 gene editing [START_REF] Mchugh | A G542X cystic fibrosis mouse model for examining nonsense mutation directed therapies[END_REF]. These mice have reduced Cftr expression and absent CFTR function in the airway and intestine. They also display classical CF manifestations such as poor growth and reduced survival due to intestinal obstruction [START_REF] Mchugh | A G542X cystic fibrosis mouse model for examining nonsense mutation directed therapies[END_REF]. We will begin testing the TMEM16A ASO effectiveness in the upcoming months on those mice. However, considering that the CF mouse lacks a spontaneous lung and pancreatic phenotype, other CF models should also be explored.

Different CF animal models have been generated (mice, rats, ferrets, rabbits, pigs, sheep, zebrafish, and drosophila) and are used depending on the desired genotype and phenotype. Most animals bear the F508del mutation, but we can find mice [START_REF] Colledge | Generation and characterization of a delta F508 cystic fibrosis mouse model[END_REF][START_REF] Mchugh | A G542X cystic fibrosis mouse model for examining nonsense mutation directed therapies[END_REF], rats [START_REF] Dreano | Characterization of two rat models of cystic fibrosis-KO and F508del CFTR-Generated by Crispr-Cas9[END_REF][START_REF] Sharma | A Novel G542X CFTR Rat Model of Cystic Fibrosis Is Sensitive to Nonsense Mediated Decay[END_REF], and sheep models [START_REF] Viotti Perisse | Sheep models of F508del and G542X cystic fibrosis mutations show cellular responses to human therapeutics[END_REF] with the G542X mutation. The most attractive candidates are the CF ferret and pig models. They have a similar lung cell biology than humans and recapitulate the complete CF phenotype observed in patients, from inflammatory and infectious lung disease to in utero meconium ileus and CF-related diabetes [START_REF] Mccarron | Animal and Cell Culture Models for Cystic Fibrosis: Which Model Is Right for Your Application?[END_REF][START_REF] Rosen | Animal and model systems for studying cystic fibrosis[END_REF]. On the other hand, sheep offer a valuable platform for substance delivery studies because their lungs share similar anatomic, functional, histologic, and electrophysiological features to humans. It is also helpful for in-utero investigations because of the long gestation period and the large fetus size [START_REF] Mccarron | Animal and Cell Culture Models for Cystic Fibrosis: Which Model Is Right for Your Application?[END_REF]. In this model, maternal-fetal transfer studies can be designed. However, these models require many resources and are not easy to care for and house in standard research facilities such as ours, which limits their utilization [START_REF] Rosen | Animal and model systems for studying cystic fibrosis[END_REF].

In (Riordan et al., 1989). CF is a multi-system disease that affects the respiratory tract, intestines, pancreas, genital tract, the hepatobiliary system, and exocrine glands, lea d i n gt od i v e r s ep a t h o l o g yr a n g e sa n dc l i n i c a l problems (Elborn, 2016). While most patients have multiple organ alterations, the leading causes of both morbidity and mortality in more than 90% of patients remain chronic progressive pulmonary disease and respiratory failure (Elborn, 2016). In CF patients, the lack of CFTR chloride channel activity leads to progressive pulmonary obstruction associated with critical and constant neutrophil-dominated endobronchial inflammation and overwhelming bacterial infection (Figure 1). On a pulmonary level, scientists developed many new symptomatic therapies with either anti-inflammatory properties, antibiotics, or molecules improving mucociliary clearance (mucolytics) in order to treat inflammation, infection, or mucus abnormalities (Figure 2A). The discovery of these new drugs was made possible by the accumulation of knowledge in these three areas. After the discovery of CFTR, researchers aimed for the development of therapies that can correct the disease's origin. Their work mainly focused on infection, rather than on anti-inflammatory drugs or mucus abnormalities. The proportion of published articles on infection is more than 70% compared to those published on inflammation or mucus. This proportion reaches more than 80% when focusing on publications on antibiotics compared to those on antiinflammatory drugs and mucolytics (Figures 2B,D).

In the allocation of priorities, the anti-inflammatory drugs have been, for long, the "poor relatives" in basic research compared to the modulators of CFTR activity.

These drug modulators targeting CFTR are designed to reestablish, at least partially, the CFTR expression, and improve its activity. So far, many of these treatments got through to the market, and these therapies are upgrading patients' life quality through short-and long-term improvements in clinical outcomes (Lopes-Pacheco, 2019). Despite this, the main treatments remain symptomatic, focusing on different dysregulated clinical manifestations observed in CF patients (pancreatic insufficiency, intestinal malabsorption, and lung deterioration). However, their use is limited by insufficient basic scientific knowledge (Figure 2C), which has reduced the number of medicinal products currently on the market (Lopes-Pacheco, 2019). A deeper understanding of the natural evolution of CF pathology brought about new treatment tactics in order to improve pulmonary functions and increase life expectancy. CFTR chloride channel is also involved in the FIGURE 1 | Progression of CF pathophysiology in bronchial epithelial cells. In healthy airways, sodium (Na + ) absorption and chloride (Cl -) secretion control hydration of the airway surface layer (ASL). In CF airways, impaired Cl -secretion due to the CFTR absence or loss of function leads to unregulated Na + absorption and result in inadequate hydration of ASL, causing mucociliary clearance and bacterial killing impairment. As a result, mucus obstructs the lung airways and provides a nidus for bacterial infection and inflammation. The bacteria adhere to the surface and continue to grow, ultimately forming a biofilm. The inflammation of the CF lung is characterized by exaggerated secretion of pro-inflammatory cytokines by the airway epithelial cells, leading to the infiltration of polymorphonuclear neutrophils that release reactive oxygen species (ROS) and proteases. Neutrophil released elastase in the CF airway secretions correlates with lung function deterioration and respiratory exacerbations. The acidification of the ASL and the increase of its salt concentration, along with the increase of proteases levels, have been shown to impair the bactericidal activity of numbers of anti-microbial peptides (AMPs).

regulation of other channels such as the epithelial sodium channel (ENaC).

Other channels are directly or indirectly linked to CFTR, such as the calcium-activated chloride channels ANO1 (also called TMEM16a) (Benedetto et al., 2017)( Figure 3). Therefore a deregulated CFTR activity leads to an abnormal mucus composition and alteration of the airway surface liquid (ASL) hydration that could participate in the inflammatory process in CF airways [START_REF] Puthia | A dual-action peptide-containing hydrogel targets wound infection and inflammation[END_REF]. Recent publications have also highlighted that a loss of CFTR-mediated bicarbonate secretion and pH acidification impairs airways host defense by increasing mucus viscosity and reducing bacteria-killing (Shah et al., 2016). Current studies have established that the CFTR function is not restricted to ion transport regulation. Results have suggested a significant role of CFTR as a surface receptor for the internalization of Pseudomonas aeruginosa (P. aeruginosa) via endocytosis and consequent bacteria removal from the airway (Pier, 2000). In the CF airways, the permanent presence of bacteria might participate in the inflammatory process contributing to a vicious cycle between airway mucus obstruction, chronic infection, and exaggerated inflammation (Figure 4). Nowadays, it remains unclear how and why this vicious cycle is initiated, even though different elements suggest that different inflammatory pathways are deregulated in CF airways independently from infection [START_REF] Bardin | miR-636: A Newly-Identified Actor for the Regulation of Pulmonary Inflammation in Cystic Fibrosis[END_REF]. However, mucus alterations could be one of the triggers of this process. Mucins tethering to the apical bronchial surfaces lead to acidification of ASL, thus reducing the anti-bacterial properties of CF airways [START_REF] Song | Hyperacidity of secreted fluid from submucosal glands in early cystic fibrosis[END_REF]Quinton, 2008;[START_REF] Adam | Cystic fibrosis airway epithelium remodelling: involvement of inflammation[END_REF].

Finally, it is essential to bear in mind that mucus alteration, infection, and inflammation are elements that are very carefully intertwined and difficult to separate in the process of an inflammatory response (Figure 4). Multiple hypotheses explain the early events leading to the CF lung pathophysiology progression (Jacquot et al., 2008a;[START_REF] Esther | Mucus accumulation in the lungs precedes structural changes and infection in children with cystic fibrosis[END_REF].

PATHOPHYSIOLOGY IN CF AIRWAYS Inflammation

Although inflammation is a natural and protective process resulting from aggression, it plays a major role in CF lung pathology and progression. Inflammation was initially recorded by the Roman encyclopedist Aulus Cornelius Celsus in the 1 st century A.D. by some typical characteristic signs of inflammation as heat (calor), pain (dolor), redness (rubor), and swelling (tumor). Chronic and exaggerated inflammation in people with CF causes damages to lung tissues that can eventually lead to respiratory failure (Cantin et al., 2015). Many recent results show that bronchial epithelial cells play a significant role in the progression of the disease. In addition to being a physical barrier, epithelial cells secrete many inflammatory factors such as cytokines, eicosanoids, enzymes, and adhesion molecules (Roesch et al., 2018). This CF airway inflammation is characterized by an excessive production of interleukin (IL)-8 secreted by airway epithelial cells, and the presence of large numbers of neutrophils and macrophages among other inflammatory cells [START_REF] Hubeau | Quantitative analysis of inflammatory cells infiltrating the cystic fibrosis airway mucosa[END_REF]. However, it is not the only pro-inflammatory cytokine enhanced. In the airways of CF patients, TNF-a, IL-1b, IL-6, IL-8, IL-33, GM-CSF, and G-CSF are increased, also other molecules also play a major role such as the pro-inflammatory metabolites of arachidonic acid metabolism. Very recent results have highlighted the central role of other cytokines such as IL-17 (Roesch et al., 2018). In CF, the infiltration of inflammatory cells across the epithelium into the lumen can be very deleterious to epithelia and, as a consequence, requires robust regulation. Numerous works have tried to identify targets and strategies to reduce the exaggerated immune response that causes chronic inflammation without affecting the natural defenses against infection [START_REF] Muhlebach | Endotoxin activity and inflammatory markers in the airways of young patients with cystic fibrosis[END_REF]. It is unclear whether the inflammation is a direct consequence of the cftr mutation or whether it is a consequence of infection and mucus accumulation. We do not know the contribution of infection to airway inflammation, but it must act as a catalyst and becomes self-perpetuating. Different studies have demonstrated the direct implication of the CFTR protein in this process mainly in the lung but also in extra-pulmonary tissues as the intestine or pancreas [START_REF] Raia | Evidence of Chronic Inflammation in Morphologically Normal Small Intestine of Cystic Fibrosis Patients[END_REF][START_REF] Cohen | Cystic fibrosis: a mucosal immunodeficiency syndrome[END_REF][START_REF] Stoltz | Origins of cystic fibrosis lung disease[END_REF][START_REF] Bardin | miR-636: A Newly-Identified Actor for the Regulation of Pulmonary Inflammation in Cystic Fibrosis[END_REF]. Even before symptom onset, pulmonary inflammation and infection are often present in CF patients [START_REF] Muhlebach | Endotoxin activity and inflammatory markers in the airways of young patients with cystic fibrosis[END_REF]. Although which comes first has been uncertain, this aspect is well reviewed in the article from Stoltz [START_REF] Armstrong | Lower respiratory infection and inflammation in infants with newly diagnosed cystic fibrosis[END_REF][START_REF] Khan | Early pulmonary inflammation in infants with cystic fibrosis[END_REF][START_REF] Nixon | Early airway infection, inflammation, and lung function in cystic fibrosis[END_REF][START_REF] Stoltz | Origins of cystic fibrosis lung disease[END_REF]. Moreover, new models lacking CFTR, including pigs, ferrets, and rat manifest inflammatory features typically observed with CF even in absence of infection [START_REF] Rogers | Disruption of the CFTR Gene Produces a Model of Cystic Fibrosis in Newborn Pigs[END_REF][START_REF] Sun | Disease phenotype of a ferret CFTR-knockout model of cystic fibrosis[END_REF][START_REF] Tuggle | Characterization of Defects in Ion Transport and Tissue Development in Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)-Knockout Rats[END_REF]. For example, airways of CF piglets show no evidence of inflammation during the first hours after birth [START_REF] Stoltz | Cystic fibrosis pigs develop lung disease and exhibit defective bacterial eradication at birth[END_REF]. Evidence has also demonstrated that non-infected human CF airway graft is in a pro-inflammatory state [START_REF] Tirouvanziam | Inflammation and infection in naive human cystic fibrosis airway grafts[END_REF][START_REF] Tirouvanziam | Primary inflammation in human cystic fibrosis small airways[END_REF][START_REF] Perez | CFTR inhibition mimics the cystic fibrosis inflammatory profile[END_REF]Cantin et al., 2015). These data are reinforced by in vitro experiments using specific CFTR inhibitor. For example, Perez et al. have shown that Inh-172 treatment conducted in significant increase in IL-8 secretion in basal but also in response to P. aeruginosa infection [START_REF] Perez | CFTR inhibition mimics the cystic fibrosis inflammatory profile[END_REF]. All these data support the hypothesis that mutations in cftr gene make epithelial cells intrinsically more pro-inflammatory compared with healthy cells [START_REF] Perez | CFTR inhibition mimics the cystic fibrosis inflammatory profile[END_REF]Cantin et al., 2015), which, once infection is introduced, sets the stage for mucosal damage and chronic airway infection [START_REF] Tirouvanziam | Inflammation and infection in naive human cystic fibrosis airway grafts[END_REF].

Although the link between CFTR deficiency and host inflammatory response remains unclear, this aspect has long been recognized as a central pathological feature, and consequently, an important therapeutic target. Some have hypothesized that in CF, the unfolded proteins accumulation on the endoplasmic reticulum induced a proteinopathy responsible for inflammation, impaired trafficking, altered metabolism, cholesterol, and lipids accumulation, and impaired autophagy at the cellular level. Some have speculated that chloride dysregulation participated in a stress-inducing ionic imbalance in the airway, with the implication of calcium activation, which could induce an inflammatory state [START_REF] Ribeiro | Cystic fibrosis airway epithelial Ca2+ i signaling: the mechanism for the larger agonist-mediated Ca2+ i signals in human cystic fibrosis airway epithelia[END_REF]Tabary et al., 2006a). New hypotheses have emerged with the direct activation of NOD-, LRR-, and pyrin domaincontaining protein 3 (NLRP3) inflammasome and can be a key regulator of CF inflammation and a promising target [START_REF] Mcelvaney | Specific Inhibition of the NLRP3 Inflammasome as an Antiinflammatory Strategy in Cystic Fibrosis[END_REF][START_REF] Jarosz-Griffiths | Different CFTR modulator combinations downregulate inflammation differently in cystic fibrosis[END_REF].

However, since the appearance of high throughput sequencing, many studies have attempted to study the deregulated mechanisms, but the heterogeneity of samples and data makes analysis difficult. A meta-analysis of the different studies has summarized all this data (Ideozu et al., 2019). To summarize, many proteins are dysregulated, including gene from signal transduction (PI3K/Akt/ mTOR signaling pathway) and immune system (NFkBandMAP kinase pathways), but this method is more relevant to highlight the consequence than the cause of the inflammatory dysregulation. A very recent article has confirmed the implication of NLRP3 inflammation activation due to the alteration of electrolyte homeostasis induced by the over-activation of b-ENac channel in CF [START_REF] Scambler | ENaC-mediated sodium influx exacerbates NLRP3-dependent inflammation in cystic fibrosis[END_REF].

Furthermore, different authors showed more than 15 years ago that there is a deregulation of lipid metabolism in CF with an imbalance between pro-inflammatory metabolites of arachidonic acid metabolism and pro-resolving mediators form eicosanoid pathway (Freedman et al., 2004;Karp et al., 2004;Serhan, 2017;Roesch et al., 2018). Ceramide (CER) is an airway component composed of fatty acid and sphingosine that may alter the CF inflammatory response. CER is present in the cells membrane and when in contact with a specific stimulus, like a bacterial infection, CER in transmembrane signaling processes to help regulate cellular responses to infection by activating the inflammation processes. This could be an interesting alternative to treat CF inflammatory dysregulation by inhibiting CER synthesis (Mingione et al., 2020).

Although there is no consensus regarding the regulation of CER in CF cells currently, even if more recent data have demonstrated their implication on the progression of CF lung disease (Horati et al., 2020;Mingione et al., 2020). Consequently, these results have led to the proposal that upregulated inflammation is related to the molecular defect of CF with a strong implication of nuclear factor kappa B (NFkB) or mitogen-activated protein (MAP) kinase pathways with other transcription factors including NFAT, NF-IL6, AP1 and AP2 [START_REF] Tabary | Genistein inhibits constitutive and inducible NFkappaB activation and decreases IL-8 production by human cystic fibrosis bronchial gland cells[END_REF][START_REF] Tabary | Interleukin-10 Inhibits Elevated Chemokine Interleukin-8 and Regulated on Activation Normal T Cell Expressed and Secreted Production in Cystic Fibrosis Bronchial Epithelial Cells by Targeting the I(k)B Kinase alpha/beta Complex[END_REF][START_REF] Muselet-Charlier | Enhanced IL-1beta-induced IL-8 production in cystic fibrosis lung epithelial cells is dependent of both mitogen-activated protein kinases and NF-kappaB signaling[END_REF].

More recently, different articles have also associated microRNA (miRNA) dysregulation to CF inflammation [START_REF] Fabbri | Expression of miR-93 and IL-8 During Pseudomonas aeruginosa Mediated Induction of Pro-Inflammatory Responses[END_REF]Bardin et al., 2018a;[START_REF] Bardin | miR-636: A Newly-Identified Actor for the Regulation of Pulmonary Inflammation in Cystic Fibrosis[END_REF]. How the lack of CFTR expression in ionocytes, ciliated, and submucosal gland epithelial cells of the respiratory tract, boosts pulmonary inflammation is still partially comprehended. Different authors have also highlighted the central role of neutrophil in CF airway inflammation, and many believed that bronchiectasis results from the proteolytic and oxidative damage induced by these cells. Longitudinal data from the Australian Respiratory Early Surveillance Team for Cystic Fibrosis demonstrated that neutrophil elastase activity at 3 months of age was a predictor of bronchiectasis at 12 months and 3 years (Wijker et al., 2020). The central role of neutrophils and its genesis has been extensively review by Nichols et al. and Perrem et al. (Nichols and Chmiel, 2015;Perrem and Ratjen, 2019).

Understanding the initial host defense defects in CF airways could suggest new prevention strategies and treatments, the means to assess disease status and efficacy of therapeutics [START_REF] Stoltz | Origins of cystic fibrosis lung disease[END_REF]. Several mechanisms are suggested to e x p l a i ni nw h a tw a yC Fb a s a li n flammation promotes subsequent bacterial infection. One possible explanation is that serine protease, released by activated neutrophils, degrades innate immune mechanisms, including anti-microbial peptides (AMP), participates in secondary infection, and to this vicious cycle. The molecular mechanisms relating to abnormal CFTR chloride function in airway epithelial cells to excessive lung neutrophilic inflammation have not yet been fully clarified even if extensive works have already been published [START_REF] Taggart | Increased elastase release by CF neutrophils is mediated by tumor necrosis factor-alpha and interleukin-8[END_REF]Tabary et al., 2006b). Decreased neutrophil apoptosis and the high secretion of IL-8 by epithelial cells are contributing factors. In 2016, researchers discovered the leukocyte adhesion deficiency IV (LAD-IV), which is a defect in monocyte integrin activation in CF patients. The study showed that CFTR mutations could lead to a monocyte-specifica d h e s i o n deficiency (~80%) and impairment in transmigration into the alveolar space, which could explain the extreme infiltration of neutrophil since monocytes play a crucial part in inflammation and its resolution. Thus, failing to recruit monocytes in CF patients' lungs may explain the excessive production of cytokines, the impaired inflammation resolution, and pathogen capture impairment (Sorio et al., 2016). The continuous driven recruitment of neutrophils and other immune cells and their implication in non-resolving inflammation have been already discussed in different reviews (Cantin et al., 2015;Nichols and Chmiel, 2015;Roesch et al., 2018).

Whether CFTR dysfunction causes directly or indirectly, a more important predisposition to infection and whether the inflammation occurs separately from the infection has yet to be determined. The development of new anti-inflammatory strategies in CF remains limited due to the limited researches in this area compared to infection (Figure 2D).

Bacterial Infection

Respiratory infections in CF occur from childhood. In progressive lung diseases like CF, typical pathogens (P. aeruginosa, Streptococcus aureus, Burkholderia cepacia, Achromobacter xylosoxidans) colonize the airways (Palser et al., 2019). More than 50% of children diagnosed at birth have shown positive P. aeruginosa cultures by five years of age (Palser et al., 2019). If P. aeruginosa is neither spontaneously cleared nor eradicated with antibiotic therapy, the CF lung environment facilitates the infection.

The presence of pathogens triggers inflammatory processes in the airways contributing to the destruction of the cell barrier. Since inflammation is a natural process of defense and the eradication of pathogens, limiting it too much or for a long term could be counterproductive. For this reason, antibiotics are more frequently recommended than anti-inflammatory drugs in CF lung disease treatment and could indirectly serve to diminish airway inflammation (Oermann et al., 1999). The anti-inflammatory drugs that could alter the natural defense of the lung are only prescribed during exacerbations. Constant development and ideal usage of new anti-microbial compounds are vital for improving the CF patients survival chance and quality of life (Waters and Smyth, 2015). As a result of long-term antibiotic treatment, the decrease in infection and inflammation is associated with lung function improvements and pulmonary exacerbations reduction (Waters, 2018).

In a normal situation, the airways can defend themselves by forming a physical barrier between the outside and the inside. Also, the lung is capable of secreting cytokines that will allow the recruitment of inflammatory cells, but it is also capable of secreting anti-bacterial molecules. Thus, many natural AMPs, contained in the airways, are part of the innate immune response to the airway defense (Hancock et al., 2016). AMPs exhibit microbicidal activities on a broad spectrum of microbes, but bacteria appear to be the most targeted pathogens [START_REF] Scott | Cationic antimicrobial peptides and their multifunctional role in the immune system[END_REF][START_REF] Zasloff | Antimicrobial peptides of multicellular organisms[END_REF]. AMPs can kill bacteria rapidly in a few minutes. If most of the AMPs kill targeted pathogens via an electrostatic action on their membranes, some of them kill by more sophisticated mechanisms such as the IIA secretory phospholipase A2 (sPLA2-IIA) which kills bacteria through selective hydrolysis of their membrane phospholipids [START_REF] Van Hensbergen | Type IIA Secreted Phospholipase A2 in Host Defense against Bacterial Infections[END_REF], or by interfering with intracellular targets in bacteria [START_REF] Geitani | Cationic antimicrobial peptides: alternatives and/or adjuvants to antibiotics active against methicillin-resistant Staphylococcus aureus and multidrugresistant Pseudomonas aeruginosa[END_REF][START_REF] Wang | Antimicrobial peptides: Promising alternatives in the post feeding antibiotic era[END_REF]. Except for very few examples, little is known about the specificity of AMPs toward Gram-positive vs. Gram-negative bacteria. The sPLA2-IIA is one of the rare AMPs that target Gram-positive bacteria that exerts its bactericidal effect at much lower concentrations than other molecules [For details, see the review [START_REF] Van Hensbergen | Type IIA Secreted Phospholipase A2 in Host Defense against Bacterial Infections[END_REF]].

AMPs represent an essential part of the host defenses against infections and also as a potential therapeutic tool, as has been shown in infections animal models [START_REF] Morrison | Characterization of the Mouse Beta Defensin 1, Defb1. Mutant Mouse Model[END_REF][START_REF] Piris-Gimenez | In Vivo Protective Role of Human Group IIA Phospholipase A2 against Experimental Anthrax[END_REF]. This effect was also supported by studies in patients with infectious diseases showing that altered AMP expression and/or gene polymorphisms were associated with increased infections [START_REF] Rivas-Santiago | Susceptibility to Infectious Diseases Based on Antimicrobial Peptide Production[END_REF]. On the other hand, unfavorable circumstances for AMPs actions as abnormal salt concentration or acidification, and inactivation by proteases, in ASL of CF patients (Figure 1), have been shown to inactivate AMPs bactericidal functions which may explain increased airway infections (Bals et al., 2001;Lecaille et al., 2016;Simonin et al., 2019). Normalizing ASL pH by inhibition of the persistent proton secretion, mediated by ATPase H + /K + transporting non-gastric alpha2 subunit (ATP12A), might enhance innate airway defense in CF newborns during the onset of S. aureus infection. A recent study showed that the hydrophobic Nterminal domain of Cg-BigDef1 (a big defensin from oysters) exhibits salt-stable interactions with bacterial membranes opening the doors to eventual drug developments when physiological salt concentrations inhibit the anti-microbial activity of b-defensins such as in CF disease [START_REF] Loth | The Ancestral N-Terminal Domain of Big Defensins Drives Bacterially Triggered Assembly into Antimicrobial Nanonets[END_REF].

In parallel to their anti-microbial functions, several AMPs have been shown to play immuno-modulatory roles, in particular by interacting with the inflammatory reaction produced by host cells. Several studies have shown that AMPs can target host cells involved in innate immunity and modulated their production of inflammatory mediators, including cytokines. Although it is not always easy to dissociate these actions as most AMPs exhibit both functions, depending on their concentrations, the host cell targets, and the environments. However, AMPs have been shown to impair the inflammatory reaction induced by invading pathogens by different mechanisms [START_REF] Masera | Corticostatins/defensins inhibit in vitro NK activity and cytokine production by human peripheral blood mononuclear cells[END_REF][START_REF] Finlay | Can innate immunity be enhanced to treat microbial infections?[END_REF][START_REF] Mcinturff | Granulysin-derived peptides demonstrate antimicrobial and antiinflammatory effects against Propionibacterium acnes[END_REF].

The anti-inflammatory potential of AMPs correlates with their capability of attracting and recruiting neutrophils and other inflammatory cells. They may also have direct or indirect effects on their maturation, differentiation, degranulation, or apoptosis [START_REF] Lai | AMPed up immunity: how antimicrobial peptides have multiple roles in immune defense[END_REF]. AMPs also act by blocking neutrophils apoptosis, therefore prolonging their lifetime, and ultimately their phagocytic functions [START_REF] Nagaoka | Modulation of Neutrophil Apoptosis by Antimicrobial Peptides[END_REF]. AMPs can also potentiate the effects of inflammatory cells such as macrophages while limiting other tissue damage [START_REF] Brook | Neutrophil-derived alpha defensins control inflammation by inhibiting macrophage mRNA translation[END_REF].

Mucus Alteration

In healthy people, ASL is a thin liquid film covering the airways and participating in mucociliary clearance and airways desiccation (Figure 1). Historically, studies suggested that different secretory cells (goblet cells, submucosal glands cells, and serous cells) contribute to ASL production (Tarran et al., 2001). The recent finding of the airway "ionocyte" could similarly result in a revised understanding of ASL production (Plasschaert et al., 2018). This group has identified by RNA sequencing all the RNAs present inside airway cells and by a new method, called pulse-seq, has discovered this scarce cell type.

They created the term "ionocytes" due to the cell's likeness to ionocytes in charge of regulating ion transport and hydration in the fish gills and frog skin. In the airway, ASL consists of two main layers: 1) the apical layer consisting of a water-based polymeric mucus, and 2) a periciliary layer (PCL) that bathes the epithelium (Atanasova and Reznikov, 2019). Normal mucus is made of 97% water and 3% proteins, lipids, and salt. The m u c u sg e ll a y e ra c t sa sap h y s i c a lb a r r i e rt op r e v e n tm o s t pathogens from accessing the cells (Button et al., 2012). The mucus hydration and the mucin concentration dramatically affects its viscoelastic properties, which, in turn, determines how effectively it is cleared from the distal airways toward the trachea by ciliary action and cough (Fahy and Dickey, 2010).

The commonly accepted explanation for airway disease in CF is the "low volume" hypothesis. A reduced volume of the periciliary fluid layer (PCL) causes failure of mucociliary clearance, the 'lungs' innate defense mechanism. In addition to having altered physical properties, the mucus composition is modified and will participate in the CF pathophysiology by altering host defense proteins (Henderson et al., 2014). An increase in mucin secretion and an abnormal composition of mucus are implied by the formation of endobronchial mucus plaques and plugs. Mucus present in bronchia becomes the primary site of airflow obstruction, and subsequently for chronic infection, and persistent inflammation leading to early small airways disease succeeded by bronchiectasis development. Increased mucus and airway obstruction are hallmark features of multiple respiratory diseases and contribute, especially in CF, to a complicated, inflammatory process [START_REF] Puthia | A dual-action peptide-containing hydrogel targets wound infection and inflammation[END_REF]. A chronic cycle of infection and inflammation could be initiated, resulting in airways structural integrity damages and leading to bronchiectasis development (Chalmers et al., 2017). More recent studies from Esther et al. have shown that the increase of mucus burden and inflammatory markers without infection suggest that mucolytic therapies could serve as preventive therapy for CF lung pathology [START_REF] Esther | Mucus accumulation in the lungs precedes structural changes and infection in children with cystic fibrosis[END_REF]. More, mucus composition and properties also depend on the levels of mucin production by epithelial cells that can be increased by bacteria suggesting a complex role of inflammation, infection, and mucus, especially in CF pathology [START_REF] Mohamed | A Crucial Role of Flagellin in the Induction of Airway Mucus Production by Pseudomonas aeruginosa[END_REF]. The up-regulation of airway mucin genes by inflammatory/immune response mediators at the transcriptional and/or posttranscriptional level is one of the major contributors to mucin overproduction. The MUC5AC gene is transcriptionally up-regulated by several inflammatory mediators, including LPS, IL-9, neutrophil elastase, TNF-a,a n d IL-1b [START_REF] Song | Interleukin-1b and Tumor Necrosis Factor-a InduceMUC5ACOverexpression through a Mechanism Involving ERK/p38 Mitogen-activated Protein Kinases-MSK1-CREB Activation in Human Airway Epithelial Cells[END_REF]. IL-8-induced binding of RNA-binding proteins to the 3-untranslated region of MUC5AC is a potential mechanism for regulating MUC5AC gene expression at the posttranscriptional level [START_REF] Bautista | IL-8 Regulates Mucin Gene Expression at the Posttranscriptional Level in Lung Epithelial Cells[END_REF]. Several studies have shown that PMA induces a matrix metalloproteasemediated release of transforming growth factor- [START_REF] Shao | Tumor necrosis factor -converting enzyme mediates MUC5AC mucin expression in cultured human airway epithelial cells[END_REF]. Eicosanoids mediate inflammation and mucus secretion in chronic pulmonary inflammatory diseases [START_REF] Garcia-Verdugo | A role for 12R-lipoxygenase in MUC5AC expression by respiratory epithelial cells[END_REF]. Some studies in the field have shown a substantial increase of eicosanoid levels, including PGE2 and LTB4 in CF airways [START_REF] Bautista | IL-8 Regulates Mucin Gene Expression at the Posttranscriptional Level in Lung Epithelial Cells[END_REF] and CF bronchial epithelial cells (BECs) stimulated by LPS from P. aeruginosa [START_REF] Medjane | Impact of CFTR DeltaF508 mutation on prostaglandin E2 production and type IIA phospholipase A2 expression by pulmonary epithelial cells[END_REF]. On the other hand, this bacterium stimulates mucus production through the induction of several mucins such as MUC5AC and MUC2 both in cultured BECs and in a mouse model of lung infection by P. aeruginosa. This induction mainly involves the stimulation of BECs by flagellin through the TLR5 and Naip pathways and is accompanied by the secretion of IL-8 by BECs, which amplify mucus production [START_REF] Mohamed | A Crucial Role of Flagellin in the Induction of Airway Mucus Production by Pseudomonas aeruginosa[END_REF].

Thus, we can suggest that in CF airways, mucus abnormalities offer a niche that favors bacterial infections, which in turn amplify mucus accumulation via a vicious circle that can participate in the exacerbation of the severity of CF disease. This amplification can occur either directly via virulence factors (such as flagellin and LPS) of infecting bacteria or via cytokines and eicosanoids produced by CF airways during infection.

Proteases and Lipids Imbalance

Current studies on mucolytic agents therapy used in CF have been demonstrated to increase markedly neutrophil elastase (NE) activity in CF sputum. Serine proteases, including NE, cathepsin G, and proteinase 3, are the three most major proteases found in the CF lung. These proteases are not only secreted by BECs, but also by monocytes, lymphocytes, granulocytes, and, more importantly, neutrophils [START_REF] Pelaia | Effects of hydrogen peroxide on MAPK activation, IL-8 production and cell viability in primary cultures of human bronchial epithelial cells[END_REF][START_REF] Hunt | Alpha-1 Antitrypsin-A Target for MicroRNA-Based Therapeutic Development for Cystic Fibrosis[END_REF]. Different approaches have exposed their participation in intracellular and extracellular activities, including inflammation, tissue remodeling, mucin expression, bacterial killing, and neutrophil chemotaxis. NE, as i g n i ficant product of neutrophils granule degranulation, is extensively studied in CF and is implicated in cleavage and inactivation of CFTR protein (Chalmers et al., 2017). Besides, NE also upregulates IL-8 and participates in activating cysteinyl cathepsins and matrix metalloproteases.

In the CF airway, different articles have described the protease and anti-protease imbalance, which could be explained by two different mechanisms [START_REF] Galli | Fibrosis, Oxidative stress and antioxidant therapy in cystic fibrosis[END_REF][START_REF] Causer | Circulating biomarkers of antioxidant status and oxidative stress in people with cystic fibrosis: A systematic review and meta-analysis[END_REF]. Firstly, CFTR is also a transporter of glutathione (GSH), a protease that is the main non-enzymatic antioxidant present in the ASL [START_REF] Rahman | Oxidative stress and regulation of glutathione in lung inflammation[END_REF]. Antioxidants are an essential protective response to tissue injury and occur mainly in an inflammatory environment. An absence of GSH in the extracellular medium disequilibrates this balance and induces an oxidative environment. This environment is intended to fight bacteria and viruses that may bepresent.Thegoalofthisprocessistobreakupandeliminatethe injured tissues and, thus, promote tissue repair for the inflammatory process resolution. When this natural response arises in an uncontrolled way, the outcome is extreme tissue damage that could induce chronic inflammation, as observed in CF (Figure 1). During inflammation, reactive oxygen species (ROS) such as the superoxide anion are liberated by phagocytes and are t h ou gh ttobet h em a inc a u s eoft is s u ed a m a ge .

In CF, the presence of numerous inflammatory cells that release many oxidants will have a significant role in the deregulation of the pro-and anti-inflammatory balance. Lung cells are vulnerable to the damaging effects of ROS and release inflammatory mediators, thereby amplifying lung inflammation. ROS are extraordinarily reactive, and when produced near the cell membranes, they diminish intracellular GSH and cause lipid peroxidation, which may harshly disrupt its function and may lead to cell death or DNA damage in alveolar epithelial cells. So, when ROS production increases, the redox balance of the airways is altered, and this can lead to bronchial hyperactivity and to further inflammation and participates in CF comorbidity. GSH is a sulfhydryl containing tripeptide (L-gglutamyl-L-cysteinyl-glycine) that scavenges oxidants and could, therefore, participate in the control of the inflammatory process by reducing oxidative stress [START_REF] Rahman | Oxidative stress and regulation of glutathione in lung inflammation[END_REF][START_REF] Ehre | An Improved Inhaled Mucolytic to Treat Airway Mucoobstructive Diseases[END_REF]. Therefore, a CFTR deficiency leads to an increased accumulation of intracellular GSH in the epithelial lining fluid compared with plasma. Secondly, different dysregulated parameters such as infection, inflammation, and hypoxia, increase the free radicals derived from oxygen and nitrogen. This pro-oxidative environment may directly exert its effects by activating transcription factors such as NFkB and MAP kinase pathways responsible for the coordinated expression of numerous genes involved in inflammation, cell death, proliferation, as well as cytoprotection and antioxidant defenses [START_REF] Pelaia | Effects of hydrogen peroxide on MAPK activation, IL-8 production and cell viability in primary cultures of human bronchial epithelial cells[END_REF].

CFTR-deficient tracheal epithelial cells are characterized by high GSH levels that decrease the intracellular content of ceramide (CER). CER deficiency occurring in CF seems to be responsible for the increased activation of the pro-inflammatory transcriptional nuclear factor NFkB that, in turn, is responsible for the abnormally high inflammatory response in CF respiratory epithelial cells [START_REF] Vilela | Inhibition of IL-8 release from CFTR-deficient lung epithelial cells following pre-treatment with fenretinide[END_REF][START_REF] Aureli | Unravelling the role of sphingolipids in cystic fibrosis lung disease[END_REF]). An increasing number of studies indicate that sphingolipids play an important regulatory role in CF concerning pulmonary inflammation. In different models, it has been shown that de novo sphingolipid synthesis is an inflammation responsive pathway. It is enhanced by inflammatory mediators, both at transcriptional and enzyme activity level, and the accumulation of its metabolite CER potentiates inflammation in a vicious circle [START_REF] Caretti | Anti-inflammatory action of lipid nanocarrier-delivered myriocin: therapeutic potential in cystic fibrosis[END_REF]. Sphingosine-1-phosphate (S1P), generated in the nucleus by phosphorylation of SphK2 ((Sphingosine Kinase 2), modulates HDAC (histone deacetylases) activity either by direct binding or through activation of nuclear ROS, and, regulates cell cycle and pro-inflammatory gene expression [START_REF] Fu | Nuclear lipid mediators: Role of nuclear sphingolipids and sphingosine-1-phosphate signaling in epigenetic regulation of inflammation and gene expression[END_REF]. The accumulation of CER causes Cftr -deficient mice to suffer from constitutive age-dependent pulmonary inflammation, death of respiratory epithelial cells, deposits of DNA in bronchi, and high susceptibility to severe P. aeruginosa infections [START_REF] Teichgräber | Ceramide accumulation mediates inflammation, cell death and infection susceptibility in cystic fibrosis[END_REF]. Aggregates accrual, formed by misfolded mutant CFTR and a miscellaneous of sequestered proteins within, induces inflammation and oxidative stress, impairing proteins and lipids transport, and consequently inflammatory statement (Mingione et al., 2020).

HISTORY OF "CLASSICAL" ANTI-INFLAMMATORY DRUGS

A better understanding of the molecular mechanisms involved in inflammation has led to the development of new antiinflammatory therapeutic strategies. In CF, the intertwining of inflammation, infection, and airway mucus obstruction complicates therapeutic approaches. Thus, anti-inflammatory treatments, combined with antibiotic therapies and airway clearance techniques, play an essential role in patient care, particularly during periods of exacerbations and hospitalization.

Steroid Anti-Inflammatory Treatments

Glucocorticoids (GC), a class of corticosteroids (CS), are potent anti-inflammatory molecules frequently applied in the treatment of "inflammatory" pulmonary diseases. GC target many of the proteins involved in inflammation, including IL-1b and IL-8 and NFkB and activator protein (AP-1) [START_REF] Tabary | Selective up-regulation of chemokine IL-8 expression in cystic fibrosis bronchial gland cells in vivo and in vitro[END_REF][START_REF] Barnes | Transcription factors in airway diseases[END_REF]. Until recently, CS were the main anti-inflammatory CF treatments and were mainly used during exacerbations through inhaled or oral administrations (Balfour-Lynn and Welch, 2014; [START_REF] Lands | Oral non-steroidal anti-inflammatory drug therapy for lung disease in cystic fibrosis[END_REF].

Since the first Prednisone clinical trials [START_REF] Auerbach | Alternate-day prednisone reduces morbidity and improves pulmonary function in cystic fibrosis[END_REF], oral CS have been shown to diminish the lung inflammation and reduce the development of the pathology in CF patients. However, the use of CS is still controversial in the CF context due to medium-and long-term use. The side effects include growth impairment, cataract formation, glucose intolerance, and osteoporosis (Balfour-Lynn and Welch, 2014). Nonetheless, oral CS are promptly used during an exacerbation to decrease inflammation in CF lungs.

Even though the use of GC in CF is common, the signaling pathways remain partially described. Interestingly, we have published that the NFkB signaling pathway was significantly involved and refractory to the action of GC in glandular epithelial cells [START_REF] Tabary | Selective up-regulation of chemokine IL-8 expression in cystic fibrosis bronchial gland cells in vivo and in vitro[END_REF]. Moreover, we have confirmed these results in airway neutrophils from CF patients [START_REF] Corvol | Distinct cytokine production by lung and blood neutrophils from children with cystic fibrosis[END_REF].

Even though inhaled CS have a better safety profile, their efficacy has not yet been demonstrated (Balfour-Lynn and Welch, 2014). The inhaled steroids withdrawal impact was established in a multicentric randomized, double-blind placebo-controlled trial, including CF children and adults [START_REF] Balfour-Lynn | Multicenter randomized controlled trial of withdrawal of inhaled corticosteroids in cystic fibrosis[END_REF]. This study failed to show any beneficial effect of inhaled CS in CF patients treated for six months.

Finally, GC remain interesting molecules, especially during exacerbations, as they significantly reduce inflammation. However, their use in CF can only be limited to specific cases.

Non-Steroid Anti-Inflammatory Treatments

As GC have significant side effects, alternative molecules have been proposed. For a few years, Ibuprofen, a non-steroidal antiinflammatory drug (NSAID), has emerged and was proposed to the CF patients as a GC alternative. Most of NSAID (such as Aspirin) are known to block cyclo-oxygenase (COX) enzymes that produce prostaglandins from free arachidonic acid [START_REF] Kurumbail | Structural basis for selective inhibition of cyclooxygenase-2 by anti-inflammatory agents[END_REF]. Ibuprofen, discovered by Stewart Adams laboratory in 1961, was sold initially as Brufen to treat rheumatoid arthritis [START_REF] Balfour-Lynn | Multicenter randomized controlled trial of withdrawal of inhaled corticosteroids in cystic fibrosis[END_REF][START_REF] Halford | 50th anniversary of the discovery of ibuprofen: an interview with Dr Stewart Adams[END_REF]. In CF, Ibuprofen acts directly on neutrophil activation, inhibiting their mobility and recruitment in the airways [START_REF] Konstan | Effect of ibuprofen on neutrophil migration in vivo in cystic fibrosis and healthy subjects[END_REF]. High-dose of Ibuprofen can reduce the development of CF patients' lung disease, especially in children [START_REF] Lands | Oral non-steroidal anti-inflammatory drug therapy for cystic fibrosis[END_REF][START_REF] Lands | Oral non-steroidal anti-inflammatory drug therapy for lung disease in cystic fibrosis[END_REF]. A meta-analysis from a current update of a regular review has been published on the Cochrane database [START_REF] Lands | Oral non-steroidal anti-inflammatory drug therapy for lung disease in cystic fibrosis[END_REF]. Multiple unwanted effects were a matter of concern due to the high doses usage, which has limited the Ibuprofen use in CF. Recent results have described that obvious benefits of Ibuprofen therapy outbalance the low risk of gastrointestinal bleeding, although long-term safety results are limited. In low doses, some shreds of evidence indicate that Ibuprofen may cause inflammation [START_REF] Lands | Oral non-steroidal anti-inflammatory drug therapy for lung disease in cystic fibrosis[END_REF]. Nonetheless, these outcomes are still a subject of debate among scientists who suspect the inappropriate use of Ibuprofen for CF patients [START_REF] Lands | Oral non-steroidal anti-inflammatory drug therapy for lung disease in cystic fibrosis[END_REF]. The association of Ibuprofen with infections is more complicated in that it confers risk in some situations but benefits in others, therefore its usage might require close monitoring [START_REF] Varrassi | Ibuprofen Safety at the Golden Anniversary: Are all NSAIDs the Same? A Narrative Review[END_REF].

Macrolides

Among the most exciting new anti-inflammatory drug treatments established in the last few years in the CF context the macrolides [START_REF] Southern | Macrolide antibiotics for cystic fibrosis[END_REF]. Macrolides were discovered in 1952 and were initially isolated from cultures of Streptomyces erythraea. The frequently used macrolides have 14 (Clarithromycin, Erythromycin, and Roxithromycin) or 15 (Azithromycin) atoms attached to their macrocyclic rings and were named macrolides in regards to the presence of macrocyclic lactone ring. Macrolides are interesting original antibiotics because of their double action of not only reducing infections but also reducing inflammation. The macrolides were used as antibiotics to treat different infectious diseases, including numerous airway pathology as pneumonia, CF, bronchitis, pharyngitis [START_REF] Zalewska-Kaszubska | Anti-inflammatory capabilities of macrolides[END_REF]. Surprisingly, in 1987, a Japanese group has reported a spectacular effect in panbronchiolitis patients' lifespan when treated with Erythromycin antibiotic [START_REF] Kudoh | Clinical effects of low-dose long-term erythromycin chemotherapy on diffuse panbronchiolitis[END_REF]. This pathology is a typical and representative disease of chronic respiratory tract infection in Japan, characterized by chronic inflammation localized predominantly in the respiratory bronchioles with inflammatory cells such as monocytes, macrophages, neutrophils and, T lymphocytes.

The molecule showing the most interesting effects in CF patients is Azithromycin, with an improvement of lung parameters, a decrease of P. aeruginosa infection, and hospitalization duration (C l e m e n te ta l . ,2 0 0 6 ; [START_REF] Saiman | Effect of azithromycin on pulmonary function in patients with cystic fibrosis uninfected with Pseudomonas aeruginosa: a randomized controlled trial[END_REF][START_REF] Nichols | Pulmonary Outcomes Associated with Long-Term Azithromycin Therapy in Cystic Fibrosis[END_REF]. Prolonged use of small dose Azithromycin was related to a beneficial impact on lung disease expression, well ahead of P. aeruginosa infection. A metanalysis of these researches proved substantial improvement or maintenance of the forced expiratory volume in one second (FEV1, a measure of lung function) and forced vital capacity (FVC) in treated patients vs. controls after 12 months of therapy. Even though there was no decline in the intravenous antibiotic therapy necessity or the hospitalization duration of any of these studies, a positive effect on the restoration of Cl -efflux in CF has also been shown [START_REF] Saint-Criq | Restoration of chloride efflux by azithromycin in airway epithelial cells of cystic fibrosis patients[END_REF].

Moreover, some scientists demonstrated that macrolides operate by limiting pro-inflammatory cytokines and provoking direct alterations in the neutrophils function [START_REF] Equi | Long term azithromycin in children with cystic fibrosis: a randomised, placebo-controlled crossover trial[END_REF][START_REF] Southern | Azithromycin for cystic fibrosis[END_REF][START_REF] Haydar | Azithromycin Polarizes Macrophages to an M2 Phenotype via Inhibition of the STAT1 and NF-kB Signaling Pathways[END_REF]. However, they failed to reduce the inflammation in BECs in CF patients (Saint-Criq et al., 2012). One recently published article has demonstrated that Azithromycin could modify the M2 phenotype macrophage and, therefore, indirectly modify the inflammatory process by inhibiting NFkBa c t i v a t i o nb y increasing IKKb expression in J774 murine macrophages [START_REF] Haydar | Azithromycin Polarizes Macrophages to an M2 Phenotype via Inhibition of the STAT1 and NF-kB Signaling Pathways[END_REF].

However, some macrolides, such as Clarithromycin, can induce neutrophil extracellular trap (NET) generation, a mechanism implicated in innate immunity and some inflammatory processes. NETosis is a mechanism by which neutrophils extrude their DNA and protein contents to form NET, including AMPs. The physiology and the formation of the NET have been extensively described in the review from [START_REF] Ravindran | Neutrophil Extracellular Trap Formation[END_REF]. In the fetal stage and early childhood, neutrophilic inflammation in the peri-bronchial regions is present in CF patients who have mucus excess and obstructive secretions but no persistent bacterial infections. Various microbial components like inflammatory cytokines, lipid mediators, and extracellular DNA found in CF patients induce NET formation [START_REF] Henke | Mucolytics in cystic fibrosis[END_REF]. In CF airways, neutrophils are recruited to the airway upon infection and exacerbate the disease by producing NETs, which can increase mucus viscosity and consequently participate in the airway obstruction. The excess of NETs and their cytotoxic components, associated with hypervisquous mucus, exacerbate CF NET produced by Clarithromycin and inhibit Acinetobacter baumannii infection by acting on its growth and biofilm formation in an LL-37-dependent manner [START_REF] Konstantinidis | Immunomodulatory Role of Clarithromycin in Acinetobacter baumannii Infection via Formation of Neutrophil Extracellular Traps[END_REF][START_REF] Khan | Progression of Cystic Fibrosis Lung Disease from Childhood to Adulthood: Neutrophils, Neutrophil Extracellular Trap (NET) Formation, and NET Degradation[END_REF]. Clarithromycin also enhances the antibacterial defense of fibroblasts and improves their wound healing capacity through the upregulation of LL-37 on NET structures [START_REF] Arampatzioglou | Clarithromycin Enhances the Antibacterial Activity and Wound Healing Capacity in Type 2 Diabetes Mellitus by Increasing LL-37 Load on Neutrophil Extracellular Traps[END_REF]. Although Azithromycin and Chloramphenicol show that neutrophils pretreatment with these macrolides decreases the NETs release. Moreover, Azithromycin showed a concentration-dependent effect on respiratory burst in neutrophils, whereas Chloramphenicol did not affect degranulation, apoptosis or respiratory burst. So, these antibiotics modulate the ability of neutrophils to release NETs influencing human innate immunity [START_REF] Bystrzycka | Azithromycin and Chloramphenicol Diminish Neutrophil Extracellular Traps (NETs) Release[END_REF]. The macrolide immunomodulatory role depends on the macrolide used and the pathology involved.

As a final point, conventional anti-inflammatory treatments for CF are limited and have not been explicitly developed for this pathology, and could induce counterproductive effects. Research in this field is still limited compared to antibiotics, but despite this, new molecules or strategies are being evaluated.

NOVEL ANTI-INFLAMMATORY APPROACHES

Better insight into the pathways involved has led to the development of new therapeutic approaches that are currently being evaluated under cell experiments or clinical trials. These new strategies aiming at the CF inflammation are designed to treat different dysregulated aspects such as channel modulators, oxidative stress, cytokines secretion, lung remodeling, and the regulation of dysregulated pathways.

New Channel Modulators

CFTR Channel

The discovery of the CFTR gene in 1989 resulted in insights on how CFTR mutations induce CF pathology and encouraged many researchers to develop new drugs or strategies to correct the mutation or increase the protein activity (Riordan et al., 1989). Genetic therapy using adeno-associated virus (AAV) or other strategies aiming to correct the CFTR gene was very promising because CF is a monogenic disease. Nonetheless, the subsequent realization tempered expectations because the airways are well defended and are not absorptive surfaces. The natural barrier of mucus considerably impairs gene transfer into the lungs, and the epithelium renewing necessitates numerous administrations. For these reasons, only one study has demonstrated a significant but moderate effect on CF patients. Thus, further optimizations or other strategies are needed and in progress [START_REF] Alton | Repeated nebulisation of non-viral CFTR gene therapy in patients with cystic fibrosis: a randomised, double-blind, placebocontrolled, phase 2b trial[END_REF][START_REF] Alton | Preparation for a first-in-man lentivirus trial in patients with cystic fibrosis[END_REF].

These data provided the grounds for pharmacologic modulations of chloride transport, by targeting mutant CFTR and/or alternative ion channels as anoctamin-1 (ANO1) that can compensate for CFTR malfunction. This excitement has now proven to be warranted because numerous new therapies approved by the FDA or EMA are now either in the pipeline or available for CF patients (Figure 5). This finding contributes to the innovation of genetic disease pharmacotherapy with Vertex Pharmaceuticals as a leader in the CF research field. Fundamental CF research has set the stage for a better molecular understanding of CFTR mutations by supplying structural pieces of information to design new approaches for the pharmacology dynamic even if the different drugs proposed were obtained by high-throughput screening [START_REF] Callebaut | Molecular modelling and molecular dynamics of CFTR[END_REF]. Till date, two CFTR-directed molecule classes have been developed: "potentiator" compounds increasing mutated CFTR activity at the cell surface and, "corrector" drugs improving altered protein processing and trafficking to the cell surface (Wainwright et al., 2015;Rowe et al., 2017;[START_REF] Davies | VX-659-Tezacaftor-Ivacaftor in Patients with Cystic Fibrosis and One or Two Phe508del Alleles[END_REF](Figure 6). The first generation of the compounds has either been limited to a few patients with specific mutations (Ivacaftor) or was addressed to a larger group (Orkambi) and demonstrated moderate effects in CF [START_REF] Mayer | Lumacaftor-ivacaftor (Orkambi) for cystic fibrosis: behind the 'breakthrough[END_REF]. For this reason, the U.K. National Institute for Health and Care Excellence (NICE) issued a draft guidance against recommending Orkambi. Recently, the FDA has approved an auspicious combination of molecules (Elexacaftor-Tezacaftor-Ivacaftor called Trikafta) to restore the function of p.Phe508del CFTR protein in CF patients even if patients had a single p.Phe508del allele. The combination of drugs relative to the control resulted in a percentage of predicted FEV1 that was more than 14 points higher and a rate of pulmonary exacerbations that was 60% lower through 24 weeks of treatment [START_REF] Keating | VX-445-Tezacaftor-Ivacaftor in Patients with Cystic Fibrosis and One or Two Phe508del Alleles[END_REF]Middleton et al., 2019). Unfortunately, a few pieces of information are available for the inflammatory aspects of these treatments. Although, recent evidence showed that the inflammation and lung status hampers these medications and can hinder their effects. Only one article has demonstrated that CFTR modulators can reduce excessive pro-inflammatory response following LPS (lipopolysaccharide) stimulation of CF monocytes [START_REF] Jarosz-Griffiths | Different CFTR modulator combinations downregulate inflammation differently in cystic fibrosis[END_REF]. Moreover, in this article, the authors have also demonstrated that IL-8, IL-1b, and TNF-a (Tumor necrosis factor-a) decreased significantly in the serum of CF patients treated with Ivacaftor and Tezacaftor treatment. It is not known whether the observed effects are due to the restoration of Cl -efflux, GSH (glutathione), or CFTR protein interactions present at the membrane.

Other new classes of mutation are in development, such as CFTR amplifiers, CFTR stabilizers, and read-through agents

Mitri et al.

Anti-Inflammatory Approaches in Cystic Fibrosis (Figure 6). CFTR amplifiers upregulate the expression, and indirectly, the activity of mutant CFTR. PTI-428 and PTI-CH are the two amplifiers who seem promising in pre-clinical and clinical studies. PTI-428 can enhance lung function in CF patients receiving Orkambi with no significant adverse effects. CFTR stabilizer as Cavosonstat inhibits the enzyme that is involved in regulating how much CFTR protein is p r e s e n ta tt h ec e l ls u r f a c e [START_REF] Donaldson | Pharmacokinetics and safety of cavosonstat (N91115) in healthy and cystic fibrosis adults homozygous for F508DEL-CFTR[END_REF]. It could potentially increase the benefits of other medications that target the CFTR function. Read-through drugs can help the ribosome skip over the early stop sequence in order to read the mRNA remaining information and generate CFTR protein.

These therapies may be of interest to class I mutations where there is no production of mRNA or CFTR protein.

Ataluren was developed as a potential treatment for these mutations, but its development was terminated due to failed clinical trial outcomes [START_REF] Shoseyov | Ataluren for the treatment of cystic fibrosis[END_REF]. This approach needs to be completed in the future evaluation of CF trials to understand the effects better and investigate the mechanism complex. It can be assumed that earlier treatment using these drugs may avoid structural damages and give rise to more efficient and prolonged results. We can imagine that the improvement of various dysregulated parameters will have longterm effects on the inflammation present in CF patients, even if indirectly. A recent article has highlighted that by Tobramycin or the AMP, 6K-F17 could restore the effects of Orkambi on p.Phe508del-CFTR protein, suggesting a significant role of i n f e c t i o ni nt h eC Fp a t h o l o g y [START_REF] Laselva | Anti-Infectives Restore ORKAMBI((R)) Rescue of F508del-CFTR Function in Human Bronchial Epithelial Cells Infected with Clinical Strains of P. aeruginosa[END_REF]. Furthermore, using this approach, they have demonstrated that the active AMP can down-regulate the expression of pro-inflammatory cytokines like IL-8, IL-6, and TNF-a in p.Phe508del-CFTR human BECs [START_REF] Laselva | Anti-Infectives Restore ORKAMBI((R)) Rescue of F508del-CFTR Function in Human Bronchial Epithelial Cells Infected with Clinical Strains of P. aeruginosa[END_REF]. Some exciting improvements in chloride efflux have been demonstrated using Sildenafil, a phosphodiesterase type 5 (PDE5) inhibitor. This drug recues p.Phe508del-CFTR trafficking in vitro experiments and decreases sputum elastase activity and, consequently, the inflammatory process [START_REF] Lubamba | Inhaled phosphodiesterase type 5 inhibitors restore chloride transport in cystic fibrosis mice[END_REF][START_REF] Taylor-Cousar | Pharmacokinetics and tolerability of oral sildenafil in adults with cystic fibrosis lung disease[END_REF]. In parallel to Vertex's studies, many other companies are interested in similar approaches to develop CFTR modulators that either restore the CFTR protein to the membrane or activate it (Figure 5). This research work has been essential over the last ten years, and many other molecules are currently being evaluated and at a different stage.

More recently, another promising strategy has been proposed to modulate post-transcriptionally activity of CFTR regulated by acting through miRNA. Distinct groups have proved that wildtype and mutated p.Phe508del human CFTR is regulated by miR-101-3p, miR-145-5p, miR-223-3p, miR-494-3p,and miR-509-3p (Glasgow et al., 2018). The approaches to inhibit the effect of these miRNAs have demonstrated an increase in CFTR protein expression and activity in BECs (De Santi et al., 2020). This approach is exciting, but further researches are needed to understand the subtility of this regulation better.

ENaC Channel

Since CFTR negatively regulates the activity of the ENaC sodium channel, different strategies have been proposed to decrease its activity. The first proposed molecule was Amiloride, which acts as a potassium-sparing diuretic, showing some benefit in both animal studies and clinical trials. Unfortunately, its efficacy was limited due to its short half-life [START_REF] Zhou | Preventive but not late amiloride therapy reduces morbidity and mortality of lung disease in betaENaC-overexpressing mice[END_REF]. This approach was repeated with the use of a new ENaC blocker called AZD5634 from AstraZeneca and BI1265162 from Boehringer Ingelheim. A phase Ib study and a phase II study to test, respectively, the safety and effectiveness of AZD5634 and BI 1265162 are underway in CF adults. Nowadays, a more recent and exciting approach, using aerosol antisense oligonucleotide (ASO) targeting ENaC mRNA (Ionis ENAC 2.5Rx), has demonstrated some interesting and impressive results on mice by restoring inflammation and inhibiting ENaC activity (Crosby et al., 2017). A first clinical study with this therapy is currently ongoing.

In the same way, Arrowhead asks to open a phase I/II trial into inhaled small interference RNA (iRNA) therapy. The drug, called ARO-ENaC, is an investigational RNA therapy designed to lower the production of the epithelial sodium channel alpha subunit (aENaC) in the lungs of CF patients. ARO-ENaC is an iRNA molecule intending to block the production of ENaC channels. It works by targeting and destroying the aENaC mRNA molecules, which are genetic messengers that carry the necessary information for making aENaC proteins and consequently ENaC activity.

ANO1 Channel

Since functional CFTR rescue remains limited, with mutationdependent effects, alternative strategies have been suggested to compensate for the CFTR deficiency and were proposed as a potential CF therapeutic target. Such a strategy was the stimulation of calcium-activated chloride channels (CaCCs) such as the Anoctamin 1 channel (ANO1) (Figure 3). In the nineties, Knowles et al. have discovered that adenosine '5'triphosphate and uridine-5'-triphosphate stimulated Cl - secretion in both standards andC Fr e s p i r a t o r ye p i t h e l i a l , offering a potential by-pass mechanism for defective CFTR (Knowles et al., 1991). These activators transduce a signal through P2Y2 receptors that lead to the release of intracellular calcium and activate the CaCCs. An analog called Denufosol was developed. Different studies have demonstrated that this drug can increase Cl -secretion through a CaCC, inhibit sodium absorption via the epithelial sodium channel called ENaC, and stimulate epithelial ciliary beat frequency (Accurso et al., 2011). Based on these data, 'Denufosol' clinical trials begun in 2001 using a wet nebulization direct airway delivery approach. Unfortunately, the last phase III had failed to demonstrate any benefit, and the project was dropped, but the idea of developing this approach remained (Moss, 2013). At the time of this study, CaCCs were poorly known. Their identity remained elusive for over 20 years until 2008 (Nilius and[START_REF] Nilius | Amazing chloride channels: an overview[END_REF]Caputo et al., 2008;Schroeder et al., 2008;Yang et al., 2008). When ANO1, the principal CaCC present in the airways, was identified in 2008, it allowed for more targeted approaches. Attractively, ANO1 channel has, at the apical membrane of epithelial cells, the same expression pattern as CFTR channels, and this protein was shown to be essential in the activity of CFTR as a chloride channel (Benedetto et al., 2017;Benedetto et al., 2019). Besides, ANO1 is implicated in HCO 3 different permeability, proliferation, wound healing, inflammation, and its expression decreased in CF patients (Veit et al., 2012;Jung et al., 2013;Ruffin et al., 2013). Moreover, a recent article highlighted that ANO1 inhibition decreased ASL height. The authors have also demonstrated that ANO1 is not required for MUC5AC expression, the main protein of the mucus (Simoes et al., 2019) .F o rt h i sr e a s o n ,an o v e lA N O 1p o t e n t i a t o rw a s developed (ETX001), and airway epithelial function and mucus transport were evaluated in the human cells and animal models. This approach confirmed previous results and demonstrated that this drug could increase epithelial fluid secretion and enhance mucus clearance (Danahay et al., 2020).

Recently, our group has proposed a particular strategy using an ASO specific to ANO1 to reestablish ANO1 expression in the context of CF. This strategy "hijacks" the miRNA regulatory system and allows highly targeted effects. We have demonstrated that ASO-ANO1 could be used to inhibit the fixation of miR-9 on ANO1 mRNA by a target site blocker, and consequently to activate the alternative chloride channel to compensate CFTR Cl - deficiency regardless of the mutation (Sonneville et al., 2017). We have also shown that with this strategy, we can improve tissue repair on cell lines but also on CF primary patient cells. We have likewise demonstrated that with this approach, we can activate mucociliary clearance on primary cells but also CF mice. Although we have not studied the effects of ANO1 modulation of inflammation, preliminary studies have already shown that activating ANO1 limits the secretion of IL-8 (Veit et al., 2012).

Novel Anti-Cytokines Approaches

A pathophysiology pulmonary characteristic of CF is a severe neutrophil accumulation, which is correlated with high levels of pro-inflammatory cytokines (IL-8, IL-6, TNF-a), and low levels of anti-inflammatory mediators like IL-10 (Jacquot et al., 2008b). For numerous years, different approaches, as curcumin or vitamin D, have been proposed to limit IL-8 secretion and neutrophils influx [START_REF] Gaggar | The role of matrix metalloproteinases in cystic fibrosis lung disease[END_REF][START_REF] Olszowiec-Chlebna | Vitamin D inhibits pro-inflammatory cytokines in the airways of cystic fibrosis patients infected by Pseudomonas aeruginosa-pilot study[END_REF]. Some pre-clinical data have demonstrated that using antibodies, like antibodies directed against intercellular adhesion molecule (ICAM)-1 and IL-8, could be a promising target. The most advanced therapy using SB-656933, an oral CXCR2 antagonist, was already tested in CF patients and has demonstrated along with safety some exciting results in the modulation of airway inflammation [START_REF] Moss | Safety and early treatment effects of the CXCR2 antagonist SB-656933 in patients with cystic fibrosis[END_REF]. However, another study using SCH527123 (MK-7123, Navarixin), a CXCR1/2 antagonist, was also attempted in chronic obstructive pulmonary disease (COPD) but was abandoned because of a severe decline in neutrophil number [START_REF] Rennard | CXCR2 Antagonist MK-7123. A Phase 2 Proof-of-Concept Trial for Chronic Obstructive Pulmonary Disease[END_REF]. By contrast, a phase II clinical trial has already been carried out in patients with ulcerative colitis and demonstrated inhibition of ozone-induced airway inflammation in humans [START_REF] Lazaar | SB-656933, a novel CXCR2 selective antagonist, inhibits ex vivo neutrophil activation and ozone-induced airway inflammation in humans[END_REF]. Numerous other modulators of cytokines in the context of CF have been proposed, but only in vitro experiments have been performed [START_REF] Lampronti | beta-Sitosterol Reduces the Expression of Chemotactic Cytokine Genes in Cystic Fibrosis Bronchial Epithelial Cells[END_REF][START_REF] De Fenza | Exploring the effect of chirality on the therapeutic potential of Nalkyl-deoxyiminosugars: anti-inflammatory response to Pseudomonas aeruginosa infections for application in CF lung disease[END_REF]. Cytokine modulation shows that cytokines have a significant role in limiting infections, although these approaches are confusing. A recent publication has highlighted the role of an IL-1 signaling pathway in sterile neutrophilic inflammation and mucus hypersecretion and has suggested that treatment with IL-1 receptor antagonist as Anakinra could be promising to prevent lung inflammation (Balazs and Mall, 2019).

The possibility of increasing gene expression and protein activity by the use of ASO has become more and more promising in the last years. However, long-term efficacy, safe delivery, and side effects of long-term treatment must be evaluated in order to be applied in patients with CF [START_REF] Bardin | Emerging microRNA Therapeutic Approaches for Cystic Fibrosis[END_REF][START_REF] Vencken | Nebulised lipid-polymer hybrid nanoparticles for the delivery of a therapeutic anti-inflammatory microRNA to bronchial epithelial cells[END_REF]. Fabbri et al. have developed this original concept by modulating the IL-8 expression by increasing miR-93 in BECs during P. aeruginosa infection [START_REF] Fabbri | Expression of miR-93 and IL-8 During Pseudomonas aeruginosa Mediated Induction of Pro-Inflammatory Responses[END_REF]. More recent results have highlighted that other miRNA involved in CF pathology, like miR-199a-3p or miR-636, could be targeted to control the CF lung inflammatory process (Bardin et al., 2018a;[START_REF] Bardin | miR-636: A Newly-Identified Actor for the Regulation of Pulmonary Inflammation in Cystic Fibrosis[END_REF]. Other interesting approaches have been performed to modulate the cascade of inflammation targeting NFkB activity by using, for example, Angelicin derived from different angiosperms or Sulindac, an NSAID [START_REF] Rocca | New use for an old drug: COX-independent anti-inflammatory effects of sulindac in models of cystic fibrosis[END_REF]. Unfortunately, these approaches are not specific, and the risk of side effects remains high.

New Development in Antibiotic Approaches

"Synthetic" Antibiotics

In CF, antibiotics are utilized for various applications, such as initial infection prevention, eradication (for early infection), control (for chronic infection), and finally, pulmonary exacerbations treatment. The antibiotics are given in three different primary ways: oral, inhalation, or intravenous. The choice of antibiotics depends on the nature of the pathogen to be eliminated, the age of the patient, and the nature of other pathogens present such as H. influenza, S. aureus,orP. aeruginosa infections.

P. aeruginosa is an opportunistic Gram-negative pathogen and is one of the main reasons for morbidity and mortality in CF and immunosuppressed patients. In order to eradicate new P. aeruginosa infections, antibiotic regimens are now a care standard around the world. Different groups assessed the effectiveness of inhaled Tobramycin, Aztreonam, and Colistin as well as oral Ciprofloxacin in eradicating new P. aeruginosa infection (Waters, 2018;[START_REF] Pang | Antibiotic resistance in Pseudomonas aeruginosa: mechanisms and alternative therapeutic strategies[END_REF], although P. aeruginosa eradication is now much more challenging as a result of its impressive capability to resist antibiotics. These organisms become embedded in an exopolysaccharide biofilm, which protects the organism from phagocytosis and reduces the efficacy of anti-microbial drugs [START_REF] Doring | Prevention of Pseudomonas aeruginosa infection in cystic fibrosis patients[END_REF]. Once this change has occurred, the mucoid P. aeruginosa could acquire multi-drug resistance, and this bacterium is virtually impossible to eradicate [START_REF] Southern | Macrolide antibiotics for cystic fibrosis[END_REF]. If the P. aeruginosa infection cannot be cleared, the affected person is faced with an increased treatment burden, accelerated decline in lung function, increased symptom severity, and increased mortality [START_REF] Nixon | Early airway infection, inflammation, and lung function in cystic fibrosis[END_REF].

Recently, there has been a growing number of "new" antibiotics, of different classes and formulations, for pulmonary infection treatments in CF patients (Waters and Smyth, 2015). In order to limit toxicity and reduce side effects while directly targeting the lungs, many studies took an interest in using aerosols as a method of administration. In this frame of mind, Levoflo x a c i nw a sd e v e l o p e df o rC Fp a t i e n t st ot a r g e tP. aeruginosa infections [START_REF] Chirgwin | Novel Therapeutic Strategies Applied to Pseudomonas aeruginosa Infections in Cystic Fibrosis[END_REF][START_REF] Epps | State of the art in cystic fibrosis pharmacology-Optimization of antimicrobials in the treatment of cystic fibrosis pulmonary exacerbations: I. Anti-methicillin-resistant Staphylococcus aureus (MRSA) antibiotics[END_REF]. This drug, derived from the fluoroquinolone family, inhibits topoisomerases, which is essential for the synthesis of bacterial DNA. In the same way, inhaled Zitreonam is now available to treat P. aeruginosa infections in CF patients. Although its aerosolized formulation was proven to be beneficial, the formulation for intravenous injections induces significant lung inflammation, which has limited its use. Another example of the existing improvement of drugs is Tobramycin, presented as a dry powder. Inhaled tobramycin provides, in less than 5 minutes, a rapid action directly at the site of the lung infection.

In order to increase the efficacy of P. aeruginosa eradication and have a less often resistance development in comparison to the existing "classical" antibiotics, recent P. aeruginosa suggested treatment is the use of a combination of antibiotics and the development of new ones. Also, they can be associated with an alternative strategy such as EDTA (Respirion) or inhaled glycopolymer (SNP113).

Thus, a new carbapenem antibiotic called Doripenem has been developed with wide spectrum activity against bacteria through bacterial cell wall synthesis inhibition. Different authors have shown in vitro that this molecule has more significant activity than other antibiotics of the same family on strains isolated from CF patients [START_REF] Traczewski | In Vitro Activity of Doripenem against Pseudomonas aeruginosa and Burkholderia cepacia Isolates from both Cystic Fibrosis and Non-Cystic Fibrosis Patients[END_REF][START_REF] Riera | Pseudomonas aeruginosa carbapenem resistance mechanisms in Spain: impact on the activity of imipenem, meropenem and doripenem[END_REF]. A clinical phase III study showed that patients infected with P. aeruginosa and treated with Doripenem had higher recovery rates in comparison to Imipenem-treated patients but, no clinical trial with CF patients is in progress [START_REF] Chastre | Efficacy and safety of intravenous infusion of doripenem versus imipenem in ventilator-associated pneumonia: a multicenter, randomized study[END_REF]. In the same way, Plazomicin (a semisynthetic aminoglycoside) and POL7001 (a protein epitope mimetic) came out as an interesting strategy against P. aeruginosa [START_REF] Cigana | Efficacy of the Novel Antibiotic POL7001 in Preclinical Models of Pseudomonas aeruginosa Pneumonia[END_REF]. These drugs have demonstrated in vitro some exciting effects on the multidrug-resistant P. aeruginosa isolated from CF patients [START_REF] Cigana | Efficacy of the Novel Antibiotic POL7001 in Preclinical Models of Pseudomonas aeruginosa Pneumonia[END_REF].

"Natural" Approaches

For many years an original approach using bacteriophages has been advanced. Bacteriophages were discovered in 1915 and can kill bacteria by causing lysis [START_REF] Summers | Bacteriophage Therapy[END_REF]. Bacteriophage therapy was applied extensively in the 1930s and 1940s before antibiotics, and it is still being used in Eastern Europe. Nevertheless, after antibiotics became broadly accessible, phage therapy was renounced in Western countries. Many phages can target P. aeruginosa and have demonstrated some exciting effects on mice by decreasing the bacteria burden in the lungs or preventing infection [START_REF] Morello | Pulmonary Bacteriophage Therapy on Pseudomonas aeruginosa Cystic Fibrosis Strains: First Steps Towards Treatment and Prevention[END_REF]. Even if clinical studies have shown relative effectiveness, treatments using phages remain negligible so far. Various reasons have limited the treatments with bacteriophages. The idea of introducing a living organism into the body is difficult to accept and remains an important psychological barrier. Moreover, early tests showed that the preparation generated impurities and that these preparations were not very stable [START_REF] Morello | Pulmonary Bacteriophage Therapy on Pseudomonas aeruginosa Cystic Fibrosis Strains: First Steps Towards Treatment and Prevention[END_REF]. Although the use of phages in combination with quorum sensing inhibitors seems interesting, this approach remains marginal [START_REF] Pang | Antibiotic resistance in Pseudomonas aeruginosa: mechanisms and alternative therapeutic strategies[END_REF], and only a phase Ib/II trial is planned to test the safety and tolerability of AP-PA02 in adults with CF. AP-PA02 is a type of phage intended to control P. aeruginosa infections in CF patients. In in vitro studies, AP-PA02 can kill more than 80% of P. aeruginosa strains from CF people, and some first results are encouraging (Law et al., 2019).

Another "natural strategy" is inhaled nitric oxide (NO) for which an initial phase II study is underway. NO is a gas derived from nitrogen with anti-microbial properties. Some in vivo studies have validated this approach to eradicate the infection and to decrease mucus viscoelastic [START_REF] Rouillard | Antibiofilm and mucolytic action of nitric oxide delivered via gas or macromolecular donor using in vitro and ex vivo models[END_REF].

In the late 1970s, various studies showed that iron played an essential role in bacterial growth and was involved in particular in DNA replication, energy production, and pathogen-host interaction [START_REF] Payne | The critical role of iron in hostbacterial interactions[END_REF]. Recent results demonstrated that the iron content of human sputum is considerably high in CF, which facilitates chronic infections in the lungs of CF patients [START_REF] Reid | Increased airway iron as a potential factor in the persistence of Pseudomonas aeruginosa infection in cystic fibrosis[END_REF]. These observations resulted in the development of novel therapeutic strategies in order to limit the amount of iron present in the airways. Gallium is a compound that shares the same properties with iron. It has demonstrated in vitro and in vivo anti-Pseudomonas properties [START_REF] Tovar-Garcıá | Characterization of gallium resistance induced in a Pseudomonas aeruginosa cystic fibrosis isolate[END_REF]. The FDA has already approved the intravenous administration of Gallium. Clinical studies, in phase II for intravenous and a phase I for an inhaled strategy, are ongoing to evaluate its efficiency in treating P. aeruginosa infections in CF patients [START_REF] Tovar-Garcıá | Characterization of gallium resistance induced in a Pseudomonas aeruginosa cystic fibrosis isolate[END_REF].

During the last decades, AMPs naturally emerged as a potential therapy to cure infections with antibiotic resistance, in CF included. Treatments of bacterial infections by antibiotics result in a worldwide spread of dissemination of antibiotic resistance, both in the community and clinical settings. Besides, the development of new antibiotics is costly and timeconsuming. It is hence of great importance to note that AMPs can treat methicillin-resistant S. aureus and multidrugresistant P. aeruginosa that are resistant to conventional antibiotics [START_REF] Geitani | Cationic antimicrobial peptides: alternatives and/or adjuvants to antibiotics active against methicillin-resistant Staphylococcus aureus and multidrugresistant Pseudomonas aeruginosa[END_REF]. Studies showed that treatments of antibiotic-resistant bacterial strains with AMPs were associated with almost no induced resistance to AMPs, which may encourage their use as potential replacement therapy for antibiotics. AMPs can exert anti-inflammatory actions either by suppressing the production of pro-inflammatory cytokines or by stimulating that of anti-inflammatory cytokines by host cells (Figure 7). Cathelicidin LL-37 (one of the most studied AMPs) enhances the production of the anti-inflammatory cytokine IL-1R by the human peripheral blood-derived mononuclear cells and macrophages [START_REF] Choi | Human cathelicidin LL-37 and its derivative IG-19 regulate interleukin-32-induced inflammation[END_REF], and similar results were observed with LL-37 and beta-defensin-3 (hBD-3) [START_REF] Mookherjee | Systems biology evaluation of immune responses induced by human host defence peptide LL-37 in mononuclear cells[END_REF][START_REF] Smithrithee | Human b-defensin-3 increases the expression of interleukin-37 through CCR6 in human keratinocytes[END_REF]. Besides their direct actions on host cells involved in the initiation/modulation of inflammation, a number of AMPs, such as LL-37, Magainin-2, and bactericidal-permeability-increasing (BPI), can neutralize the activity of bacterial toxins such as LPS, thus participating in maintaining a balance between pro-and anti-inflammatory cytokines [START_REF] Sun | Inhibitory Effects of Antimicrobial Peptides on Lipopolysaccharide-Induced Inflammation[END_REF][START_REF] Skovbakke | Anti-inflammatory Properties of Antimicrobial Peptides and Peptidomimetics: LPS and LTA Neutralization[END_REF].

Most of the reported studies in the field have focused on the r o l e so fA M P si nt h em o d u l a t i on of cytokine production. However, cytokines are only the tip of the iceberg in the inflammatory process, and other mediators of inflammation, such as eicosanoids, deserve to be investigated to identify their relative role in the modulation of inflammation by AMPs. Indeed, studies have reported that AMPs such as LL-37 modulates the production of eicosanoids, including leukotriene B4 (LTB4) and thromboxane A2 (TXA2) by macrophages [START_REF] Agier | Review paper Cathelicidin impact on inflammatory cells[END_REF]. TXB2 and LTB4 are metabolites of arachidonic acid conversion by COX and lipoxygenase (LOX), respectively, and known to induce platelet aggregation and neutrophils r e c r u i t m e n ta tt h es i t eo fi n f e c t i o n [START_REF] Yeung | 12-lipoxygenase: a potential target for novel anti-platelet therapeutics[END_REF]. It has been shown that LL-37 AMP blocks the expression of pro-inflammatory pathways involved, such as NF-kB in the presence of LPS [START_REF] Agier | Review paper Cathelicidin impact on inflammatory cells[END_REF]. However, further studies are awaited to decipher the importance of the AMPs/eicosanoids network in the inflammatory reaction and potential implication in inflammatory diseases such as COPD, asthma, and CF. Similar anti-inflammatory effects were observed with WALK11.3 (an AMP with amphipathic helical conformation) in the mouse alveolar macrophage cell line RAW264.7 [START_REF] Shim | Anti-Inflammatory Action of an Antimicrobial Model Peptide That Suppresses the TRIF-Dependent Signaling Pathway via Inhibition of Toll-Like Receptor 4 Endocytosis in Lipopolysaccharide-Stimulated Macrophages[END_REF]. They revealed the ability of this peptide to inhibit the expression of several inflammatory mediators, including NO, COX-derived metabolites, IL-1b, IL-6, interferon (IFN)-b,a n dT N F -a (Figure 7). The chicken cathelicidin-2 (CATH-2), the known ortholog of the human LL-37, has been shown to reduce inflammation in parallel to its anti-microbial activity against P. aeruginosa-resistant strains from CF patients [START_REF] Banaschewski | The Antibacterial and Anti-inflammatory Activity of Chicken Cathelicidin-2 combined with Exogenous Surfactant for the Treatment of Cystic Fibrosis-Associated Pathogens[END_REF]. The ability of CATH-2 to downregulate inflammation occurred through the anti-microbial-independent process, as this down-regulation was observed by silencing the inflammatory response that arises from killed bacteria. It is now clear that AMPs play a key role in host defense toward infectious by invading pathogens and represent a potential therapeutic tool to control infections by antibioticresistant bacterial strains. They also have the potential to protect the host from harmful inflammation that may result from these infections. Drug design and structure-relationship studies will greatly improve our knowledge of AMPs and the relative importance of their bactericidal vs anti-inflammatory functions, which will be of great help to optimize their potential therapeutic use in disease characterized by both chronic infection and inflammation such as CF.

All these data suggested that AMPs could be useful for clinical applications in the view of the protective function against pathogens. A series of clinical trials have started mostly in the pediatric population, and some compounds have been used as topical treatments but not known in the CF context. Different AMPs are under evaluation for the treatment of acute skin infection as Bralicidin, Omiganan, LTC109 (phase II clinical trial), or Pexiganam (phase III clinical trial). Other strategies and applications are currently under study. For example, in sepsis, Talactoferrin was tested by systemic injection in phase II clinical study [START_REF] Guntupalli | A phase 2 randomized, double-blind, placebo-controlled study of the safety and efficacy of talactoferrin in patients with severe sepsis[END_REF]. Initial results showed a significant decrease in mortality after 28 days of treatment. However, phase II/III oral Talactoferrin was stopped for problems of safety and efficacy [START_REF] Vincent | Talactoferrin in Severe Sepsis: Results From the Phase II/III Oral tAlactoferrin in Severe sepsIS Trial[END_REF]. In the case of meningococcemia, rBPI21 pre-clinical trial has demonstrated some anti-bacterial and anti-LPS effects. Encouraging results led to the initiation of a phase III study in children with severe meningococcal sepsis [START_REF] Giroir | Bactericidal/permeabilityincreasing protein-lessons learned from the phase III, randomized, clinical trial of rBPI21 for adjunctive treatment of children with severe meningococcemia[END_REF]. The study outcome showed a reduction in complications with a shorter hospitalization also suggests the possibility to treat with rBPI21 other patients, including CF. The therapeutic applications of P. aeruginosa have been summarized in a recent publication [START_REF] Magrone | Antimicrobial Peptides in Human Disease: Therapeutic Approaches. Second of Two Parts[END_REF]). An alternative therapeutic pathway for the use of AMPs has been envisaged by indirectly promoting their expression through the use of natural compounds. Several compounds have been identified as the use of Apigenin to enhance the expression and activity of b-3 defensin and cathelicidin in mice [START_REF] Hou | Topical apigenin improves epidermal permeability barrier homoeostasis in normal murine skin by divergent mechanisms[END_REF]. Similar effects have been observed with vitamin D on in vitro studies to increase b-2 defensins and LL-37 on keratinocytes [START_REF] Kim | The effect of calcipotriol on the expression of human beta defensin-2 and LL-37 in cultured human keratinocytes[END_REF].

The use of natural or synthetic antibiotics can have a significant influence on the emergence of new pathogens. It is well established now that microbiota composition and dynamic impact the host immunity, health, and diseases [START_REF] Belkaid | Role of the Microbiota in Immunity and Inflammation[END_REF]. However, a new concept is now progressively emerging, suggesting that the innate immune response of the host can also modulate, at least in part via AMPs, the microbiota composition. For example, recent studies reported the involvement of sPLA2-IIA in the selection of species in pathologies characterized by polymicrobial infections such as CF. P. aeruginosa is known to progressively colonize CF airways to become the dominant pathogen at later stages of CF. This pathogen induces the production by CF airways of sPLA2-IIA, which in turn eradicate S. aureus, therefore helping in its gradual elimination from CF airways and its substitution by P. aeruginosa [START_REF] Pernet | Pseudomonas aeruginosa eradicates Staphylococcus aureus by manipulating the host immunity[END_REF]. This effect is mostly due to the intrinsic resistance of P. aeruginosa and high susceptibility of S. aureus to sPLA2-IIA, respectively. Finally, it emerges that AMPs represent valid substitutes of antibiotics when a condition of antibiotic resistance is established.

Alternative Strategies

Anti-Proteases

CF "anti-protease therapies" can be separated into two separate groups of drugs: some to increase anti-protease and some to inhibit protease expression. CFTR is an essential apical GSH transporter in the lung, and can indirectly participate in the inflammatory process by reducing oxidative stress. Evidence supporting the occurrence of oxidative stress in CF is established and extensively described [START_REF] Galli | Fibrosis, Oxidative stress and antioxidant therapy in cystic fibrosis[END_REF][START_REF] Causer | Circulating biomarkers of antioxidant status and oxidative stress in people with cystic fibrosis: A systematic review and meta-analysis[END_REF]. Some interesting works have demonstrated that oxidative stress could suppress CFTR expression [START_REF] Cantin | Oxidant stress suppresses CFTR expression[END_REF]. Oxidative stress has a major role in the development of lung pathology in CF children and will, in addition to having a role in lung remodeling, have a role in the pulmonary microbiota [START_REF] Shi | Meta-analysis Reveals Potential Influence of Oxidative Stress on the Airway Microbiomes of Cystic Fibrosis Patients[END_REF]. A recent metanalysis has positively correlated the expression of antioxidants with body mass index and lung function in CF [START_REF] Causer | Circulating biomarkers of antioxidant status and oxidative stress in people with cystic fibrosis: A systematic review and meta-analysis[END_REF]. The malabsorption of nutrients with antioxidants properties in CF, participate in the imbalance in favor of oxidative stress and disrupt redox signaling, and, finally, molecular damages even if some data appears to be conflicting [START_REF] Shamseer | Antioxidant micronutrients for lung disease in cystic fibrosis[END_REF][START_REF] Siwamogsatham | Relationship Between Fat-Soluble Vitamin Supplementation and Blood Concentrations in Adolescent and Adult Patients With Cystic Fibrosis[END_REF]. Therefore, multiple studies have been carried out to check the anti-protease supplementation in CF [START_REF] Galli | Fibrosis, Oxidative stress and antioxidant therapy in cystic fibrosis[END_REF]. Some studies have focused on especially serine proteases via two distinct administration routes: aerosolized and intravenously [START_REF] Mckelvey | Targeting Proteases in Cystic Fibrosis Lung Disease. Paradigms, Progress, and Potential[END_REF]. In CF, exocrine pancreatic insufficiency and reduced bile acids induce critical antioxidants malabsorption, including carotenoids (b-carotene), tocopherols (vitamin E), coenzyme Q10, and selenium. Supplementation of antioxidant micronutrients (vitamin E, C, D, b-carotene, and selenium) may, therefore, potentially help maintain an oxidant-antioxidant balance, and this aspect has been extensively reviewed [START_REF] Sagel | Effect of an antioxidant-rich multivitamin supplement in cystic fibrosis[END_REF][START_REF] Ciofu | Antioxidant supplementation for lung disease in cystic fibrosis[END_REF]. In the same approach, LAU-7b, an oral drug, is a derived form related to vitamin A. This compound can reduce the lung inflammatory response of CF people. In parallel, a phase II clinical study to test the effectiveness and safety of LAU-7b in CF patients is underway [START_REF] Lands | Oral non-steroidal anti-inflammatory drug therapy for lung disease in cystic fibrosis[END_REF]. LAU-7b, also called, Fenretidine, work to increase docosahexaenoic acid (DHA) and consequently CER concentration. Some authors supported that the decrease of CER concentration contributes to the persistent bacterial infection and the constitutive MAP kinases and NFkB activation [START_REF] Guilbault | Fenretinide corrects newly found ceramide deficiency in cystic fibrosis[END_REF][START_REF] Guilbault | Cystic fibrosis fatty acid imbalance is linked to ceramide deficiency and corrected by fenretinide[END_REF].

Human a-1 antitrypsin (A1AT) is still the most studied drug by far. Different clinical trials were already achieved. An inhaled a1-proteinase inhibitor is known to reduce NE burden in some patients with CF. A phase I in non-CF bronchiectasis and an IIa clinical study with purified A1AT products given through inhalation in CF subjects were just finalized and have demonstrated safety and efficacy [START_REF] Gaggar | Inhaled alpha1-proteinase inhibitor therapy in patients with cystic fibrosis[END_REF][START_REF] Watz | Safety and efficacy of the human neutrophil elastase inhibitor BAY 85-8501 for the treatment of non-cystic fibrosis bronchiectasis: A randomized controlled trial[END_REF]. In the conclusion of the second study, the daily a-1 hydrophobic chromatography process delivered for three weeks was safe, well-tolerated, and effective in raising the a1-PI levels in the sputum of subjects with CF. However, the effects were transient and difficult to predict due to the proteases' variability in CF patients' lungs. The administration by airway routeway effectively increased the concentration of A1AT in sputum. The current study was not powered to assess changes in FEV1 or biomarkers in sputum, and further clinical are needed.

In parallel, A1AT gene therapy is emerging. Some recent data have demonstrated encouraging results in the inhibition of miRNA, which targets the A1AT gene called SERPINA1 [START_REF] Hunt | Alpha-1 Antitrypsin-A Target for MicroRNA-Based Therapeutic Development for Cystic Fibrosis[END_REF]. This strategy aims to by-pass protein regulation systems of the most abundant inhibitor of NE in the airways. It is an alternative to the delivery of recombinant by using miRNAtargeted therapies. It was found that dual miRNA and adenoassociated viral (AAV)-based therapy engendered the long-term knockdown of circulating Z-A1AT and could be a new strategy in CF [START_REF] Mueller | Sustained miRNA-mediated knockdown of mutant AAT with simultaneous augmentation of wild-type AAT has minimal effect on global liver miRNA profiles[END_REF]. This approach was fully described in a review published [START_REF] Hunt | Alpha-1 Antitrypsin-A Target for MicroRNA-Based Therapeutic Development for Cystic Fibrosis[END_REF]. The other approach is to directly activate SERPINA1 using gene therapy by using viral vectors like retrovirus or adenovirus, but numerous side effects have been observed [START_REF] Gregory | Implications of the innate immune response to adenovirus and adenoviral vectors[END_REF]. Their use remains challenging, especially in the CF field.

Another strategy proposed is to use serine protease inhibitors such as secretory leukoprotease inhibitor (SLPI) which act locally to maintain a protease/anti-protease balance, thereby preventing protease-mediated tissue destruction. SLPI is a well-characterized member of the trapping gene family of proteins and is produced by respiratory tract epithelial cells and phagocytic neutrophils. Different approaches have been proposed to increase the antiprotease activity by nebulizing SLPI, but the efficacy is currently being evaluated alone or in association with other strategies [START_REF] Mcelvaney | Pharmacokinetics of recombinant secretory leukoprotease inhibitor aerosolized to normals and individuals with cystic fibrosis[END_REF][START_REF] Quabius | SLPI and AnxA2 expression in neoplasm-free palatine tonsils is associated with smoking habit of individuals[END_REF]. Currently, novel protease inhibitor drugs, which have promising interest in the CF context, are in development (DX-890, AZD9668, POL6014, Grifols T6006-201) in order to improve their resistance against inactivation.

Promoting tissue repair represents another strategy by focusing on the proteins involved. Matrix metalloproteinases (MMP) are a group of distinct metalloendopeptidase enzymes that regulate various inflammatory and repair processes. They are either secreted or anchored to the cell surface, and therefore their activity is directed against membrane proteins or extracellular proteins, including inflammatory mediators. In CF patients, different articles have demonstrated that MMP is upregulated in the sputum of patients and is related to tissue damage [START_REF] Delacourt | Imbalance between 95 kDa type IV collagenase and tissue inhibitor of metalloproteinases in sputum of patients with cystic fibrosis[END_REF][START_REF] Gaggar | The role of matrix metalloproteinases in cystic fibrosis lung disease[END_REF]. Various proinflammatory cytokines induce them at the transcription level. They might include the activation of a diverse group of intracellular signaling pathways (such as p38 MAPK or ERK 1/ 2 MAPK), causing the activation of nuclear signaling factors like AP1, NFkB, and STAT (signal transducer and activator of transcription). Activation of MMP can be induced by proteases or oxidants and are controlled by tissue inhibitor of metalloproteases (TIMP). There have been increasing interests in modulating MMP activity to enhance disease outcomes, and different clinical studies are in progress with promising effects in CF. A phase II study with Andecaliximab/GS-5745 in CF adults is in progress and was tolerated in patients with ulcerative colitis or Crohn's disease, and could be an exciting approach to control pulmonary degradation.

The approaches using protease inhibitors are very varied, and many studies are still in progress. Although these therapies have been shown to improve patients' health outcomes, they can only be considered in combination with other therapeutic targets.

Eicosanoids Pathway

Alterations in the metabolism of fatty acids present in membrane lipids may have an essential role in the inflammatory CF pulmonary disease. The arachidonic acid (AA): docosahexaenoic acid (DHA) ratio in blood serum, pulmonary airways, and rectal biopsies are increased in CF patients with either pancreatic sufficiency or pancreatic insufficiency, as compared with healthy control subjects (Freedman et al., 2004). AA is stored in cell membranes and is released from membrane lipids by various PLA2 proteins. Some interesting studies have highlighted the implication of sPLA2 in the pathogenicity of CF mice showing that reduced CFTR expression increased cytosolic PLA2a (cPLA2a) activity. A review has summarized the state of the art of fatty acid metabolism in CF [START_REF] Strandvik | Fatty acid metabolism in cystic fibrosis[END_REF]. These effects improved mucus secretion and accumulation in airway epithelia independent of CFTR chloride transport function [START_REF] Medjane | Impact of CFTR DeltaF508 mutation on prostaglandin E2 production and type IIA phospholipase A2 expression by pulmonary epithelial cells[END_REF][START_REF] Dif | Critical role of cytosolic phospholipase A2{alpha} in bronchial mucus hypersecretion in CFTR-deficient mice[END_REF]. Therefore, cPLA2a hasbeenproposedasan appropriate new target for therapeutic intervention in CF [START_REF] Dif | Critical role of cytosolic phospholipase A2{alpha} in bronchial mucus hypersecretion in CFTR-deficient mice[END_REF]. Small lipid mediators were produced in the course of inflammation resolution and generated varied responses, which are cell types and tissue specific. A large number of these molecules modulate inflammation processes and provide essential functions in chemoattraction, aggregation, and degranulation of inflammatory cells. They are also implicated in tissue and vascular permeability, bronchoconstriction, and mucus production. Some of the lipid mediators include lipoxins (LX), resolvins, protectins, and maresins, which are generated by the activity of lipoxygenases lipoxin A4 (LXA4).

Interestingly, inhibitors of the 12R-lipoxygenase have demonstrated an essential role in mucin expression. The inhibitors decreased MUC5AC mucin expression by the inhibition of the ERK/SP1 dependent mechanism [START_REF] Garcia-Verdugo | A role for 12R-lipoxygenase in MUC5AC expression by respiratory epithelial cells[END_REF]. LXA4 has been described as a significant signal for the inflammation resolution and is generated at a low level in the CF patients' lungs. LXA4 and RvD1 activate a GPCR termed ALX/FPR2. This pro-resolving receptor is recognized by annexin A1, an endogenous anti-inflammatory peptide. A recent article provides evidence that the miR-181b, overexpressed in CF cells, may be considered as a new strategy to decrease the anti-inflammatory process in CF via the normalization of the expression receptordependent LXA4 [START_REF] Pierdomenico | microRNA-181b is increased in cystic fibrosis cells and impairs lipoxin A4 receptor-dependent mechanisms of inflammation resolution and antimicrobial defense[END_REF]. The LXA4 inhalation consequences have been examined in a pilot study of asthmatic and healthy adult subjects. The drug was welltolerated, and no harmful effect was observed [START_REF] Christie | The effects of lipoxin A4 on airway responses in asthmatic subjects[END_REF]. Some impressive results were observed in the topical treatment of infantile eczema [START_REF] Wu | Efficacy and safety of 15(R/S)-methyl-lipoxin A(4) in topical treatment of infantile eczema[END_REF]. Together with data showing beneficial actions of LXA4 in the CF context, these results highlight additional studies to check whether the upregulation of the lipidic mediators' pathway can be considered as an appropriate tactic to fight inflammation in CF patients [START_REF] Higgins | Physiological impact of abnormal lipoxin A(4) production on cystic fibrosis airway epithelium and therapeutic potential[END_REF].

Similarly, the LTB4 produced by resting BECs has been proposed as a target. Inflammatory stimuli increase the production of LTB4 and might also contribute to progressive pulmonary destruction in CF. Bronchial epithelial LTB4 acts as a potent chemoattractant for neutrophils via the cell surface integrins upregulation. When these cells are activated and present at the site of inflammation, they can also participate in the secretion of LTB4. LTB4 synthesis includes lipid peroxidation by 5-lipoxygenase, and produce numerous ROS, and consequently, pro-inflammatory activation. A clinical trial with Montelukast (BIIL 284), a leukotriene receptor agonist, counting a small number of patients, has provided contentious results in CF patients. This therapy has demonstrated a notable decrease in serum eosinophil cationic protein levels and eosinophils without any significant improvement in FEV1, and FEF25-75%. Also, this strategy has shown a significant decrease in cough, serum, and sputum levels of eosinophil cationic protein and IL-8 chemokine. Moreover, an increase in serum and sputum levels of IL-10 has been observed. The trial was stopped early due to a significant increase in the risk of severe pulmonary events in patients receiving the active drug [START_REF] Schmitt-Grohe | Leukotriene receptor antagonists in children with cystic fibrosis lung disease : anti-inflammatory and clinical effects[END_REF]. A more recent drug, Acebilustat (CTX-4430), has been evaluated in CF patients. This drug has shown anti-inflammatory activity via the LTA4 hydrolase inhibition and LTB4 modulation. In two-phase I clinical trials, Acebilustat decreased the production of LTB4 and pro-inflammatory cytokines in healthy volunteers and CF patients, and in phase II, optimal dose and duration were identified for future studies [START_REF] Elborn | Phase I Studies of Acebilustat: Biomarker Response and Safety in Patients with Cystic Fibrosis[END_REF][START_REF] Elborn | EMPIRE-CF: A phase II randomized placebo-controlled trial of oncedaily, oral acebilustat in adult patients with cystic fibrosis -Study design and patient demographics[END_REF].

Cannabinoid-Derived Drug

Ajulemic acid (JBT-101, Lenabasum) is a cannabinoid-derived molecule that preferably binds to the active CB2 receptor and is non-psychoactive. In some pre-clinical trials done on human l u n gc e l l so b t a i n e df r o mC Fp atients, it was shown that Lenabasum stopped the production of both TNF-a and IL-6, two crucial pro-inflammatory cytokines that trigger inflammation. In phase I and II clinical trials, this drug demonstrated favorable safety and tolerability. Recently, a group has also shown significant efficacy in mice models of inflammation and fibrosis [START_REF] Burstein | Ajulemic acid: potential treatment for chronic inflammation[END_REF]. Therefore, phase II was initiated. It will be used to test safety, tolerability, pharmacokinetics, and efficacy of JBT-101 in 70 subjects ≥ 18 and < 65 years of age with documented CF. Treatment of CF patients with Lenabasum twice daily has been able to decrease the number of acute lung exacerbations as well as a reduction of inflammatory cells and mediators present in the sputum. A new clinical trial is undergoing and seeks to enroll more than 400 CF patients over numerous clinical sites.

Mucus Therapies

In the lungs, the abnormal production of mucus has been assumed to participate actively in the early CF pathogenesis [START_REF] Ehre | Cystic fibrosis: an inherited disease affecting mucin-producing organs[END_REF]. For many years, researchers and clinicians have been trying to understand the origin of mucus abnormalities and found mucoactive drugs molecules to control CF bronchial obstruction. Mucoactive drugs are regularly used as a therapeutic option and are defined by their activity as mucolytics, expectorants, and cough facilitating drug. The expectorants, such as hypertonic solution (HSS), increase the ASL layer and decrease mucus adhesiveness. Mucolytics, such as both N-acetylcysteine (NAC) and recombinant human DNase (rhDNase), reduce sputum viscosity. Medications such as inhaled mannitol, rhDNase (Dornase), and hypertonic HSS have proven efficacy in CF and indirectly reduced inflammation in airways of CF patients [START_REF] Tarrant | Mucoactive agents for chronic, non-cystic fibrosis lung disease: A systematic review and meta-analysis[END_REF]. The low volume hypothesis would estimate that approaches increasing the ASL height will increase mucociliary clearance, and consequently reduce lung infection. In order to increase the ASL height and fluidity, an HSS (3 to 7% NaCl) has been proposed to treat CFTR deficiency for better mucociliary clearance. Recently, Wark & McDonald have performed a meta-analysis of 17 different clinical trials of HSS and concluded that, after four weeks, a small enhancement in the lung function was observed but was not sustained at 48 weeks. HSS might also have a little impact on improving life quality in adults [START_REF] Wark | Nebulised hypertonic saline for cystic fibrosis[END_REF]. New clinical trials are in progress in order to establish who may benefit most and whether this benefit is sustained in the longer term (https://www.cff.org/Trials/Finder).

In the same manner, a meta-analysis was performed with mannitol, which is a naturally occurring sugar alcohol. When inhaled mannitol creates a change in the osmotic gradient. It leads to water movement into the CF airway hydrating the ASL, and enhancing mucociliary clearance. In the different studies, there was no evidence showing that the mannitol treatment for over six months is related to an enhancement of lung function in CF patients compared to control [START_REF] Nevitt | Inhaled mannitol for cystic fibrosis[END_REF]. Recently, different groups have observed expression, biochemical and biophysical alterations of the mucous present in the airways of CF patients [START_REF] Rhim | Terminal glycosylation in cystic fibrosis (CF): a review emphasizing the airway epithelial cell[END_REF]. More, they observed that abnormal glycosylation of the airway mucins is associated with bacterial infection and inflammation. The effects of altered host mucin glycosylation affect P. aeruginosa adhesion and so pathogenicity. A review from Ventalakrishan et al. has extensively described this feature [START_REF] Venkatakrishnan | Host mucin glycosylation plays a role in bacterial adhesion in lungs of individuals with cystic fibrosis[END_REF]. Different therapeutic approaches have been proposed to correct this observation by using, for example, mannose-biding lectin, which recognizes bacterial glycoconjugates and participates in an effective defense against pathogens (Moller- [START_REF] Moller-Kristensen | Deficiency of Mannose-Binding Lectin Greatly Increases Susceptibility to Postburn Infection with Pseudomonas aeruginosa[END_REF].

Another strategy used in CF is to disrupt the high DNA content present in the airway mucus of CF patients. DNA is a polyanion compound responsible for the viscosity and adhesiveness of the pulmonary secretions. DNA release and accumulation in ASL occur as a result of tissue destruction caused by inflammatory cells on bacteria and epithelial cells. The strategy is to use a recombinant human deoxyribonuclease I (rhDNase), an enzyme that selectively cleaves DNA, hence decreasing mucus viscosity [START_REF] Puchelle | Physical and functional properties of airway secretions in cystic fibrosis-therapeutic approaches[END_REF]. Nebulized rhDNase hydrolyzes extracellular DNA within the mucus and transforms it from an adhesive gel into a liquid form of fluid through dilution within minutes. In contrast to mannitol or HSS, rhDNase has shown some significant effects on the improvement of lung function of CF patients and is considered as an effective treatment for the liquefaction of viscous mucus in CF. However, individual responses are unpredictable [START_REF] Yang | Dornase alfa for cystic fibrosis[END_REF].

The only approved reducing agent for human use is Nacetylcysteine (NAC), a well-known antioxidant GSH drug. This drug ameliorates the redox imbalance in neutrophils present in the blood and inhibits their recruitment in the airways of CF patients [START_REF] Tirouvanziam | High-dose oral N-acetylcysteine, a glutathione prodrug, modulates inflammation in cystic fibrosis[END_REF]. NAC is also used in CF as an aerosolized mucus solution to break down disulfide bonds between mucin proteins in order to fluidify mucus [START_REF] Duijvestijn | Systematic review of N-acetylcysteine in cystic fibrosis[END_REF]. Some evidence demonstrated that NAC has excellent anti-bacterial properties, the capacity to intervene with biofilm formation and, to disturb the adherence of respiratory pathogens to respiratory epithelial cells [START_REF] Blasi | The effect of N-acetylcysteine on biofilms: Implications for the treatment of respiratory tract infections[END_REF]. In CF patients, NAC has been proven to be safe at large doses with negligible interaction with other drugs. NAC was investigated in CF despite its partial effectiveness as an inhaled mucolytic agent because the extremely oxidizing CF airway environment consumes aerosolized antioxidants quickly [START_REF] Tirouvanziam | High-dose oral N-acetylcysteine, a glutathione prodrug, modulates inflammation in cystic fibrosis[END_REF][START_REF] Cantin | Antioxidants in cystic fibrosis. Conclusions from the CF antioxidant workshop[END_REF]. Finally, inhaled NAC is being used as a mucolytic drug in CF for several decades, although the positive results remain limited. Newer agents targeting other components of CF mucus are currently in development or clinical trials (NAC 40630) and exhibit an exciting effect on mucus [START_REF] Blasi | The effect of N-acetylcysteine on biofilms: Implications for the treatment of respiratory tract infections[END_REF].

Another original approach is undergoing with OligoG CF-5/ 20. OligoG is an alginate oligosaccharide derived from natural seaweed. It is administrated using a dry powder inhaler and also developed as a liquid to use with a nebulizer. Studies have shown that this dry power drug is capable of reducing the mucus thickness in the lungs. In addition, this drug enhances the efficiency of antibiotics and may facilitate mucus clearance in CF patients. The drug could detach CF mucus by calcium chelation [START_REF] Ermund | OligoG CF-5/20 normalizes cystic fibrosis mucus by chelating calcium[END_REF]. Initiated in 2018, phase II includes more than 120 patients from European and Australian sites. It aims to determine the optimal dose of OligoG and to describe long-term safety and efficacy, with FEV1 as a primary endpoint.

Recently, numerous articles have been published to describe new regulation mechanisms of the different proteins present in the mucus and especially on mucins expressed in the airways. The epigenetic regulation role of MUC5AC and MUC5B, the main mucins expressed in the airways, has been thoroughly researched in COPD and have highlighted the implication of methylation and miRNA. Different specific therapies are in progress to modulate the miRNA, and new treatment ways are in progress in CF [START_REF] Bardin | Emerging microRNA Therapeutic Approaches for Cystic Fibrosis[END_REF].

CONCLUSION

Although current anti-inflammatory drugs (corticosteroids and Ibuprofen) in CF patients have shown little effectiveness, the creation and improvement of new anti-inflammatory drugs for CF lungs has been overlooked for a long time. In the last decade, most of the research fields in CF therapy, have focused mainly on the discovery of new CFTR activators. Despite this, basic researches that are now in the evaluation phase have shown that new approaches could be very promising in resolving efficiently the CF lungs' ongoing inflammatory vicious cycle. However, treatment complexity is challenging. Currently available treatments offered to CF patients certainly help reduce inflammation, but in indirect and non-specific pathways, by targeting the viscosity of the mucus, reducing infection, or activating Cl -efflux. As the traditional approaches have shown their limitations, it seems essential to us that original work should continue in order to identify innovative approaches that would be more specific. The identification of critical

Introduction

Cystic fibrosis (CF), an autosomal recessive genetic multiorgan disease, is caused by an absent or dysfunctional CF transmembrane conductance regulator (CFTR) channel that mainly mediates chloride anion transport across the apical membrane of epithelial cells. On the pulmonary level, CF leads to persistent pulmonary infections, chronic inflammation, and mucus plugging in the airways, causing irreversible lung damage. To date, more than 2000 different mutations in the CFTR gene have been identified [1]. A classification system groups mutations into six classes according to the functional consequences they generate on the CFTR protein: ( 1 6) class VI: decreased CFTR stability at plasma membrane. Today, many symptomatic therapeutics (antibiotics, mucus thinners, bronchodilators, supplements to prevent malnutrition, etc.) are available to treat patients with CF, which has lengthened their life expectancy from 5 years in 1960 to over 50 years. New curative treatments aimed at rescuing CFTR dysfunctionality have emerged. There are four FDA-approved CFTR modulators developed by Vertex Pharmaceuticals (Kalydeco ® , Orkambi ® , Symdeko ® , and the latest Trikafta ® ) [2], although the proven efficacy of these correctors and potentiators is limited only to particular mutations. There are still 15% of patients without any CFTR-directed therapeutics. Hence, there is an interest in finding an alternative strategy to treat patients with CF independently of CFTR mutations. Alternative ion channels have been suggested to bypass CFTR dysfunction [3,4], such as ENaC [5], the solute carrier 26A9 (SLC26A9) [6], and calcium-activated chloride channel (CaCCs), including anoctamin-1 (ANO1 or TMEM16A) [4]. Such approaches might be efficient therapies for all patients, regardless of their CF mutations.

Anoctamin-1

CaCCs were described for the first time in 1981 in Rana pipiens eggs [7] and then in Xenopus laevis oocytes [8]. Today, CaCCs have been identified in many cellular types (neurons, epithelial cells, smooth muscle cells, pancreatic cells, etc.) and have been shown to play essential roles in cellular functions [9][10][11]. Among CaCCs is the anoctamin family (ANO for anion channel and OCTA for their eight predicted transmembrane domains or TMEM16), consisting of 10 proteins (ANO1-10). ANOs might play an essential role in development due to their temporal and spatial differential expression in many developing tissues. We can separate ANOs into two groups: ANO1-2 and the rest. However, ANO2 has different biophysical characteristics to ANO1 and its expression is limited to the vomeronasal epithelium [12,13]. In 2008, ANO1 was identified as a CaCC by three independent research groups [14][15][16].

Structure of ANO1

Since its discovery 11 years ago, many studies have been published describing the topological structure of ANO1. In 2011, a chemical crosslinking study performed by Sheridan et al. revealed that ANO1 exists as a dimer [17]. The dimeric structure of ANO1 was also confirmed by Fallah et al. using biochemical techniques such as native PAGE and chemical crosslinking [18]. Furthermore, Brunner et al. published the X-ray structure of calcium-activated ANO1 and showed that both subunits of ANO1 are symmetrical and have identical conformation [19]. However, two research groups recently proposed, on the basis of high-resolution single-particle cryoelectron microscopy, a common structure of ANO1 which is concurrent to the finding of earlier biochemical studies on ANO1 showing homodimers in the structure of anoctamins (Figure 1) [20,21]. Each subunit contains N-and C-terminal domains in this homodimer, a transmembrane unit consisting of 10 membrane-spanning α-helices, and extracellular components. Helices α1-α10 traverse the entire membrane, and a few of the helices are bent and tilted with respect to the plane [19]. Adjacent to the cavity, there are calcium-binding pockets, particularly α3-α7; using the site-directed mutagenesis approach, it was found that the acidic residues E654, E702, E705, E734, and D738 group together to form two calcium ion-binding sites and, thus, regulate the opening of the channel [22,23] (Figure 1). Each monomeric subunit is regulated independently [24,25]. The N-terminal domain also participates in an intrasubunit interaction with the cytoplasmic C terminus, the Cα1 helix. In this proposed cryo-EM structure, the channel is supposed to be nonconducting because ANO1 reduces calcium in a time-dependent manner, pushing the channel into an inactive state [20,26]. Despite several attempts by researchers to decipher the structure of ANO1 in the open state, the fully open pore structure of ANO1 has not been elucidated [27].

Regulation and Activation of ANO1

Most of the natively expressed CaCCs are sensitive to cytosolic calcium in very low concentrations (100-600 nM), as well as membrane potential [28][29][30]. The CaCCs expressed in many tissues show synergistic activation in response to intracellular calcium and voltage [31][32][33]. The synergistic regulatory mechanism is due to the calcium-binding site localized within the transmembrane segment of the channel, which makes CaCCs sensitive to membrane potential [33][34][35]. A similar regulatory mechanism has been reported with ANO1, as it is also sensitive to both calcium and voltage. According to a few studies, the required calcium concentration for the activation of ANO1 ranges from 0.4-0.6 µM at a positive membrane potential, and this value increases as the membrane potential deviates to the negative side [16,36,37]. Studies have also shown that ANO1 is activated in the presence of other divalent ions such as Ba 2+ , Sr 2+ , and Ni 2+ , along with calcium [38][39][40]. The molecular mechanism responsible for the channel's gating under the influence of calcium and voltage is unknown. However, experiments in isolated membranes have shown direct activation of ANO1 in response to increased intracellular calcium concentration [39]. However, the specific protein or mechanism responsible for channel activity's slow and inevitable breakdown is yet unknown. Notably, studies have shown that this reduction in the activity of isolated membrane ANO1 over time can be prevented in the presence of ATP and calmodulin via an unknown mechanism [41,42]. Concomitantly, ANO1 conductivity was significantly reduced in the presence of calmodulin inhibitor trifluoperazine or inhibitory peptides [41]. However, contrary to this finding, Terashima et al. (2013) did not find any modulation in the activity of the ANO1 channel in the presence of calmodulin and proposed that calmodulin may not be essentially required for the activation of ANO1 [33]. Overall, these contrary findings indicate that further investigation is required to elucidate the exact role of calmodulin in the activation of ANO1.

Phosphorylation is another possible mechanism known to be involved in the regulation of ANO1 [42]. The potential phosphorylation sites in ANO1 have already been identified in the analysis of ANO1 amino-acid sequencing [43] (Figure 1). However, studies have shown that phosphorylation does not always have a stimulatory effect on ANO1, and contrary results have also been reported [44]. Hence, inclusive studies are required to trace the specific phosphorylation site and how this modulates channel activity in an in vivo environment.

Phospholipid phosphatidylinositol-(4,5)-bisphosphate (PIP 2 )-mediated regulation of ANO1 has also been reported in the literature [45,46]. Studies have shown that decreased PIP 2 in rat PASMCs has a negative impact on the activation of ANO1. However, Ta et al. showed a significant increase in channel activity in HEK-293 expressing ANO1 when treated with diC8-PIP 2 [START_REF] Ta | Contrasting effects of phosphatidylinositol 4,5bisphosphate on cloned TMEM16A and TMEM16B channels[END_REF]. Earlier studies have established that the activation of cells with phospholipase C causes an increase in the production of IP 3 , which in turn triggers the release of calcium from intracellular space and may activate ANO1. Subsequently, the release of intracellular calcium delays the release of IP 4 ; thus, a similar model may work as an inhibitory feedback mechanism of ANO1 [START_REF] Xie | Regulation of Ca2+-dependent Clconductance in a human colonic epithelial cell line (T84): Cross-talk between Ins(3,4,5,6)P4 and protein phosphatases[END_REF]. Some fatty acids such as stearic, arachidonic, oleic, and docosahexaenoic acid were also found to have inhibitory effects on ANO1 activity in a concentration-and voltage-dependent manner, probably due to altered lipid channel-protein interaction [START_REF] Antollini | Fatty Acid Regulation of Voltage-and Ligand-Gated Ion Channel Function[END_REF].

Expression of ANO1

ANO1 is ubiquitously expressed in all secretory exocrine glands in the epithelia (salivary gland, mammary gland, pancreas, kidneys, and gut), notably in airway epithelial cells [START_REF] Huang | Inhibition of intracellular Ca2+ release by a Rho-kinase inhibitor for the treatment of ischemic damage in primary cultured rat hippocampal neurons[END_REF]. In humans, ANO1 is expressed throughout the respiratory tract from the large bronchi to the alveoli [START_REF] Kunzelmann | Airway epithelial cells-functional links between CFTR and anoctamin dependent Cl-secretion[END_REF]. Moreover, ANO1 is present in interstitial Cajal cells that generate the pacemaker activity of gastrointestinal smooth muscles [START_REF] Pedemonte | Structure and function of TMEM16 proteins (anoctamins)[END_REF][START_REF] Huang | Studies on expression and function of the TMEM16A calcium-activated chloride channel[END_REF], as well as in smooth muscles of the respiratory and reproductive systems [START_REF] Kunzelmann | Modulating Ca(2)(+) signals: A common theme for TMEM16, Ist2, and TMC[END_REF] and sensory neurons. It is also expressed in proliferating cells of different types [START_REF] Pinto | Regulation of TMEM16A by CK2 and Its Role in Cellular Proliferation[END_REF].

Physiological and Physiopathological Functions of ANO1

Due to its presence in many cell types, the ANO1 channel is involved in several important physiological and pathophysiological processes such as the regulation of smooth muscle contraction, nociception, and fluid secretion in the salivary glands [START_REF] Huang | Inhibition of intracellular Ca2+ release by a Rho-kinase inhibitor for the treatment of ischemic damage in primary cultured rat hippocampal neurons[END_REF][START_REF] Manoury | TMEM16A/anoctamin 1 protein mediates calcium-activated chloride currents in pulmonary arterial smooth muscle cells[END_REF] (Figure 2). ANO1 hyperactivity plays a role in asthma and neuropathic pain, while it is also involved in cell migration and proliferation in different cancers [START_REF] Qu | The Ca(2+) -activated Cl(-) channel, ANO1 (TMEM16A), is a double-edged sword in cell proliferation and tumorigenesis[END_REF][START_REF] Wanitchakool | Role of anoctamins in cancer and apoptosis[END_REF][START_REF] Britschgi | Calciumactivated chloride channel ANO1 promotes breast cancer progression by activating EGFR and CAMK signaling[END_REF][START_REF] Wang | Cell-specific mechanisms of TMEM16A Ca(2+)-activated chloride channel in cancer[END_REF][START_REF] Kim | Anoctamin-1 affects the migration and invasion of anaplastic thyroid carcinoma cells[END_REF][START_REF] Song | Inhibition of ANO1/TMEM16A induces apoptosis in human prostate carcinoma cells by activating TNF-alpha signaling[END_REF]. Ruffin et al. demonstrated a significant decrease in cell proliferation and migration when ANO1 is decreased [63]. In contrast, a decrease in ANO1 activity is associated with dry eye and dry mouth syndromes, Sjögren syndrome, and CF (Figure 2).

Rock et al. showed the importance of ANO1 in the development of the murine trachea [START_REF] Rock | The transmembrane protein TMEM16A is required for normal development of the murine trachea[END_REF]. All homozygous mice for the ANO1 null allele died within 1 month of birth and exhibited severe tracheomalacia with gaps in the tracheal cartilage rings along the entire length of the trachea, as well as a mucus build-up leading to airway obstruction and contributing to neonatal mortality [START_REF] Rock | The transmembrane protein TMEM16A is required for normal development of the murine trachea[END_REF][START_REF] Rock | Transmembrane protein 16A (TMEM16A) is a Ca2+-regulated Cl-secretory channel in mouse airways[END_REF]. Impaired mucociliary clearance has also been demonstrated in ANO1 KO mice, suggesting that ANO1 could participate in mucociliary clearance regulation in the airways [START_REF] Ousingsawat | Loss of TMEM16A causes a defect in epithelial Ca2+-dependent chloride transport[END_REF].

One study showed that ANO1 overexpression decreases proinflammatory cytokine interleukin-8 (IL-8) secretion in CF bronchial epithelial cells [START_REF] Veit | Proinflammatory cytokine secretion is suppressed by TMEM16A or CFTR channel activity in human cystic fibrosis bronchial epithelia[END_REF], while a different study showed that ANO1 is upregulated in inflammatory conditions and is associated with airway goblet cell metaplasia [START_REF] Scudieri | Association of TMEM16A chloride channel overexpression with airway goblet cell metaplasia[END_REF]. Huang et al. showed that ANO1 is specifically expressed in mucus cells and poorly expressed in ciliated cells. The induction of mucus cell hyperplasia with IL-4, a potent inducer of mucus secretion and mucus cell metaplasia in the airways, increases the number of cells co-expressing ANO1 and mucin 5AC (MUC5AC, predominant mucin produced by airway epithelial cells) [START_REF] Huang | Calcium-activated chloride channel TMEM16A modulates mucin secretion and airway smooth muscle contraction[END_REF]. Scudieri et al. suggested that ANO1 expression is particularly required under mucus hypersecretion conditions to ensure adequate water and electrolyte secretion [START_REF] Scudieri | Association of TMEM16A chloride channel overexpression with airway goblet cell metaplasia[END_REF].

ANO1 is also involved in bicarbonate ion secretion following the direct association of ANO1 and calmodulin at high cytosolic calcium concentrations [START_REF] Jung | Dynamic modulation of ANO1/TMEM16A HCO3(-) permeability by Ca2+/calmodulin[END_REF].

ANO1 in Cystic Fibrosis

Overview

A study showed that the activity of CaCCs, in general, was increased in the nasal epithelium in vivo of patients with CF [START_REF] Knowles | Activation by extracellular nucleotides of chloride secretion in the airway epithelia of patients with cystic fibrosis[END_REF]. In 1986, another team demonstrated the presence of chloride current at the apical membrane of epithelial cells of CF in the presence of ionized calcium [START_REF] Frizzell | Altered regulation of airway epithelial cell chloride channels in cystic fibrosis[END_REF]. Moreover, another study showed a decrease in ATP-induced chloride efflux in the primary bronchial epithelial cell line [START_REF] Blouquit | Ion and fluid transport properties of small airways in cystic fibrosis[END_REF]. The identification of ANO1 as a CaCC later on and its involvement in many deregulated processes in patients with CF made it a real therapeutic target. In the last few years, more research has been dedicated to ANO1 in CF.

Recent data reported the absence of calcium-induced chloride currents in epithelial cells of ANO1 KO mice [START_REF] Benedetto | Epithelial Chloride Transport by CFTR Requires TMEM16[END_REF]. A subsequent study showed that CFTR expression requires ANO1 in plasma membranes, indicating a close relationship between the two chloride channels [START_REF] Benedetto | Transport properties in CFTR-/-knockout piglets suggest normal airway surface liquid pH and enhanced amiloride-sensitive Na(+) absorption[END_REF]. Moreover, ANO1 expression and chloride activity were decreased in CF [63]. According to several observations ((1) ANO1's absence decreases airway secretion, (2) ANO1 is ubiquitously expressed in all the tissue affected by CF, including airway epithelial cells where ANO1 is a secondary chloride channel, (3) ANO1 provides a chloride pathway that is CFTR-independent, and (4) ANO1 is involved in HCO 3

-secretion, which is highly important for fluid secretion and mucus hydration [START_REF] Quinton | Role of epithelial HCO 3 (-) transport in mucin secretion: Lessons from cystic fibrosis[END_REF]), increasing ANO1 activity can probably compensate for CFTR deficiency (Figure 3). Different approaches targeting ANO1 have been developed to bypass CFTR dysfunction. In CF airways, dysfunctional CFTR leads to compromised chloride efflux. Sodium entry is upregulated, leading to a dehydrated air surface liquid (ASL) and impaired mucociliary clearance favoring mucostasis, causing chronic inflammation and infection. In healthy airways, ANO1 is colocalized with CFTR within the apical membrane of epithelial cells, contributing to ion and water homeostasis. In CF ciliated cells, the expression of ANO1 is also diminished. Modulating ANO1, as an alternative CF therapy, could compensate for defective CFTR and, thus, enhance fluid secretion by ciliated epithelial cells, regulating ASL height and pH.

Drug Approaches Targeting ANO1 in Cystic Fibrosis

Many molecules have been used to modulate ANO1's activity, but few were shown to be efficient and, most importantly, specific to ANO1 (Table 1).

Long before discovering ANO1's function in the airways, a clinical trial targeting CaCCs, in general, took place. Inspire Pharmaceuticals developed denufosol (INS37217), the first CFTR-independent drug for CF lung therapy, carried into clinical trials in 2001 [START_REF] Deterding | Safety and tolerability of denufosol tetrasodium inhalation solution, a novel P2Y2 receptor agonist: Results of a phase 1/phase 2 multicenter study in mild to moderate cystic fibrosis[END_REF][START_REF] Deterding | Phase 2 randomized safety and efficacy trial of nebulized denufosol tetrasodium in cystic fibrosis[END_REF][START_REF] Goss | Experience using centralized spirometry in the phase 2 randomized, placebo-controlled, double-blind trial of denufosol in patients with mild to moderate cystic fibrosis[END_REF][START_REF] Kellerman | A review of studies with inhaled P2Y(2) agonists that led to Phase 3[END_REF]. The inhaled P2Y2 receptor agonist activated P2Y receptors and led to intracellular calcium activation of chloride efflux through CACCs. This increased airway epithelial chloride efflux in vitro, increasing airway fluid volume. However, the second phase III clinical trial of denufosol was disappointing as it did not achieve statistical significance for its primary efficacy endpoint in improving forced expiratory volume in 1 s (FEV1). This failure was due to (1) denufosol targeting all CaCCs, (2) its short half-life in vivo due to rapid degradation by ectonucleotidases, and (3) increased intracellular calcium stimulating goblet cell exocytosis that might have led to an increase in mucus in the airways. Therefore, CaCC activators need to target CaCCs directly without elevating cytoplasmic calcium for more targeted therapy and efficacy [START_REF] Moss | Pitfalls of drug development: Lessons learned from trials of denufosol in cystic fibrosis[END_REF].

ANO1 identification paved the way for the development of specific activator molecules which would, without modifying the calcium signaling, obtain a more sustained activation over time, leading to better efficiency.

Another novel drug is an ANO1 potentiator (ETD002) developed by Enterprise Therapeutics (based in the University of Sussex Innovation Center, UK) and acquired by Roche (Genentech) in October 2020. This inhaled molecule demonstrated an upregulation of ANO1, which boosts epithelial fluid secretion and mucus clearance in primary CF bronchial epithelial cells and ovine models. Unlike denufosol, intracellular calcium measurements checked that ETD002 did not affect calcium mobilization, coherent with a direct effect on ANO1. A phase I study to test the safety of ETD002 in healthy participants is in progress [START_REF] Danahay | TMEM16A: An Alternative Approach to Restoring Airway Anion Secretion in Cystic Fibrosis?[END_REF]. The mechanism via which ETD002 potentiates ANO1 activity is still unclear.

Our team has also worked on an innovative alternative approach using a locked nucleic acid (LNA)-enhanced antisense oligonucleotide (ASO). A previous study demonstrated that microRNA (miR-9) contributes to the downregulation of ANO1 expression and activity by directly targeting its 3'UTR. ASO ANO1 binds to the 3'UTR target site of ANO1 mRNA, preventing miR-9 from gaining access to that site [START_REF] Sonneville | MicroRNA-9 downregulates the ANO1 chloride channel and contributes to cystic fibrosis lung pathology[END_REF]. ASO ANO1 has increased ANO1 expression, chloride activity, and mucus clearance in primary human CF cells and CF mice. Recent studies have suggested that ANO1 plays an essential role in mucus production [START_REF] Benedetto | TMEM16A is indispensable for basal mucus secretion in airways and intestine[END_REF]. To date, we have not observed any significant increase in mucin 5AC (MUC5AC, the main component of respiratory mucus produced by goblet cells) or mucin 5B (MUC5B, gel-forming mucin that plays a key role in mucociliary clearance) expression in vitro. Currently, ANO1 activation as a therapeutic target is subject to controversial opinions. Centeio et al. found an upregulation of ANO1 expression in submucosal glands, airway smooth muscles, and pulmonary blood vessels in CF and asthmatic inflamed lungs [105]. Moreover, activating ANO1 with Eact, which also activates other channels, induced mucus production in airway goblet cells and bronchoconstriction in ovalbumin-sensitized asthmatic mice [START_REF] Ramu | Counteracting suppression of CFTR and voltage-gated K+ channels by a bacterial pathogenic factor with the natural product tannic acid[END_REF], whereby activating ANO1 could worsen the pathology in inflammatory airway diseases. Instead, the authors demonstrated that ANO1 inhibition by niclosamide significantly reduced goblet cell metaplasia and mucus production in asthmatic mice, as well as inhibited MUC5AC expression in Calu-3 human submucosal cells, suggesting that ANO1 inhibition might be beneficial in inflammatory airway diseases [105]. It is also important to note that a transcriptome meta-analysis revealed that CF and asthma pathways are highly divergent [106]. Furthermore, the same group reported a defect of mucus secretion and accumulation in secretory cells in 2018 when ANO1 was knocked out in ciliated airway epithelial cells and intestinal goblet cells, highlighting the vital role of ANO1 in mucus secretion [START_REF] Benedetto | TMEM16A is indispensable for basal mucus secretion in airways and intestine[END_REF]. Another study showed that ANO1 inhibitors reduced both mucus secretion and airway hyperactivity [START_REF] Huang | Calcium-activated chloride channel TMEM16A modulates mucin secretion and airway smooth muscle contraction[END_REF].

On the other hand, using a human respiratory basal cell line (BCi-NS1.1), Simões et al. showed that MUC5AC production does not require ANO1, and their simultaneous upregulation is only circumstantial under cell proliferation [107]. The authors also showed a decrease in ASL when inhibiting ANO1. Furthermore, while replying to Olschewski et al.'s concerns on increasing ANO1 activity, Danahay et al. declared that positive modulation of ANO1 induces no bronchospasm in the conscious sheep model nor affects vascular smooth muscle contraction (unpublished observations) [108,109]. Overall, ANO1's possible role in mucus production remains obscure and evokes controversial opinions over the beneficial or deleterious results of stimulating the channel in CF.

It is critical to emphasize the importance of this CFTR-independent strategy regarding the 10-15% of struggling patients, notably class I patients and nonresponders, who cannot benefit from Vertex's molecules and should not be forgotten. This strategy might also be combined with CFTR modulators for additive/synergistic actions to enhance clinical outcomes.

In a recent case report, seven male patients between 17 and 39 years of age, treated with Trikafta ® , reported testicular pain [110]. It is suggested that this may be related to CFTR function restoration in the male reproductive tract. In the same manner, we can probably expect not only an ANO1 activation in the airways but also in multiple tissues affected by CF and in which ANO1 is expressed, such as the male reproductive tracts, the liver, and gallbladder, the proximal digestive tract, and the gastrointestinal tract (https://www.proteinatlas.org; accessed on October 2021). Hence, patients with CF can be treated on multiple fronts by using an alternative non-CFTR chloride pathway to compensate for defective CFTR. Moreover, ANO1 might help repair and regenerate CF airways because ANO1 upregulation is correlated with enhanced proliferation [63]. Furthermore, relieving gastrointestinal (GI) symptoms in patients with CF was set as a "top 10" priority by the CF community [111]. Since the role of ANO1 in gut motility has been demonstrated in mice homozygous for the null ANO1 allele [112,113], we hypothesize that activating ANO1 might have a therapeutic effect in patients with CF who suffer from intestinal obstruction syndromes. Hence, improving their nutritional status is also associated with better clinical outcomes [114].

Conclusions

Although there are still some reservations concerning ANO1's role in mucus production, enhancing ANO1's activity constitutes an up-and-coming alternative for treating patients with CF independently of CFTR mutations, alone or in combination with CFTR potentiators and correctors for better clinical efficacy.

quatre modulateurs CFTR approuvés par la FDA et produits par Vertex Pharmaceuticals réduisent la gravité de la maladie chez des sous-ensembles de patients, il existe un besoin clinique non satisfait de thérapies pour les personnes présentant des mutations non compatibles avec ces médicaments mais aussi pour les patients non répondeurs. Pour certains de ces patients (environ 15%), la protéine n'est même pas produite, ce qui rend les thérapies actuelles inefficaces. TMEM16A est un canal qui transporte le chlorure et le bicarbonate en réponse au calcium. TMEM16A est exprimé de façon ubiquitaire dans toutes les glandes sécrétrices exocrines des épithéliums, notamment dans les cellules épithéliales des voies respiratoires. Il est activé indirectement par les nucléotides triphosphate (UTP ou ATP), libérés lors de stimuli mécaniques comme l'étirement imposé aux cellules par la respiration ou l'impact de particules sur la muqueuse. Ces nucléotides se lient aux récepteurs purinergiques situés à la face apicale des cellules épithéliales (Schroeder, Cheng, Jan, & Jan, 2008), ce qui déclenche l'augmentation du calcium intracellulaire, pouvant ainsi constituer une voie de transport alternative à CFTR chez l'ensemble des patients atteints de mucoviscidose indépendamment de leurs mutations.

Cependant chez les patients atteints de mucoviscidose l'expression et l'activité de ces transporteurs sont négativement régulées par miR-9 qui se fixe sur la région 3'UTR de l'ARNm de TMEM16A et empêche ainsi sa traduction (Ruffin et al., 2013). Dans le but de pouvoir cibler l'ensembles des patients atteints de mucoviscidose en ciblant un canal alternatif, nous avons donc développé un oligonucléotide compétitif de miR-9 (ASO TMEM16A), permettant de se fixer de façon très spécifique sur la région 3'UTR de l'ARNm de TMEM16A, empêchant ainsi la fixation de miR-9 sur celuici. L'ASO synthétisé porte avec des groupements LNA (locked nucleic acid) et des groupements phosphorothioates afin de le rendre résistant aux RNAses pour améliorer la demi-vie et d'augmenter la spécificité.

L'utilisation du ASO TMEM16A a permis de montrer que nous étions capables d'augmenter l'expression et l'activité chlorure de TMEM16A. De plus, nous avons montré que cette molécule avait un effet majeur sur la clairance mucociliaire et la réparation, deux paramètres altérés dans la mucoviscidose (Sonneville et al., 2017).

Ces résultats ont été montrés sur des différents modèles portant la mutation principale F508del : des lignées cellulaires, des cellules épithéliales bronchiques primaires de patients atteints de mucoviscidose cultivées en interphase air-liquide et sur un modèle murin muté pour cftr porteurs de la mutation F508del (Sonneville et al., 2017).

Mon projet de thèse a pour but de poursuivre la preuve de concept, afin d'amener la molécule déjà brevetée vers la clinique. Cystic fibrosis (CF) is an autosomal recessive genetic multiorgan disease resulting from a dysfunctional or absent CFTR chloride and bicarbonate ion channel. In the last decade, correctors and potentiators of CFTR protein have demonstrated good clinical outcomes for patients with specific CFTR gene mutations. Nevertheless, due to the diversity of the mutations, there are still patients for whom those treatments are unsuitable and require alternative CFTR-independent strategies. Although CFTR is the main chloride channel in the lungs, anoctamin-1 (TMEM16A or ANO1), a calcium-activated chloride channel, could compensate for the chloride-CFTR deficiency. In a previous study, TMEM16A expression and activity were decreased in CF bronchial epithelial cells due to microRNA-9 overexpression, which induces translational repression. We have then developed an antisense oligonucleotide (ASO TMEM16A) that prevents microRNA-9 from binding to the TMEM16A mRNA, thereby increasing its expression and activity in the epithelia.

This work aims to investigate in vitro and in vivo effects of ASO TMEM16A on CF-deregulated parameters and prepare for future clinical studies.

Results show that ASO TMEM16A enhances mucociliary clearance and TMEM16A chloride activity in a specific manner, even in primary cells of patients with CF that do not benefit from any treatment. Moreover, ASO TMEM16A does not induce inflammatory cytokine expression nor alter intracellular calcium mobilization or cell proliferation. These specificity results were confirmed by "in vitro pharmaceutical profiling", a standard method used by the industry. While studying different administration routes on mice, we showed that ASO TMEM16A is still detectable 30 days after subcutaneous injection. Additionally, acute administrations of 50 times the effective dose did not show behavioral, macroscopic, or histopathologic changes in mice. In addition, none of the blood markers were affected. Studies on CF mice, which generally die early of intestinal obstruction upon weaning, show that injection of ASO TMEM16A significantly increases the lifespan of the mice up to 200 days. In addition, results show an improvement in male CF mice fertility, a known CF phenotype in male patients. Altogether all these results demonstrated that this strategy could be applied to all patients with CF, regardless of their mutations (i.e., mutation-agnostic treatment). This novel strategy can be combined with CFTR modulators or TMEM16A potentiators to obtain additive effects and target multiple fronts, such as pulmonary symptoms and other manifestations observed in patients with CF.

Cystic fibrosis -Anoctamin 1 -Antisens oligonucleotide -Alternative therapy La mucoviscidose (CF) est une maladie génétique autosomique récessive résultant d'un dysfonctionnement ou de l'absence du canal chlorure CFTR. Ces dernières années, les correcteurs et les potentiateurs de la protéine CFTR ont donné de bons résultats cliniques chez les patients présentant des mutations spécifiques du gène CFTR. Cependant, en raison de la multiplicité des mutations, il existe encore des patients pour lesquels ces traitements ne sont pas adaptés et qui ont besoin de stratégies alternatives indépendantes de CFTR. Bien que CFTR soit le principal canal chlorure au niveau pulmonaire, l'Anoctamine 1 (TMEM16A ou ANO1), un canal chlorure activé par le calcium, pourrait compenser la déficience en CFTR. Au niveau bronchique, chez les patients atteints de mucoviscidose, l'expression et l'activité de TMEM16A ont été montrées comme diminuées et corrélées à une surexpression du microARN-9 qui va induire une répression traductionnelle de TMEM16a. Dans ce but de rétablir cette expression et cette activité de TMEM16a, nous avons développé un oligonucléotide antisens (ASO TMEM16A) qui empêche le microARN-9 de se lier à l'ARNm de TMEM16A, au niveau des épithélia.

Ce travail vise à étudier les effets in vitro et in vivo de l'ASO TMEM16A sur les paramètres dérégulés dans la mucoviscidose afin de préparer les futures études cliniques.

Les résultats montrent in vitro que l'ASO TMEM16a améliore la clairance mucociliaire et l'activité chlorure lié à TMEM16a de manière spécifique même dans les cellules primaires de patients atteints de mucoviscidose qui ne pourraient être traités. Aussi, l'ASO TMEM16A n'induit pas l'expression de cytokines proinflammatoires et n'altère ni la mobilisation du calcium intracellulaire ni la prolifération cellulaire. Ces résultats de spécificité ont été confirmés par le "profilage pharmaceutique in vitro", une méthode standard utilisée par l'industrie.

En étudiant différentes voies d'administration chez la souris, nous avons montré que l'ASO TMEM16A est détectable 30 jours après injection sous-cutanée qui permettait une bonne répartition dans les tissus. L'administration aiguë de 50 fois la dose efficace n'a pas montré de changements comportementaux chez la souris, ni de changements macroscopiques ou histologiques. En outre, tous les marqueurs sanguins se sont révélés normaux.

Des études sur des souris CF, qui meurent normalement d'une obstruction intestinale au moment du sevrage, démontrent que l'injection d'ASO TMEM16A augmente significativement la durée de vie des souris jusqu'à 200 jours. En outre, les résultats montrent une amélioration de la fertilité des souris CF mâles, également décrite chez les hommes atteints de mucoviscidose.

L'ensemble de nos résultats montrent que cette stratégie pourrait s'appliquer à tous les patients atteints de mucoviscidose, quelles que soient leurs mutations. Elle peut être associée à des modulateurs de CFTR ou à des potentiateurs de TMEM16a pour obtenir des effets additifs et cibler plusieurs fronts tels que les symptômes pulmonaires, et les autres atteintes observées chez les patients atteints de mucoviscidose.

Mucoviscidose -Anoctamine 1 -Oligonucleotide antisens -Thérapie Alternative

Thesis

  Results Valorization .................................................................................... 216 Training and Seminars ........................................................................................... 219

Figure 1 :

 1 Figure 1: Global Genetic Epidemiology for Cystic Fibrosis. ......................................

Figure 2 :

 2 Figure 2: Chromosomal Localization of the CFTR Gene. .........................................

Figure 3 :

 3 Figure 3: Classes of CFTR Mutations. .....................................................................

Figure 4 :

 4 Figure 4: Overall Structure of CFTR. ........................................................................

Figure 5 :

 5 Figure 5: CFTR mRNA Expression Levels in Several Tissues. ................................

Figure 6 :

 6 Figure 6: CFTR mRNA Expression in Lungs. ...........................................................

Figure 7 :

 7 Figure 7: Ion Transport in The Airway Epithelial Cells. .............................................

Figure 8 :

 8 Figure 8: CFTR Protein Function and Dysfunction in CF Lung Pathophysiology. ....

Figure 9 :

 9 Figure 9: Clinical Manifestations of Cystic Fibrosis. .................................................

Figure 10 :

 10 Figure 10: Progression of CF Pathophysiology in Bronchial Epithelial Cells. ...........

Figure 11 :

 11 Figure 11: CF Therapies and Patient Survival Over The Decades. ..........................

Figure 12 :

 12 Figure 12: Cystic Fibrosis Drug Development Pipeline. ...........................................

Figure 13 :

 13 Figure 13: Cystic Fibrosis Discontinued Drug Development. ...................................

Figure 14 :

 14 Figure 14: Simplified Model of TMEM16A Protein. ...................................................

Figure 15 :

 15 Figure 15: Electrophysiological Properties of TMEM16A Channel in Response to Increasing Concentration of Ca2+. ...........................................................................

Figure 16 :

 16 Figure 16: TMEM16A RNA and Protein Expression Profile. .....................................

Figure 17 :

 17 Figure 17: TMEM16A Expression Summary in Different Types of Cancers. ............

Figure 18 :

 18 Figure 18: The Positive Impacts of Activating or Inhibiting TMEM16A in Different Pathologies. .............................................................................................................

Figure 19 :

 19 Figure 19: Speculative Effects of TMEM16A Upregulation in CF Airways. ...............

Figure 20 :

 20 Figure 20: MiRNA Biogenesis. .................................................................................

Figure 21 :

 21 Figure 21: TMEM16A mRNA Regulation by miRNA-9. ............................................

Figure 22 :

 22 Figure 22: Summary of Approved Oligonucleotide Drugs. .......................................

Figure 23 :

 23 Figure 23: Ribonucleotide Structure. ........................................................................

Figure 24 :

 24 Figure 24: Mechanism of Action of ASOs. ................................................................

Figure 25 :

 25 Figure 25: LNA-enhanced Target Site Blockers Mechanism. ...................................

Figure 26 :

 26 Figure 26: ASO Mediated Toxicities. ........................................................................

Figure 27 :

 27 Figure 27: Splisence pipeline. ..................................................................................

Figure 28 :

 28 Figure 28: ASO TMEM16A Mechanism. ..................................................................

Figure 29 :

 29 Figure 29: TMEM16A, miRNA-9 and ASO TMEM16A Sequences. .........................

Figure 30 :

 30 Figure 30: Body Weight Curve of CF Mice. ............................................................

Figure 31 :

 31 Figure 31: WT and CF Mice Enamels. ...................................................................

Figure 32 :

 32 Figure 32: TMEM16A ASO Effect on Mice Teeth Enamels. ...................................

Figure 33 :

 33 Figure 33: Fecal Pro-inflammatory Lipocalin. .........................................................

Figure 1 :

 1 Figure 1: Global Genetic Epidemiology for Cystic Fibrosis. Adapted from (Shteinberg et al., 2021).

Figure 2 :

 2 Figure 2: Chromosomal Localization of the CFTR Gene. CFTR gene located at chromosome 7 at region q31.2 contains 27 exons spanning 250 kb of genomic DNA. From (Prasad, 2018).

Figure 3 :

 3 Figure 3: Classes of CFTR Mutations. Adapted from (Boyle & De Boeck, 2013).

Figure 4 :

 4 Figure 4: Overall Structure of CFTR. From (Lopes-Pacheco et al., 2021).

Figure 7 :

 7 Figure 7: Ion Transport in The Airway Epithelial Cells. CFTR drives Cl -and HCO 3 - secretion and regulates Na + absorption by inhibiting ENaC on the apical surface. CFTR also positively regulates the Cl -channels (ANO1/TMEM16A and SLC26A9) and the anion exchanger SLC26A4, increasing Cl -and HCO 3 -secretion and therefore increasing ASL hydration and pH. Na + and water follow the electrochemical gradient through the paracellular pathway. On the basolateral membrane, Na-K-2Cl cotransporter-1 (NKCC1) accumulates Cl - intracellularly, supported by electrochemical gradients established by the basolateral Na + /K + ATPase. K + recycling across the basolateral membrane is governed by the K + channel and serves to adjust membrane potential. From (Saint-Criq & Gray, 2017).

Figure 8 :

 8 Figure 8: CFTR Protein Function and Dysfunction in CF Lung Pathophysiology.The CFTR protein is not just a Cl -channel. It is also involved in HCO 3 -, glutathione (GSH), and SCN -transport, regulation of ENaC, immune cells, and metabolism of lipids. In CF, the loss of functional CFTR results in bronchial obstruction (in yellow), inflammation (in orange), and infection (in green), leading to lung injury. From[START_REF] Hanssens | CFTR Protein: Not Just a Chloride Channel?[END_REF].

Figure 9 :

 9 Figure 9: Clinical Manifestations of Cystic Fibrosis. Created with BioRender.com.

  Postural drainage and percussion, to help clear mucus from the lungs,  Nonsteroidal anti-inflammatory drugs to improve weight gain and reduce declines in lung function,  Lung transplantation may eventually be considered for patients with end-stage lung disease. Digestive problems caused by CF are managed with these medications:  Oral ingestion of pancreatic enzymes to help digestion and food absorption,  High food intake, and vitamin and mineral supplements, particularly the lipid-soluble vitamins A, E, K, and sometimes D, to prevent malnutrition,  Enemas and mucolytic agents to treat intestinal obstructions,  Patients experiencing gastrointestinal obstruction may also require surgery.

Figure 11 :

 11 Figure 11: CF Therapies and Patient Survival Over The Decades. From (Elborn, 2013).

( 2 .

 2 8 percentage points) and Symdeko (4.0 percentage points). The concentration in sweat chloride significantly decreased to -55.2 mmol/L change from baseline compared to placebo. The results are similar for the respiratory domain of the CFQ-R.The improvements with Trikafta (22.5 score increase) significantly exceed the modest improvements obtained with Orkambi (5.1 score increase) and Symdeko (2.2 score increase)[START_REF] Tice | Modulator Treatments for Cystic Fibrosis: Effectiveness and Value; Final Evidence Report and Meeting Summary[END_REF].

Figure 14 :

 14 Figure 14: Simplified Model of TMEM16A Protein. The TMEM16A channel protein contains ten transmembrane ion domains with carbon and nitrogen terminals on the cell's inner membrane. The intracellular loop between TMD-6 and TMD-8 contains six amino acids (N650, E654, E702, E705, E734, and D738) that form the Ca 2+ binding pockets of the dimeric channel (red dotted circle). The depicted location of two phosphorylation sites is denoted with the letter P. Two TMEM16A proteins assemble to form the TMEM16A channel. Created with BioRender.com.

Figure 17 :

 17 Figure 17: TMEM16A Expression Summary in Different Types of Cancers. For each cancer, color-coded bars indicate the percentage of patients (maximum 12 patients) with high and medium TMEM16A protein expression levels. Adapted from (proteinatlas.org/ENSG00000131620-ANO1/pathology).

Figure 19 :

 19 Figure 19: Speculative Effects of TMEM16A Upregulation in CF Airways.In healthy airways, TMEM16A is colocalized with CFTR within the apical membrane of epithelial cells, contributing to ion and water homeostasis. In CF airways, dysfunctional CFTR leads to compromised ions efflux. Na + entry is upregulated, leading to a dehydrated ASL and impaired MCC favoring mucostasis, causing chronic inflammation and infection. In CF, the expression of TMEM16A is also diminished. Modulating TMEM16A, as an alternative CF therapy, could compensate for defective CFTR and, thus, enhance fluid secretion by epithelial cells, regulating ASL height and pH. Adapted from(Mitri et al., 2021).

Figure 20 :

 20 Figure 20: MiRNA Biogenesis. MiRNA is transcribed by RNA polymerase II (RNAP II) to form double-stranded hairpin loop structure called pri-miRNA cleaved by nuclease Drosha to form pre-miRNA. Exportin transports the pre-miRNA to the cytoplasm, where it is further processed by Dicer to form a single-stranded mature miRNA. The mature miRNA is uploaded in the RNA-induced silencing complex (RISC) where it associates with Argonaute 2 protein.This miRNA-RISC complex interacts with the seed region of the mRNA and regulates the mRNA translation by either mRNA cleavage or by steric hindrance. From[START_REF] Dhuri | Antisense Oligonucleotides: An Emerging Area in Drug Discovery and Development[END_REF].

Figure 21 :

 21 Figure 21: TMEM16A mRNA Regulation by miRNA-9. In CF, miRNA-9 coupled to an RNAinduced silencing complex (RISC) binds to the 3'UTR region of the TMEM16A mRNA and inhibits the protein translation. Created with BioRender.com.

Figure 22 :

 22 Figure 22: Summary of Approved Oligonucleotide Drugs. To date, nine single-stranded ASO therapies (and Milasen, n-of-1 approval) with a variety of mechanisms of action were approved for the treatment of different diseases, in five organs, with different administration routes. Adapted from (Scharner & Aznarez, 2021).

Figure 23 :

 23 Figure 23: Ribonucleotide Structure. The general structure of a ribonucleotide consists of a phosphate backbone, a ribose sugar group, and a nucleobase, in which the nucleobase can either be adenine, guanine, cytosine, or uracil. Adapted from (Jens Kurreck, 2003).

  an allele-independent manner (Mipomersen prevents production of apolipoprotein B leading to decreased levels of very low-density lipoprotein (VLDL) and LDL in autosomal dominant familial hypercholesterolemia; Volanesorsen inhibits apolipoprotein C3 reducing triglyceride levels by 70-80% in autosomal recessive hereditary familial chylomicronaemia syndrome). On the other hand, in the case of Inotersen, allele-specific mRNA degradation targets only the mutant allele encoding the transthyretin protein in autosomal dominant hereditary transthyretin amyloidosis; thus, the correct protein can resume its function properly.

Figure 24 :

 24 Figure 24: Mechanism of Action of ASOs. ASOs act by either causing (1) RNA cleavage or (2) RNA blockage. Phosphorothioate (PS) modification induces mRNA cleavage. Peptide nucleic acids (PNA), 2'-O-methyl (2'-O-Me) and 2'-O-methoxyethyl (2'-O-MOE) modifications, phosphorodiamidate morpholino (PMO), locked nucleic acid (LNA) act on mRNA to sterically block its translation or these ASOs can act as splice switching oligonucleotides to modulate splicing pattern. Adapted from (Dhuri et al., 2020).

  single-stranded non-coding RNA molecule (containing about 21-26 nucleotides) that regulates the gene expression by either degrading the target mRNA or preventing its translation. The miRNA usually binds to RISC, which uses the miRNA as a template for recognizing complementary mRNA. Hybridization usually occurs on miRNA recognition elements (MREs) at the 3' UTR of the target mRNA(De Santi et al., 2020;[START_REF] Quemener | The powerful world of antisense oligonucleotides: From bench to bedside[END_REF]. To prevent miRISC complex repression, the synthesized TSB binds specifically to the miRNA recognition elements of the target RNA, preventing the miRNA from gaining access to the site, hence, restoring the RNA translation. Contrary to anti-miRNAs that directly inhibit miRNAs by binding to them with stable high-affinity bonds, TSB blocks the target site of a specific RNA without affecting other miRNA targets.

Figure 25 :

 25 Figure 25: LNA-enhanced Target Site Blockers Mechanism. LNA competes effectively with the miRISC complex for the miRNA binding site. From (qiagen.com).

3. 3 .

 3 Metabolism and Excretion Metabolism is the process in which the drug becomes chemically modified and broken down by the body. ASOs are metabolized by nucleases ubiquitously expressed by cells in most tissues. Nucleolytic cleavage occurs first by endonucleases in an internal region of the ASO as it recognizes the 2′-hydroxy group on the ribose, then exonucleases recognize the 3' and 5' hydroxy group and cleave the terminal sites of the ASO. The metabolites are inactive, suggesting that they do not have any other pharmacological effects.

Figure 26 :

 26 Figure 26: ASO Mediated Toxicities. From (Hammond et al., 2021).

Finally,

  Sonneville et al. approach was based on an LNA ASO acting as a TSB that competes specifically with miRNA-9 for binding to miRNA recognition elements of the TMEM16A mRNA. By preventing miRNA-9 from gaining access to TMEM16A mRNA, the authors increased TMEM16A expression and, thus, restored Cl -efflux and MCC in CF primary cells(Sonneville et al., 2017) (Figure28, Figure29).

Figure 28 :

 28 Figure 28: ASO TMEM16A Mechanism. In CF, miRNA-9 binds to the 3'UTR region of the TMEM16A mRNA and inhibits protein translation. The TMEM16A ASO competes with miRNA-9 and binds to the target region. TMEM16A ASO prevents miRNA-9 from gaining access to the target, thereby enabling protein translation and re-establishing TMEM16A expression and activity at the cell surface. Created with BioRender.com.

  Figure 29: TMEM16A, miRNA-9 and ASO TMEM16A Sequences. Adapted from (targetscan.org).



  TMEM16A ASO increases Cl -efflux and enhances mucociliary clearance  TMEM16A ASO does not alter intracellular calcium mobilization  TMEM16A ASO does not induce inflammation or cell proliferation  TMEM16A ASO is not toxic in vivo  TMEM16A ASO is detectable in mice 30 days after subcutaneous injection  TMEM16A ASO considerably increases the CF mouse lifespan  TMEM16A ASO improves fertility in CF male mice Enhancing TMEM16A activity constitutes a cutting-edge alternative for treating patients with CF independently of CFTR mutations, alone or in combination with CFTR potentiators and correctors to better improve clinical efficacy.IntroductionCystic fibrosis (CF) is an autosomal recessive genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene 1 . More than 2,100 different mutations have been described affecting protein production or activity. The CFTR chloride (Cl -) channel, present in many epithelia, regulates transepithelial solute and water fluxes. In CF, impaired Cl -ion efflux coupled with sodium (Na + ) ion hyperabsorption results in epithelial surface dehydration and tenacious, thick mucus that obstructs the lungs, the pancreatic ducts, the biliary tract, the intestine, and the male reproductive tract. Chronic lung infections and pulmonary inflammation are CF's most common clinical manifestations, leading to progressive tissue destruction and loss of function, hence elevated morbidity and mortality.

  TMEM16A stimulation, iodide ions flow down the concentration gradient into the cells and quench YFP fluorescence upon binding; the amount of quench is directly proportional to TMEM16A ion flux. The concentration ever used at 50 mM is in the middle of the range of activation. At 200 nM, modifications in the morphology of the cells were observed, suggesting a toxic dose confirmed by the lactate dehydrogenase (LDH) method after a treatment of 24h (data not shown).

  In 2001, a strategy targeting airway epithelial CaCC with Denufosol (INS37217) was proposed to treat CF lung disease and went into clinical trials20 . This drug activated CaCC by increasing intracellular Ca 2+ concentration, inducing adverse effects such as increased mucus in the airways. Our study demonstrated that ASO TMEM16A does not activate TMEM16A through elevating intracellular Ca 2+ since no Ca 2+ mobilization was observed in TMEM16A ASO-treated cells compared to control cells (Figure3 C-D).

  Luria-Bertani medium (LB), at 37 °C, 120 rpm shaking. The bacterial growth of P. aeruginosa is measured over time by luminescence measurement with the FLUOstar OPTIMA plate reader(BMG LabTech, France). The data are analyzed with the MARS Data Analysis Software v3.01 R2 (BMG LabTech, France).For cell infection, the culture medium of CFBE41o-is replaced with a Live Cell Imaging Solution medium at 1X (Life technologies™, USA), which allows working without CO2 during infection, and supplemented with 10% SVF (Eurobio, France) without antibiotics. The CFBE41o-are then infected with P. aeruginosa. The luminescence emitted is proportional to the number of bacteria and represents bacterial growth over time. The MOI (Multiplicity of infection) used are 0.01, 0.1, 1, and 10.TMEM16A chloride channel activity assayFor Ussing experiments, the bath solution contained (mM): 120 NaCl, 25 NaHCO3,3.3 KH2PO4, 0.8 K2HPO4, 1.2 MgCl2, 1.2 CaCl2, and 10 glucose. The pH of this solution is 7.3 -7.4 when gassed with a mixture of 94% N2, 1% O2, and 5% CO2 at 37°C. Chemicals were acquired from Sigma (St. Louis, MO). Each solution was formulated as 1000x stock and used at 1x in the Ussing chamber. All experiments were performed with low Cl -solution (6 mM) in the mucosal bath. In the low Cl -solution, all sodium chloride was exchanged for sodium gluconate, and Ca 2+ was increased to 5 mM to compensate for the chelation of Ca 2+ by gluconate. Pharmacologic preparations were the following: amiloride (100μM); forskolin (20 μM); Inh-172 (10 μM) and uridine triphosphate (UTP) (150 μM). Amiloride blocks epithelial sodium channels and supports the notion that variation in short-circuit current (ΔISC) from subsequent manipulation is due to effects on chloride channel activity. Forskolin stimulates CFTR via a cAMP-mediated mechanism. Inh-172 is a selective inhibitor of CFTR and aids in determining the degree of contribution of CFTR to the ISC under experimental conditions. UTP stimulates the CaCC TMEM16A by triggering P2Y receptor-mediated increases in intracellular Ca 2+ . The ISC was measured at one sample per second. By convention, a positive deflection in ISC is defined as the net movement of an anion in the serosal to the mucosal direction44 .

  days and/or stimulated with VX-770 and VX-809 (lumacaftor/ivacaftor, Orkmabi®, Boston, Massachusetts, USA) for 24h. One day after transfection, FluoSpheres Carboxylate-Modified Microspheres, 1.0 µm, yellow-green fluorescent (505/515) (Thermo Fisher Scientific) diluted in culture medium (1/50) were added to the apical membrane of the cultures. The movement of the beads was recorded under an

Fig. 1 .

 1 Fig. 1. TMEM16A ASO increases chloride efflux. A. Representative curve of Cl -efflux assessed by halide-sensitive YFP probe in CFBE41o-cells transfected with control ASO (Ctl ASO) or TMEM16A ASO for 24h at the indicated concentrations. B. Quantification of iodine (I -) uptake in (A)(n=4). C-D. The normalized total area under the curves of Ussing-chamber assays of non-CF (NCF) (C) and CF (D) hBEC transfected with Ctl ASO or TMEM16A ASO for 1 hour each day for three days at 50 nM. E. Average traces (n = 3) from Ussing-chamber assays of (C-D). Black arrowheads show when amiloride, forskolin, CFTR inhibitor 172, or UTP were added F. The normalized total area under the curves of Ussingchamber assays of non-CF hBEC when stimulated with UTP with or without Ani9 pretreatment (1 µM) (n=3). G. Representative curve of the Cl -efflux of the different indicated cell lines treated with the Ctl ASO (dotted curves) or TMEM16A ASO (full curves) for 24h at 50 nM. H. Quantification of iodine (I -) uptake in (G)(n=3). Data are represented as mean values ± SD. **p≤0.01; ***p≤0.001; ****p≤0.0001, ANOVA followed by Dunnett's, Bonferroni's, and Tukey's posthoc test.

Fig. 2 .

 2 Fig. 2. TMEM16A ASO increases mucociliary clearance in different cells in various CFTR mutations and does not affect other CF dysregulated parameters. A. Representative examples of fluorescent bead tracking on control or TMEM16 ASO treated hBEC (F508del/F508del). Scale bar: 100 µm. B-D. Bead mean speed measured in hBEC with (B) F508del/F508del, (C) 1707-1G>A/F508del and (D) 2184DelA/W1282x mutations after the indicated treatments. (n = 5 per condition, with 100 beads tracked per sample). E. Representative curve of bioluminescence production and bacterial growth over time of P. aeruginosa cultured alone with control or TMEM16A ASO (50 nM). F. Representative curve of bioluminescence production and bacterial growth over time of P. aeruginosa cultured at MOI of 0.1, 1.0, or 10 bacteria/cell in the presence of CFBE41o-pretreated with control or TMEM16A ASO (24h, 50 nM). G. Quantification of infected CFBE41o-cell surfaces treated with control ASO or TMEM16A ASO (24h, 50 nM) by crystal violet assay. The maximum cell area was indicated by the dotted line indicating the surface of cells without bacteria (n=5). H. IL-8 secretion of the indicated cells treated with TMEM16A ASO was measured by ELISA and normalized to the secretion of cells treated with Ctl ASO. Data are represented as mean values ± SD. **p≤0.01; ***p≤0.001; ****p≤0.0001, ANOVA followed by Dunnett's, Bonferroni's, and Tukey's posthoc test.

Fig. 3 .

 3 Fig. 3. TMEM16A ASO is specific and does not induce undesired effects. A. The ASO TMEM16A's binding profile results were expressed as a percent of inhibition of control specific binding [(100-measured specific binding/control specific binding) x100] obtained in the presence of TMEM16A ASO. Binding is considered positive if it is superior to 100%. B. Cytotoxicity was evaluated by Lactate dehydrogenase (LDH) quantification in CFBE41o-treated with Ctl and TMEM16A ASO (n=3). C-D. Mean peak values of ionomycin responses (10 µM) were evaluated in 16HBE cell lines labeled with fluo-4 after TMEM16A ASO transfection (50 nM) for 24h and were reported to the (C) 16HBE WT treated with TMEM16A ASO or (D) the similar Ctl cell line (n=5). E-F. Proliferation was evaluated in 16HBE cell lines after TMEM16A ASO transfection (50 nM) for 24h and is reported to the (E) 16HBE WT treated with TMEM16A ASO or (F) the similar Ctl cell line (n=5). Data are represented as mean values ± SD. No significance was found, ANOVA coupled with Dunnett's, Bonferroni's, and Tukey's posthoc test.

Fig. 4 .Fig. 5 .

 45 Fig. 4. Subcutaneous injection allows good ASO biodistribution that persists in time and induces no toxicity. A-D. Biodistribution studies in nude mice after intravenous, subcutaneous, intraperitoneal injections or intranasal instillation of fluorescent TMEM16A ASO at different time points: (A) day 0 (DO), (B) day 7 (D7), (C) day 21 (D21) and (D) day 30 (D30), (n=3 per group). E. Representative histological images of major organs were collected from treated mice following subcutaneous injection of TMEM16A ASO at a dose of 500 mg/kg (50 times the effective dose) 14 days post-injection. Scale bar: 20 µm.

Fig

  Fig. S1. TMEM16A ASO does not induce inflammation or toxicity.A. After 24h of treatment of cells with Ctl ASO or TMEM16A ASO (50 nM), media were collected, and cytokines were assayed by multiplex analysis (Bio-Rad, France). The induction of cytokines was reported in the control results for each cell line (n=3). B-E. (B) Serum alanine aminotransferase (ALAT), (C) aspartate aminotransferase (ASAT), (D) Urea, and (E) amylase levels were determined in control and treated male and female mice after injecting TMEM16A ASO subcutaneously at 0, 10, 20, 100, 300, and 500 mg/kg at 14 days post injection (n = 3 per group). F-G. (F) Serum alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), (G) Urea, and creatinine levels were determined 200 days after TMEM16A ASO repeated injections (10 mg/kg) for long-term toxicity observations (n=5 per group). Data are represented as mean values ± SD, and Student's t-test determined statistical significance. No significant difference was found.

Figure 30 :

 30 Figure 30: Body Weight Curve of CF Mice. Mice were treated with control or TMEM16A ASO. Each point represents the mean of the group. Due to the high mortality rate of the control CF mice, we limited our time point to 100 days.

Figure 33 :

 33 Figure 33: Fecal Pro-inflammatory Lipocalin. Lipocalin was quantified by Elisa in WT and CF mice. ****P<0.0001 by unpaired t-test.

FIGURE 2 |

 2 FIGURE 2 | (A)The proportion of publications published in Pubmed (https://www.ncbi.nlm.nih.gov/pubmed) by years about "anti-inflammatory,""mucolytic," and "antibiotic" in combination with "cystic fibrosis" compared to the total number of publications in CF. (B) The proportion of publications published in Pubmed by years about "anti-inflammatory,""mucolytic," and "antibiotic" in CF. (C) The proportion of publications published in Pubmed by years about "mucus,""infection," and "inflammation" in combination with "cystic fibrosis" compared to the total number of publications in CF. (D) The proportion of publications published in Pubmed by years about "mucus,""infection," and "inflammation" in CF.

FIGURE 4 |FIGURE 3 |

 43 FIGURE 4 | Interrelation between the main dysregulated aspects in the airway of Cystic Fibrosis patients. CFTR mutations affect inflammation, mucus properties, and infection. These different aspects are very intertwined, and treating one of these elements will have consequences on the other two.

FIGURE 5 |

 5 FIGURE 5 | List of the different categories of drugs under development or clinical trials in the context of CF (adapted from https://www.cff.org/Trials/pipeline/).

FIGURE 6 |

 6 FIGURE 6 | Description of the different classes of CFTR mutations related to the different therapeutic proposed in the literature. I-ynthesis defect, II-processing defect, III-channel gating defect, IV-channel conductance defect, V-reduced CFTR production, VI-defect of stability; ER, endoplasmic reticulum.

FIGURE 7 |

 7 FIGURE 7 | General mechanisms by which AMPs exert anti-inflammatory actions on host cells. AMPs can bind to bacterial virulence factors such as LPS or LTA and prevent their interactions with host cells. AMPs are also able to interfere with host cell signaling pathways involved in the inflammatory reaction. The overall consequence is that AMPs reduce the production of inflammatory mediators by these cells that may help in the resolution of inflammation.

  ) class I: no functional CFTR protein; (2) class II: CFTR trafficking defects; (3) class III: defective channel gating; (4) class IV: decreased channel conductance; (5) class V: reduced synthesis of CFTR; (

Figure 1 .

 1 Figure 1. Schematic representation of ANO1 channel protein. The ANO1 channel protein consists of 10 transmembrane domains (TMD). The intracellular loop between TMD-7 and TMD-8 contains six amino acids (N650, E654, E702, E705, E734, and D738) that participate in forming the calcium-binding pockets of the channel. The depicted location of two phosphorylation sites (serine 471 and serine 673) is denoted with the letter P.

Figure 2 .

 2 Figure 2. Schematic representation of the positive impacts of activating or inhibiting ANO1 in different pathologies.

  Une approche alternative avait été proposée dans les années 2000 avec le Dénufosol tetrasosium qui est un agoniste sélectif de P2Y2. Cette approche permettait une augmentation de la mobilisation calcique intracellulaire induisant une activation non spécifique des canaux chlorure calcium dépendant. Les premières études cliniques ont montré que cette approche semblait améliorer la capacité respiratoire des patients sans induire de toxicité. Malheureusement, une étude clinique de phase III effectuée sur 466 patients n'a pas validé ces résultats. Différentes explications ont été avancées pour expliquer cet échec comme le choix de la cohorte d'étude, la faible demi-vie de la molécule dans les fluides biologiques ou encore le caractère transitoire des effets passant par le calcium. Dans le cadre de nos travaux, nous avons repris cette idée de cibler une protéine qui pourrait avoir une activité proche de CFTR et qui serait localisée à la membrane apicale des cellules épithéliales. La protéine TMEM16a (ou ANO1 pour anoctamine 1) a été décrite et caractérisée en 2008 par trois équipes indépendantes (4-6) comme étant un canal chlorure exprimé au niveau des organes où se trouve CFTR avec une activité chlorure qui est calcium dépendant. Une telle approche est avantageuse pour contourner les défauts de CFTR et traiter tous les patients indépendamment de leurs mutations CFTR.

  Les oligonucléotides antisens (ASO) sont de nouvelles pistes potentielles pour le développement de traitements personnalisés pour certaines maladies héréditaires dont la mucoviscidose. Un ASO est un brin unique d'acide nucléique ou d'analogues d'acides nucléiques, de 15 à 20 nucléotides de longueur avec une séquence complémentaire à une séquence nucléotidique cible. Le Target Site Blocker (TSB) est un oligonucléotide particulier qui va se fixer de façon complémentaire à un brin d'ARN sur une séquence non codante pour empêcher la fixation d'un microARN (miR).

  Nous voudrions donc continuer à déterminer les effets de cette molécule sur les différents paramètres dérégulés dans la mucoviscidose, en déterminant le champ d'application et afin de préparer de futures études cliniques. Nos premiers résultats ont montré que l'ASO TMEM16A induit une amélioration de la clairance mucociliaire. Son effet est plus efficace que l'Orkambi®, la molécule de référence pour traiter les patients de classe II, sur les cellules de patients portant des mutations de classe I et/ou de classe II, et il existe même un effet additif de l'Orkambi® et de l'ASO TMEM16a chez certains de ces patients. Nous avons aussi montré, avec une sonde YFP sensible au halogénures, une augmentation de l'efflux chlorure dans des cellules CFBE. Ceci a ensuite été confirmé sur des cellules primaires par la méthode d'Ussing. Par des approches précliniques, nous nous sommes intéressés à montrer la spécificité et la non-toxicité de la molécule. Dans une approche in vitro, nous avons effectué par une CRO (clinical research organization) un « in vitro pharmaceutical profiling » qui est une technique qui permet d'étudier l'effet d'une molécule sur des cibles sensibles et qui pourraient induire des effets secondaires importants. Cette technique a été mise au point par quatre grands groupes pharmaceutiques (AstraZeneca, GlaxoSmithKline, Novartis, and Pfizer) et sert de référence dans les approches précliniques (Bowes et al., 2012). Nous avons pu montrer par cette technique que notre molécule n'active pas ou n'inhibe pas de façon significative des protéines d'une manière non spécifique. Sur des modèles cellulaires primaires, nous avons montré que notre molécule n'induit pas de lactate déshydrogénase (LDH) qui est un marqueur de toxicité cellulaire, et que cette molécule, contrairement au Dénufosol n'induit pas les efflux chlorures via la mobilisation calcique. Nous avons également montré que nous avons une réponse qui est dose-dépendante. De plus, nous avons montré par la technologie multiplex que l'ASO TMEM16a n'induit pas une sécrétion accrue des 24 cytokines que nous avons pu tester. Comme la surexpression de TMEM16A est connue pour être impliquée dans différents type de cancers, nous avons aussi montré l'absence d'effet de l'activation de TMEM16A sur la prolifération de cellules bronchiques non-CF et CF. Nous avons complété notre approche cellulaire par une approche in vivo en continuant de travailler sur le modèle murin CF F508del. Dans un premier, par utilisation d'un  17 th ECFS Basic Science Conference, 30 th March -3 rd April 2022, Albufeira, Portugal.  23 rd French Symposium for Young Researchers in Cystic Fibrosis, 15 th February 2022, Paris, France. (Online)  21 st French Symposium for Young Researchers in Cystic Fibrosis, 14 th February 2020, Paris, France. Poster Communications  17 th Respiratory Research Day (J2R), 14 th -15 th October 2022, Reims, France.  17 th ECFS Basic Science Conference, 30 th March -3 rd April 2022, Albufeira, Portugal.  15 th European Young Investigators Meeting (EYIM), 9 th -10 th March 2022, Paris, France. (Online) (Poster + Pitch Poster)  Saint Antoine Research Center Meeting, 25 th -26 th November 2021, Lieusaint, France. (Poster + Pitch Poster)  16 th Respiratory Research Day (J2R), 15 th -16 th October 2021, Brest, France.  22 nd Doctoral School Day (JED), 27 th -28 th May 2021, Paris, France. (Online)  22 nd French Symposium for Young Researchers in Cystic Fibrosis, 2 nd April 2021, Paris, France. (Online) (Poster + Pitch Poster) Scientific Vulgarisation  Meeting with Vaincre La Mucoviscidose Association members, 27 th September 2022, Paris, France.  Short video for scientific vulgarization for Vaincre La Muciviscidose Association. July 2022.  16 th Rally Against Cystic Fibrosis, Motards du Viaduc, 2 nd -3 rd July 2022, Campagnac, France.  Meeting with patients, Pre-French Symposium with Vaincre La Muciviscidose, 14 th February 2022, Paris, France. (Online)  Meeting with patients, Pre-French Symposium with Vaincre La Muciviscidose, 1 st April 2021, Paris, France. (Online)  Meeting with Blanche Association. (Online)

Table 1 : Onset of Cystic Fibrosis Disease by Approximate Age. Adapted from

 1

  bowel obstruction formed by thick and adhesive meconium that attaches to the intestinal mucosa initiating blockage at the terminal ileum level.Meconium ileus develops in utero at first and is often the first clinical manifestation of CF. Term babies with meconium ileus have CF in 80% of cases. Meconium ileus is

	very common in patients with class I-III CFTR mutations, typically associated with
	lower CFTR function
	-efflux causing
	lower pH and thick secretions, which is the primary reason behind all gastrointestinal
	complications (De Lisle & Borowitz, 2013).
	4.2.1. Luminal Region

4.2.1.1. Luminal Obstruction

Meconium ileus is estimated to occur in 10 to 20% of neonates with CF. It is characterized by a

Table 2 : Approved CFTR modulators

 2

. (Last checked September 2022).

  CFTR missense mutation (class III) present in 4 to 5% of patients with CF (J. C. Davies

	Brand name Modulator	Approval year	Current approved ages	FEV1 changes vs placebo	CF mutation eligibility	CF population eligibility	Price in US per year per patient	Price in France per year per patient
	Kalydeko	Ivacaftor	2012	> 4 months	10.6 %	At least one gating class III mutation and the R117H mutation	3 -4 %	$311,000	250,00 €
				> 1 year in
	Orkambi	Ivacaftor + Lumacaftor	2015	US > 2 years in	2.8 %	Homozygous F508del	50%	$272,623	130,000 €
				France
						Homozygous
	Symdeko (or Symkevi)	Ivacaftor + Tezacaftor	2018	> 6 years	5.1 %	F508del or and residual function One copy of F508del	55%	$292,200
						mutation
	Trikafta (or Kaftrio)	Ivacaftor + Tezacaftor + Elexacaftor	2019 in US 2020 in FR	> 6 years	22.5 %	At least one F508del irrespective of the second mutation	90%	$311,741	270,000 €

  drug is being developed by Enterprise Therapeutics to inhibit the function of ENaC. This therapy applies to all patients and helps maintain fluid within the airways to improve mucus clearance. The phase I study was completed, and a phase II study is being planned (enterprisetherapeutics.com).

	7.4. ENaC Channel Inhibitors
	ETD001 novel inhaled ION-827359 is another inhaled ENaC inhibitor being developed by Ionis
	Pharmaceuticals and is currently in a phase 1/2a study (NCT03647228) (See chapter
	3, paragraph 5).
	Other compounds were also developed to block the ENaC sodium channel, such as
	BI 1265162 (Boehringer Ingelheim), QBW276 (Novartis Pharmaceuticals), and VX-
	371 (Vertex Pharmaceuticals Inc. and Parion Sciences), among many others.
	Therapeutics) or override premature stop signals (Icagen, SpliSense) (See chapter 3,
	paragraph 5) to treat all patients with CF regardless of their specific CFTR gene
	mutation.

7.3. Molecules in the Pre-Clinical Phase for CFTR Function Restoration Other interesting gene therapy programs aim to deliver a WT CFTR gene directly into the lungs (Arcturus therapeutics, Carbon Biosciences, Spirovant Sciences, ReCode

Table 3 : Summary of TMEM16A Inhibitors and Activators Used in CF. Modified

 3 from(Mitri et al., 2021).

	Inhibitors	Specificity	Assay	References
	Ani9	Not specific	In vitro	(Danahay & Gosling, 2020; Ramu et al., 2014)
	CCinh-A01 Not specific	In vitro, in vivo	(Genovese et al., 2019)
	DIDS	Not specific	In vitro	(Gianotti et al., 2016)
	Flufenamic acid Not specific	In vitro	(McCarty et al., 1993; Reddy & Quinton, 2002)
	Niclosamide Not specific	In vitro, in vivo	(Fischer et al., 2010)
	Niflumic acid Not specific	In vitro, in vivo	(Cabrita et al., 2019)
	Plumbagin	Not specific	In vitro	(Scott-Ward et al., 2004)
	Quercetin	Not specific	In vitro, in vivo, clinical trial (phase II)	(Pyle et al., 2010; B. Yu et al., 2019; Zhang et al., 2011)
	Tannic acid Not specific	In vitro	(NCT01348204)
	T16ainh-A01	Specific	In vitro	(Genovese et al., 2019)
	Activators
				(Accurso et al., 2011; Deterding et
	Denufosol (INS37217)	Not specific	In vitro, in vivo, clinical trial (phase III failed)	al., 2007; Deterding et al., 2005; Goss et al., 2008; Kellerman et al., 2008; Moss, 2013; Ratjen et al.,
				2012)
	Duramycine (MOLI1901)	Not specific	In vitro, in vivo, clinical trial (phase II failed)	(NCT00671736)
	Eact	Not specific	In vitro	(Genovese et al., 2019)
	ETX001	Specific	In vitro, in vivo, clinical trial (phase I)	(Danahay et al., 2020b)
	Interleukin-4 Not specific	In vitro	(Caputo et al., 2008b)
	Resveratrol Not specific	In vitro, in vivo, clinical trial	(NCT02690064, NCT04166396)
	ASO TMEM16A	Specific	In vitro, in vivo, preclinical	(Sonneville et al., 2017)

Table 4 : Main Chemical Modifications of Antisense Oligonucleotides

 4

. Adapted from

[START_REF] Dhuri | Antisense Oligonucleotides: An Emerging Area in Drug Discovery and Development[END_REF]

.

  The 2789+5G->A mutation (class V) causes skipping over exon 16, leading to a frameshift and a premature termination codon. SPL16-ASO avoids skipping over

	exon	16,	generating	a	fully	functioning	WT	CFTR
	(https://splisense.com/pipeline/).
	 The W1282X mutation (class I) generates a premature termination codon (PTC)
	and activates the nonsense-mediated RNA decay, leading to RNA degradation.
	SPL23-ASO skips over exon 23 carrying the mutation, leading to mature and
	active CFTR (Oren et al., 2022).
	In 2017, Ionis Pharmaceuticals wanted to decrease ENaC channel hyperactivity to
	decrease the Na + absorption that leads to mucus dehydration and accumulation in
	patients with CF. They identified several potent ENaC-specific ASOs and tested them
	by inhalation in mouse models of CF-like lung disease. Promising results showed that
	the ASOs could penetrate through the mucus into the lung cells and reduce the
	corresponding ENaC subunit level and ENaC function, reversing the disease.
	Reduction of any of the ENaC subunits normalized sodium channel activity, mucus

 The N1303K (class II) mutation results in the production of a misfolded protein. SPL24-N-ASO skips over exon 24, carrying the mutation, avoiding the maturation defect and proteasomal degradation, and generating mature and active CFTR (https://www.cipherbio.com/dataviz/organization/SpliSense/products). Splisence has also developed ASOs to lower mucin expression and production in mucus using an exon-skipping ASO, leading to premature termination codon and, thus, truncated protein degradation (https://splisense.com/pipeline/spl5/). Kim et al. also developed a cocktail of two PS-MOE ASO that target the splicing sites flanking exon 23 and induce efficient exon 23 skipping in the W1282X mutation. The researchers demonstrated that treating hBECs with this ASO cocktail results in nonsense-mediated RNA decay-resistant mRNA increasing CFTR-Δex23 protein and

CFTR-mediated Cl -current (Y. J.

[START_REF] Kim | Exon-skipping antisense oligonucleotides for cystic fibrosis therapy[END_REF]

.

production, inflammation, and improved lung function

(Crosby et al., 2017;[START_REF] Zhao | Antisense oligonucleotide targeting of mRNAs encoding ENaC subunits α, β, and γ improves cystic fibrosis-like disease in mice[END_REF]

. Currently, ION-827359 (or IONIS-ENaCRx), an αENaC inhibitor ASO (16 80 nucleotides in length with a PS backbone and constrained ethyl modifications), has demonstrated strong evidence of tolerability and safety to support further investigation in patients with CF (Newman et al., 2021) (NCT03647228).

  5'-triphosphate (UTP) activate Cl - efflux in normal and CF airway epithelia26 . So, before identifying the airway epithelial CaCC in 2008, Denufosol was developed as a treatment for CF lung disease and was carried into clinical trials in 200427 . However, after some promising results in CF

patients, the second phase III trial failed to demonstrate any benefits. Two points emerged to explain this failure: first, the effects of Denufosol were indirect and mobilized intracellular Ca 2+ to activate CaCC; second, the stability of the Denufosol was very low in CF sputum with a half-life of only 17 minutes 20 . Our approach using ASOs, a novel therapeutic tool that binds specifically to the targeted mRNA via complementary Watson-Crick base pairing, can prevent such issues as ASOs do not interfere with Ca 2+ mobilization.

  -transport decreases ASL pH and is related to altered mucus properties[START_REF] Budai-Szűcs | Rheological effects of hypertonic saline and sodium bicarbonate solutions on cystic fibrosis sputum in vitro[END_REF][START_REF] Yang | Normal mucus formation requires cAMP-dependent HCO3-secretion and Ca2+-mediated mucin exocytosis[END_REF]. Another recent study byMorrison et al. demonstrated that airway hydration was the main factor responsible for modulating mucus properties and that airway dehydration in CF caused mucin polymer entanglement[START_REF] Morrison | Treatment of cystic fibrosis airway cells with CFTR modulators reverses aberrant mucus properties via hydration[END_REF]. Hence, restoring water and HCO3 -efflux via TMEM16A might help decrease mucus viscosity by providing mucin disentanglement and, thus, increasing its

transportability. Overall, the above-cited factors should be closely studied to understand how increased TMEM16A function restores MCC.

  terms of feasibility, in vitro models like air-liquid interface cultures and organoids are better suited in our case to answer particular research questions. Although organoids

	ASOs	Approval Date by FDA	Approval Date by EMA	Length (nucleotide)	Chemistry	Disease	Target	Admnistration route	Target	Absorption	Distribution	Formulation	Dosing	Half-life	Mode of action	Charge	Shared common ADRs	Shared Warnings and Precautions
	Fomivirsen	26/08/1998 (withdrawn 2006 )	29/07/1999 (withdrawn 2002)		2′-H	Cytomegalovirus	IE2 mRNA	Intravitreal injection	Eye	Local eye	Retina, iris Systemic exposure: minimal quantitation) of (below limits	0.33 mg in 50 ml	Loading dosing: twice every 4 weeks dosing: once other week Maintenance	55 hours	RNase H cleavage	Negative
											CNS, plasma,		First 3 doses
											peripheral		with 14 days
											tissue		interval, the 4th
	Nusinersen	23/12/2016	30/05/2017		PS-2ʹ-MOE	Spinal muscular atrophy	Survival motor neuron-2 (SMN2) mRNA	Intrathecal injection	CNS	CNS and systematic	(skeletal muscle, liver, kidney) and	12 mg in 5 mL	dose 30 days after the 3rd dose,	163 days	Splice modulation	Negative
											fat, bone		Maintenance
											marrow and		dosing: once
											spleen		every 4 months
											Liver, kidney,
	Mipomersen	29/01/2013	Refused		2′-MOE	Familial hypercholesterole mia	Apolipoprotein B mRNA	SC injection	Liver	Systematic	bone marrow, adipose tissue, lymph nodes	200 mg in 1 mL	once weekly	30 days	RNase H cleavage	Negative	Injection site reactions, Headache,
																	Pyrexia,	Hepatotoxicity,
																	Fever,	Kidney toxicity,
	Inotersen	05/10/2018	05/07/2018		2′-MOE	Polyneuropathy of amyloidosis	Transthyretin (TTR) mRNA	SC injection	Liver	Systematic	Liver, kidney, broad tissues	284 mg in 1 mL	once weekly	32.3 days	RNase H cleavage	Negative	Cough, Respiratory infection,	Hypersensitive reactions
																	Vomiting,
	Volanesorsen	Refused	2019 conditional marketing authorization		2′-MOE	Chylomicronemia Syndrome Familial	Apolipoprotein C3 mRNA	SC injection	Liver	Systematic	Liver, kidney, broad tissues	300 mg in 1 mL	once weekly	>2 weeks	cleavage RNase H	Negative	Nausea
	Eteplirsen	19/09/2016	Refused		PMO	Duchenne muscular dystrophy	Exon 51 dystrophin pre-mRNA	IV infusion	Skeletal muscle	Systematic	Kidney, skeletal muscles	30 mg/kg	35-60 min infusion once weekly	3-4 hours	Splice modulation	Uncharged
	Golodirsen	12/12/2019			PMO	Duchenne muscular dystrophy	Exon 53 dystrophin pre-mRNA	IV infusion	Skeletal muscle	Systematic	Kidney, all tissue except CNS	30 mg/kg	35-60 min infusion once weekly	3-4 hours	Splice modulation	Uncharged
	Viltolarsen	12/08/2020			PMO	Duchenne muscular dystrophy	Exon 53 dystrophin pre-mRNA	IV infusion	Skeletal muscle	Systematic	Kidney, skeletal muscles	80 mg/kg	60 min infusion once weekly	2.5 hours	Splice modulation	Uncharged
	Casimersen	25/02/2021			PMO	Duchenne muscular dystrophy	Exon 53 dystrophin pre-mRNA	IV infusion	Skeletal muscle	Systematic	Kidney, skeletal muscles	30 mg/kg	35-60 min infusion once weekly	3-4 hours	Splice modulation	Uncharged
	Milasen	January 2018 n-of-1 approval	-		PS-2ʹ-MOE	Neuronal Ceroid Lipofuscinosis 7	Exon 6-exon 7 Major Facilitator Superfamily Domain Containing 8	Intrathecal injection	CNS	CNS and systematic		Starting at 3.5 mg and increasing every 2 weeks up to	Maintenance doses every 3 months		Splice modulation	Negative
							(MSFD8) mRNA					42 mg
			INTRODUCTION
			Cystic fibrosis (CF) is the most common lethal monogenic disorder in Caucasians estimated to affect
			one out of 2.500-4.000 newborns. It is caused by a Cystic Fibrosis Transmembrane conductance
			Regulator (CFTR) gene mutation, which encodes a chloride channel expressed at the apical
			membrane of the epithelial cells

cannot model the whole pathophysiological CF processes, they can be used for more personalized medicine approaches, as CF-derived respiratory and intestinal organoids can be generated from patient samples. With such models, we can test by swelling assays the efficacy of TMEM16A ASO on different rare CFTR mutations, alone or with different drug combinations such as TMEM16A ASO with Trikafta or ETX001 for optimal clinical outcomes, especially since CF mice models are unresponsive to CFTR modulators.

 Conclusion

This work presents a new therapeutic strategy using an ASO to restore the expression and activity of the TMEM16A, a promising alternative chloride channel to compensate for the lack of CFTR-mediated anion secretion in CF. This strategy can be applied to all patients with CF regardless of their mutations and target multiple fronts in CF pathologies, such as lung MCC, intestinal obstruction, fertility problems, and teeth abnormalities. Preclinical results show that the proposed molecule does not induce toxicity, inflammation, or calcium mobilization and is specific to the 3'UTR TMEM16A mRNA target. Further studies will be needed to move this molecule from bench to bedside.

Table 1 .

 1 Summary of ANO1 inhibitors and activators used in CF.

	Inhibitors	Specificity	Assay	References
	ANI9	Not specific	In vitro	[82,85]
	CCinh-A01	Not specific	In vitro, in vivo	[86]
	DIDS	Not specific	In vitro	[87]
	Diphenylamine-2-carboxylate
	(DPC), 5-nitro-2-(3-phenylpropylamino) benzoic	Not specific	In vitro	[88]
	acid
	Flufenamic acid	Not specific	In vitro	[89,90]
	Niclosamide	Not specific	In vitro, in vivo	[91]
	Niflumic acid	Not specific	In vitro, in vivo	[92]
	Plumbagin	Not specific	In vitro	[93]
	Quercetin	Not specific	In vitro, in vivo, clinical trial (phase II)	[94-96]
	Tannic acid	Not specific	In vitro	[97]
	T16ainh-A01	Specific	In vitro	[86]
	Activators
			In vitro, in vivo,
	Denufosol (INS37217)	Not specific	clinical trial (phase III	[77-81,98,99]
			failed)
			In vitro, in vivo,
	Duramycine (MOLI1901)	Not specific	clinical trial (phase II	[100]
			failed)
	Eact	Not specific	In vitro	[86]
	ETD002 (or ETX001)	Specific	In vitro, in vivo, clinical trial (phase I)	[101]
	Interleukin 4	Not specific	In vitro	[14]
	Resveratrol	Not specific	In vitro, in vivo, clinical trial	[102-104]
	TSB ANO1	Specific	In vitro, in vivo, preclinical	[83]

Frontiers in Pharmacology | www.frontiersin.org July 2020 | Volume 11 | Article 1096

l'obstruction intestinale, les problèmes de fertilité et les anomalies dentaires. Les résultats précliniques montrent que la molécule proposée n'induit pas de toxicité, d'inflammation ou de mobilisation du calcium et qu'elle est spécifique à sa cible 3'UTR de l'ARNm de TMEM16A. D'autres études seront nécessaires pour démontrer l'efficacité de la molécules chez les mutations de classe I et faire passer cette molécule vers la clinique.

Acknowledgments

This work is supported by grants from the "Blanche pour Vaincre la Mucoviscidose", "Vaincre la Mucoviscidose" associations and SATT Lutech. 101 CF 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).

Multiplex assays were performed by CYBIO facilities at Institut Cochin (Paris, France).

druggable molecular targets to decrease inflammation is still an unsatisfied demand that needs numerous additional researches.

Acknowledgments: We thank Satt Lutech, Vaincre la Mucoviscidose, and Blanche pour Vaincre la Mucoviscidose for their support.

FUNDING

This review was funded in part by grants from Inserm, Sorbonne Universite, Facultédes Sciences, Institut Pasteur, and the nonprofit organization Vaincre la Mucoviscidose. CM received a Ph.D. grant from Vaincre la Mucoviscidose. ZX was founded by Yangzhou University.

Funding: This research group was funded by grants from Inserm, Sorbonne University, Satt Lutech, and the French nonprofit organization "Vaincre la Mucoviscidose." C.M. received a PhD fellowship from "Blanche pour Vaincre la Mucoviscidose".

List of Tables

List of Appendices

List of Abbreviations

16HBE

Aim of The Work Abstract

Patients with cystic fibrosis (CF) bearing mutations non-responsive to current treatments need alternative CFTR-independent therapies. Here we study the effects of TMEM16A potentiation on dysregulated parameters in CF and prepare for preclinical studies by assessing the best administration route and the TMEM16A oligonucleotide's toxicity and specificity. The oligonucleotide potentiates the activity of TMEM16A, increases Cl -efflux, and enhances mucociliary clearance. TMEM16A ASO is detectable in mice 30 days after subcutaneous injection and considerably increases the CF mice's lifespan. Acute administration of 50 times the effective dose did not show any toxicity. TMEM16A ASO is specific and does not induce inflammation or alter intracellular calcium mobilization and cell proliferation. Overall, TMEM16A compensates for CFTR deficiency in CF, and this strategy could apply to all patients independently of their CFTR mutation.

Keywords: CFTR-independent therapy; TMEM16A; calcium-activated chloride channel; oligonucleotides; preclinical studies; RNA therapy The human bronchial tree is covered with a continuous layer of epithelial cells whose primary function is facilitating the gas exchange to and from the alveoli and protecting the airway from inhaled pathogens. The airway epithelium in the large airways (trachea and bronchi) forms a pseudostratified layer consisting of ciliated cells, club cells, and goblet cells. Goblet cells secrete mucins to trap pathogens, and ciliated cells are primarily responsible for mucociliary clearance. The submucosal glands are specialized structures essential for maintaining airway homeostasis. Like goblet cells, submucosal glands secret mucins in the large airways via a ciliated duct, which extends to collecting ducts formed by mucous tubules and serous acini. In the small airways, the cell types are similar, with relatively more ciliated cells, and the secretory cells shift to the Club cell type. Club cells (also known as Clara cells) have several protective roles, including airway repair, anti-inflammatory and immunomodulatory proteins secretion, and detoxification. Afterward, the airway epithelium merges with the alveolar epithelium, with type I and II pneumocytes. Basal cells are progenitor cells at high numbers within the epithelium and are crucial for regeneration. A variety of less common cell types, such as Ionocytes and neuroendocrine (PNEC) cells, are also present, among others. Recently, pulmonary ionocytes have been described; their role in ion transport, regulation of mucus, and high CFTR expression suggest that they play a critical role in CF biology and disease. CF mice model has proven to be very useful over the years for genomic studies as the mouse Cftr is 78% similar to the human orthologue. However, there are still critical physiological differences between human and mice CF pathology.

Additional results

Conclusion and Discussion

Appendices

In our study, we used the 129-Cftr tm1Eur CF mice, homozygous for the F508del mutation [START_REF] French | A delta F508 mutation in mouse cystic fibrosis transmembrane conductance regulator results in a temperature-sensitive processing defect in vivo[END_REF], with normal levels of Cftr mRNA. Unlike humans, the leading cause of death in CF mice is an intestinal obstruction which is not surprising due to the vital role of CFTR in the mice's intestinal tract. Moreover, CF mice have poor growth compared to their wild-type littermates, a reduced survival rate, and tend to die upon weaning [START_REF] Wilke | Mouse models of cystic fibrosis: Phenotypic analysis and research applications[END_REF]. Although intestinal abnormalities are no longer the first cause of death in humans, the evident intestinal pathology in CF mice validates their use to study human CF intestinal disease. Similarly, our model displays abnormal enamels, abnormal bone density (Wright et al., 1996a;Wright et al., 1996b), osteopenia [START_REF] Henaff | Enhanced F508del-CFTR Channel Activity Ameliorates Bone Pathology in Murine Cystic Fibrosis[END_REF][START_REF] Velard | Cystic fibrosis and bone disease: defective osteoblast maturation with the F508del mutation in cystic fibrosis transmembrane conductance regulator[END_REF], and male infertility caused by obstruction or absence of the vas deferens [START_REF] Durie | Characteristic Multiorgan Pathology of Cystic Fibrosis in a Long-Living Cystic Fibrosis Transmembrane Regulator Knockout Murine Model[END_REF][START_REF] Reynaert | Morphological Changes in the Vas Deferens and Expression of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) in Control, delta F508 and Knock-Out CFTR Mice During Postnatal Life[END_REF], just like in humans with CF.

The trachea, salivary gland, liver, biliary duct, gallbladder, pancreas, and kidney, which could also be affected in Cftr mice mutant [START_REF] Wilke | Mouse models of cystic fibrosis: Phenotypic analysis and research applications[END_REF], were not characterized in our study. Cystic fibrosis (CF) is the most common genetic disorder among Caucasians, estimated to affect more than 70,000 people in the world. Severe and persistent bronchial inflammation and chronic bacterial infection, along with airway mucus obstruction, are hallmarks of CF lung disease and participate in its progression. Anti-inflammatory therapies are, therefore, of particular interest for CF lung disease. Furthermore, a better understanding of the molecular mechanisms involved in airway infection and inflammation in CF has led to the development of new therapeutic approaches that are currently under evaluation by clinical trials. These new strategies dedicated to CF inflammation are designed to treat different dysregulated aspects such as oxidative stress, cytokine secretion, and the targeting of dysregulated pathways. In this review, we summarize the current understanding of the cellular and molecular mechanisms that contribute to abnormal lung inflammation in CF, as well as the new anti-inflammatory strategies proposed to CF patients by exploring novel molecular targets and novel drug approaches.

AUTHOR CONTRIBUTIONS

All authors: CM, PB, ZX, HC, LT, and OT have written and reviewed the manuscript.

Conflict of Interest:

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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