Treatment and prevention of cardiac and fibrotic disorders

Abstract

The invention relates to an oligonucleotide comprising one of the sequences selected from: (i) 5'- +T C A +G T C +T G A +T A +A G +C +T -3' (formula I); or (ii) 5'- +T C +A G T +C T G +A +T A +A G C +T -3' (formula II); wherein A, T, G and C are deoxyribonucleotide building blocks, or C is 5-methyl-2'-deoxycytidine; and wherein +T, +A, +G, and +C are bridged nucleotide building blocks and / or morpholino building blocks. Also provided are oligonucleotides and compositions comprising the same for therapeutic use.

Description

TREATMENT AND PREVENTION OF CARDIAC AND FIBROTIC DISORDERSThe project leading to this application has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 825670.FIELD OF THE INVENTION

[0001] The present invention refers to oligonucleotides, which are effective inhibitors of microRNA 21 (miR-21), compositions comprising the same, and their use in medicine, particularly in the prevention or treatment of cardiac disorders, inflammation and / or fibrotic disorders.BACKGROUND

[0002] Heart diseases represent an increasing burden for families and the healthcare system globally with prevalence, hospitalization and mortality rates far exceeding those of other diseases, with heart failure one of the leading pathological causes of mortality in the world. Myocardial infarction (Ml) is the most important cause of heart failure as Ml leads to subsequent progressive remodelling of the heart (e.g., impaired contractility, tissue necrosis, pericardial inflammation) resulting in heart failure with poor prognosis. The therapeutic pharmacologic options currently used for treating heart failure include SGLT2-inhibitors, angiotensin-modulating agents, beta-blockers, diuretics, aldosterone antagonists, combined neprilysin inhibitor with angiotensin-2-receptor blocker, vasodilators, inotropic agents or semaglutide. Although several clinical studies have shown significant decreases in heart failure-induced mortality rates for all these agents, the 5-year mortality rate remains unacceptably at almost 50%. Thus, there is a great need to develop novel and more efficient therapeutic approaches for heart failure.

[0003] Pathological hypertrophic growth of cardiomyocytes can lead to the development of cardiac remodelling, heart failure and sudden cardiac death. Hypertrophic growth of cardiomyocytes is a response to increased cardiac wall stress caused by cardiac volume and / or pressure overload. Initially, cardiac hypertrophy is a compensatory mechanism aiming to decrease wall stress and to increase cardiac output. However, prolonged cardiac hypertrophy progresses to contractile dysfunction, cardiac decompensation and finally heart failure (Hill and Olson, 2008; Barry and Townsend, 2010). The transition from physiological to pathological hypertrophy canoccur depending on many factors including myocyte loss through apoptosis or necrosis, alterations in autophagy, defects in contractile response, dysregulated calcium homeostasis, desensitization of adrenergic receptors, or cardiac fibrosis (Hill and Olson, 2008; Barry and Townsend, 2010).

[0004] Cardiac remodelling and cardiac inflammation are hallmarks of heart failure. Cardiac inflammation is a key component in cardiovascular disease, and a natural reaction to an infection or injury to the heart, generally affecting the lining of the heart or valves, heart muscle, or tissue surrounding the heart. This can, amongst others, lead to arrhythmia, heart failure and coronary heart disease. The three main types of heart inflammation have been identified as endocarditis, pericarditis, and myocarditis. While endocarditis refers to inflammation of the inner lining of the heart chambers and valves, pericarditis concerns inflammation of the pericardium. Myocarditis generally defines acute inflammation and damage of the heart muscle. Common causes may include viral and / or bacterial infections and medical conditions (e.g., autoimmune diseases (lupus)).

[0005] On the other hand, cardiopulmonary disease (pulmonary heart disease, PHD) is a heterogeneous condition that affects the heart and the lungs. Most patients with PHD have relatively mild pulmonary vascular disease and cardiac dysfunction. In these patients, it is the lungs, and not the heart that remains the primary therapeutic target. In contrast, a smaller subset of patients with chronic respiratory disease (CRD) can develop severe pulmonary vascular disease (PVD) and right heart dysfunction. These patients typically clinically deteriorate despite optimization of treatment of their underlying CRD, suggesting they have taken on a second disease. The failure to recognize the different phenotypes of pulmonary heart disease (PHD) oversimplifies the condition, which increases the potential for misapplication of therapy. Hence, there is an unmet need for effective therapies that target different patient groups.

[0006] Pulmonary inflammation is characterized by an increased responsiveness of the trachea, bronchi and alveoli to various stimuli and manifested by a widespread airway narrowing causing episodic dyspnea, coughing and wheezing and the associated debilitation of the afflicted person. Pulmonary inflammation causes may include, amongst others, lung injury, systemic diseases (e.g., fibromyalgia, lupus, rheumatoid arthritis etc.), asthma, infections (e.g., molds, bacteria) and / or radiation treatments.

[0007] Cytokines represent a major mediator of inflammation, and it has been shown that the levels of inflammatory cytokines are increased in patients with heart failure(Gullestad et al., 2012). Expression of the pro-inflammatory cytokines TNF-alpha, interleukin (IL)-1beta and IL-6 is consistently induced in cardiac fibrosis (Kong et al., 2014; Tanaka etal., 2014). For instance, IL-6 induces expression of a variety of proteins responsible for acute inflammation, while TNF-alpha exerts pleiotropic effects on a variety of cell types and has been reported to also be crucial in the process of cardiac fibrosis (Jiang et al., 2021). Although antibodies against IL-6 have been used in certain disease settings, results of clinical trials using antibodies to block TNF-alpha gave disappointing results, showing only short-term improvement that was fully reversible after the cessation of therapy or worsening of heart failure (Chung et al., 2003; Mann et al., 2004). However, the reasons of these disappointing results remain elusive. Hence, controlling inflammatory events holds a great potential for novel therapeutic treatment options. Like in cardiac inflammation, IL-6 has been implicated to play a critical role in the progress of lung inflammation and / or injury (Yu et al., 2002; Rincon and Irvin, 2012).

[0008] Treatment of cardiac and cardiopulmonary inflammation may include corticosteroids, (non-steroidal) anti-inflammatory drugs, antibiotics, intravenous immunoglobulin, and potentially even heart surgery, pericardiocentesis and / or implantable cardioverter. Various therapeutic candidates including anti-hypertensive therapies, heart rate lowering drugs, anti-inflammatory agents, as well as growth factor inhibitors have been used for the treatment of cardiac fibrosis. Antihypertensive classes, including beta-blockers and calcium channel blockers, have shown efficacy in reducing fibrosis in non-human animals; however, results in humans have been inconsistent (Roubille et al., 2014; Leask et al., 2010). While existing drugs and new chemical compounds are in various stages of development, current treatments have drawbacks such as unwanted side-effects. Moreover, despite decades of research and development in all areas related to cardiac diseases and heart failure, current treatment options primarily alleviate the symptoms and do not address the underlying root causes.

[0009] Like cardiopulmonary disease, fibrosis is generally very heterogeneous and an essential process in wound healing. However, excessive fibrosis is common in many disease conditions and plays an important role in disease pathogenesis. Diseases characterized by excessive fibrosis include, amongst others, hypertrophic cardiomyopathy, dilated cardiomyopathy (DCM), atrial fibrillation, ventricular fibrillation, myocarditis, asthma, and idiopathic pulmonary fibrosis. Cardiac fibrosis is a common pathophysiologic process in most heart diseases and characterised by scarring events in the cardiac muscle and hardening of tissues and organs that lead to cardiac fibroblast(CF) activation and their differentiation into myofibroblasts. This is followed by an increase in and excess production of extracellular matrix (ECM) proteins such as collagen and proteases including the matrix metalloproteinases (MMPs) (Jiang et al., 2021). The resultant disturbance of the ECM homeostasis leads to pathological ECM remodelling and profound structural and functional abnormalities in matrix composition and quality (e.g., matrix stiffness), as well as the heart muscle (e.g., diastolic and systolic dysfunction) (Berk et al., 2007; Kong et al., 2014). While cardiac fibrosis is associated with different cardiovascular diseases, including heart failure, hypertension, and cardiomyopathies, fibrotic scaring of the cardiac muscle most commonly occurs after myocardial injury, when CFs are converted into myofibroblasts by upregulating expression of pro-inflammatory cytokines.

[0010] As to the treatment of (cardiac) fibrosis, there are currently no drugs on the market with primarily anti-fibrotic action that have been proven to reverse the conditions (or halt their progress) associated with cardiac fibrosis. Antifibrotic drugs known from clinical studies include, for example, RASS inhibitors lisinopril, losartan, and spironolactone, inflammatory modulators, such as, etanercept, infliximab, colchicine, and atorvastatin, and TGF-beta signalling inhibitors. However, the role of statins in the treatment of chronic heart failure (HF) is still controversial. Although several retrospective studies have revealed a better prognosis for patients with HF treated with statins, two randomized clinical trials, GISSI-HF (Italian Group for the Study of Survival in Heart Failure Failure) and CORONA (Controlled Rosuvastatin Multinational Trial in Heart Failure), reported no prognostic benefit from rosuvastatin treatment. Furthermore, anti-TGF-beta antibody therapy has also been associated with serious adverse effects (Frantz et al., 2008). More recent studies have even looked at using CAR-T cells engineered in vivo to contain or express a receptor directed against the FAP protein, which is expressed by cardiac myofibroblasts (Morfino et al., 2022). However, despite the ongoing research, there is currently no anti-fibrotic drug that has clearly demonstrated the regression of fibrosis and the health improvement in clinical trials.

[0011] Accordingly, as the therapeutic and diagnostic methods relating to cardiac inflammation, cardiopulmonary inflammation, and cardiac fibrosis still require further improvement and new approaches, there is an unmet medical need for new therapeutic agents to diagnose and treat these conditions. As will be shown throughout the specification, with the present invention the inventors aim to address this need.

[0012] Antisense oligonucleotide (ASO)-based therapies have been gaining more traction over the years for the treatment of different diseases or genetic disorders including cardiovascular disease. ASOs are short, single-stranded synthetic RNA or DNA molecules that use Watson Crick base pairing to bind to the endogenous target RNA. To ensure specificity, their sequences are generally complementary to the target RNA. ASOs can be chemically modified to protect them against the action of nucleases that would otherwise degrade them and / or to increase their effectiveness. ASOs can be broadly classified into 1st, 2nd, and 3rdgeneration ASOs. The first ASOs were employed to inhibit translation of Rous sarcoma virus ribosomal RNA (Stephenson and Zamecnik, 1978). While 1stgeneration ASOs are characterised in having a modified backbone, wherein the nucleotides are linked by sulfur, methyl or amine groups to generate phosphorothioates (PS), methyl-phosphonates, and phosphoramidates, respectively, 2ndgeneration ASOs additionally carry alkyl modifications at the 2’ position of the ribose. These 2ndgeneration ASOs tend to be less toxic than PS-modified ASOs and have a slightly higher affinity for their target. 3rdgeneration ASOs tend to be even more heterogenous as they include a large number of chemical modifications that aim to improve binding-affinity, stability, and pharmacokinetics (Quemener et al., 2019). The diversity of chemical modifications, together with the sequence of the ASO, offers considerable flexibility as relates to the therapeutic approach. That is, depending on their mechanism of action, ASOs can be used to degrade target mRNA to decrease protein levels, to modify or correct splicing events, to modulate RNA translation or to target pathological coding or non-coding RNAs (Quemener et al., 2019). In particular, the role of non-coding RNAs (ncRNAs, including microRNAs (miRs)) in various diseases makes them an attractive target for an effective therapeutic strategy.

[0013] Various studies known from the art have described the role of long non-coding RNAs (IncRNAs) in cardiac pathologies. WO 2013 / 034653 describes that miR-132 and / or miR-212 may induce cardiac hypertrophy and thus constitute potential therapeutic targets for heart failure treatment. WO 2016 / 042561 describes a method of treating a lipid-related disorder by administering to the subject a therapeutically effective amount of a polynucleotide agent which is substantially complementary to a nucleotide sequence of a human miR-132. The IncRNA maternally expressed gene 3 (Meg3) was found to be mostly expressed by cardiac fibroblasts and inhibition of meg3 in vivo lead to a decrease in cardiac fibrosis and improved diastolic performance (Piccoli et al., 2017).

[0014] Other potential targets may include miRNA-21 (miR-21), a pro-inflammatory miR that has been associated with a significant role in cardiovascular disease and cancer through regulating genes involved in apoptosis, cell proliferation, cell migration, and tissue invasion (Surina et al., 2021 ; Barnett et al., 2016; Sheedy, 2015). MiR-21 targeting for the therapy of pancreatic cancer and psoriasis has previously been described (Sims et al., 2017; Wu et al., 2017; Sicard, 2013; Guinea- Viniegra et al., 2014). While the role of miR-21 in the immune system is still under investigation, it is hypothesized that miR-21 is involved in the transition between the pro-inflammatory and anti-inflammatory phases of the innate immune response via macrophage polarization. In the heart, miR-21 is highly expressed and has been identified as a cardiac fibroblast- derived miRNA (Bang et al., 2014). MiR-21 suppresses Sprouty homolog 1 (Spryl) expression, and thus enhances ERK-MAPK activity, which leads to fibroblast activation / prol iteration and cardiac fibrosis (Thum etal., 2008). While miR-21 also plays a role in cardiac hypertrophy and inhibiting its expression could reverse the cardiac hypertrophy phenotype, miR-21 has been shown to be a key regulator of the antiinflammatory response in macrophages (Sheedy, 2015).

[0015] Despite some advancement in the field of cardiac inflammation and cardiac fibrosis, and in particular the treatment and diagnosis thereof, there is still a major unmet need for effective therapies. It is particularly desired to provide compositions for therapy and diagnosis which are highly effective.SUMMARY OF THE INVENTION

[0016] The inventors have discovered that specific ASOs targeting miR-21 have the potential to improve or treat cardiac inflammation and cardiac fibrosis, and that these ASOs may also be used in the diagnosis thereof. The problem solved by the present invention lies in the provision of improved treatment therapies for the treatment of cardiac inflammation, cardiac fibrotic disorders, lung fibrotic disorders, liver fibrotic disorders and kidney fibrotic disorders. Specifically, the present invention provides synthetic antisense, LNA-containing oligonucleotides that specifically target (endogenous) miR-21. The inventors have surprisingly discovered that the synthetic ASOs of the invention provide the advantage of effectively inhibiting functional miR-21 to reduce (cardiac) inflammation and fibrosis. For instance, the synthetic ASOs of the invention provide the advantage of effectively inhibiting functional miR-21 and can beof use in the treatment and / or prevention of heart failure with reduced ejection fraction (HFrEF). The oligonucleotide analogues of the invention, in the following designated CDR076L, are mixmers comprising DNA and LNA building blocks and preferentially having internucleosidic phosphorothioate (PS) linkages. Said oligonucleotides lack significant toxicity in a human liver cell line and isolated neonatal rat cardiomyocytes. Further, CDR076L oligonucleotides exhibit superior effects compared to other oligonucleotide analogues having the same nucleotide sequence but a different distribution of LNA building blocks. As will be clear from the below disclosure, the antisense oligonucleotides (ASOs) described herein may also be referred to as anti-microRNA oligonucleotides (AMOs) given their capacity to target miR-21. It is understood that ASO and AMOs may be referred to herein interchangeably to refer to the oligonucleotides of the invention.

[0017] In a first aspect, the present invention provides an oligonucleotide comprising one of the sequences selected from (i) 5’- +T C A +G T C +T G A +T A +A G +C +T - 3’ (formula / ); or (ii) 5’- +T C +A G T +C T G +A +T A +A G C +T - 3’ (formula If); wherein A, T, G and C are deoxyribonucleotide building blocks, or C is 5-methyl-2’- deoxycytidine; and wherein +T, +A, +G, and +C are bridged nucleotide building blocks and / or morpholino building blocks.

[0018] In a second aspect provided herein is an oligonucleotide, comprising the sequence of formula la:5‘- +T*d5mC*dA*+G*dT*d5mC*+T*dG*dA*+T*dA*+A*dG*+5mC*+T -3‘; wherein dA is 2’-deoxyadenosine, dG is 2’-deoxyguanosine, dT is 2’-deoxythymidine; wherein +T is an LNA-T building block, +A is an LNA-A building block, +G is an LNA-G building block and +5mC is an LNA-5-methyl-2’-cytidine building block; wherein d5mC is 5- methyl-2’-deoxycytidine; and wherein * is a phosphorothioate linkage.

[0019] In a third aspect provided herein is a composition of the invention comprising the oligonucleotide of the invention, and optionally a pharmaceutically acceptable carrier. In some embodiments, the composition comprises a pharmaceutically acceptable carrier.

[0020] In a fourth aspect provided herein is an oligonucleotide or a composition containing said oligonucleotide of the invention for therapeutic use in a subject.

[0021] In a fifth aspect provided herein is an oligonucleotide or a composition for use in the prophylaxis and / or treatment of an inflammatory disease in a subject.

[0022] In a sixth aspect provided herein is an oligonucleotide or a composition for use in the prophylaxis and / or treatment of cardiopulmonary disorders in a subject.

[0023] In a seventh aspect provided herein is an oligonucleotide or a composition for use in the prophylaxis and / or treatment of fibrotic disorders in a subject.

[0024] In an eighth aspect provided herein is an oligonucleotide of the invention or composition of the invention for use in the diagnosis of an inflammatory disease, a cardiopulmonary disorder or a fibrotic disorder or combination thereof.

[0025] In a ninth embodiment, provided herein is a method of treating or preventing inflammatory diseases, cardiopulmonary disorders and / or fibrotic disorders in a subject in need thereof, wherein the method comprises administering to the subject an effective amount of the oligonucleotide or composition comprising the same. Also provided herein is an in vitro method of diagnosing an inflammatory disease, a cardiopulmonary, kidney or liver disorder, or a fibrotic disorder, or a combination thereof, wherein the method comprises an oligonucleotide of the invention.

[0026] It is an advantage of this invention to provide new and improved oligonucleotides. The oligonucleotides of the invention are chemically modified oligonucleotides. Another advantage of the invention is that it provides new and improved compositions comprising said oligonucleotides. A still further advantage of this invention is that a pharmaceutically acceptable composition is utilized to prevent and reduce cardiac and pulmonary inflammation and fibrotic disorders. Additional advantages of the invention will be set forth in part in the description, which follows and in part will be obvious from the description or may be learned by practice of the invention.BRIEF DESCRIPTION OF DRAWINGS

[0027] The figures shown in the following are merely illustrative and shall describe the present invention in a further way. The figures shall not be construed to limit the present invention thereto.

[0028] Fig. 1 presents the experimental set up of efficacy and toxicity screening, and in vitro results of different anti-miR-21 candidate compounds in a murine (RAW 264.7) model.

[0029] Fig. 2 presents the experimental set up of efficacy and toxicity screening, and in vitro results of different anti-mi R-21 candidate compounds in a human (TH P-1) in vitro model of (cardiac) inflammation.

[0030] Fig. 3 presents graphs showing the effect of lead candidates CDR076L-02 and -21 on (A) functional miR-21 , (B) IL-6, (C) Stat3, (D) Notch2, and (E) Nos2 expression in murine RAW 264.7 macrophages compared to placebo and miRCURY LNA-21 upon inflammatory stimulation with LPS (lipopolysaccharide).

[0031] Fig. 4 presents graphs showing CDR076-02 and -21-mediated reduction in expression of (A) functional miR-21 , (B) IL-6, (C) STAT3, and (D) Notch2 in human THP- 1 monocytes / macrophages compared to placebo upon inflammatory stimulation with lipopolysaccharide (LPS) and interferon gamma (IFN-y).

[0032] Fig. 5 presents graphs showing the experimental set up and results of a quantitative in vitro efficacy screening in a human model of cardiac fibrosis (NHCF-V cells) and toxicity studies of various anti-miR-21 oligonucleotide analogues.

[0033] Fig. 6 presents graphs relating to the efficacy screening of CDR076L-02 and - 21 in reducing expression of (A) functional miR-21 and (B)-(E) fibrotic marker genes (CDH2, COL1a1 , CTGF, FAP) in human cardiac fibroblasts compared to placebo and miRCURY LNA-21.

[0034] Fig. 7 presents graphs relating to the dose optimization of candidates CDR076L-02 and -21 in reducing expression of (A) functional miR-21 and (B)-(E) fibrotic marker genes (CDH2, COL1a1 , CTGF, FAP) in human cardiac fibroblasts compared to placebo and miRCURY LNA-21.

[0035] Fig. 8 represents (A) the experimental set up of efficacy and toxicity screening in NHLF lung fibroblasts and (B) effect of CDR076L-02 and -21 in reducing the expression of functional miR-21 and COL1a1.

[0036] Fig. 9 presents graphs showing the experimental set up of (A) cellular apoptosis assay, (B) metabolic activity assay, and (C) cell / membrane damage assay in hepatic (HepaRG) and (D) Heatmap summarising results of various assays (apoptosis, metabolic assay, cell damage) in renal cells (RPTEC).

[0037] Fig. 10 presents graphs demonstrating low in vitro cytotoxicity of CDR076L-02 and -21 in (A) human hepatocytes (HepaRG) and (B) human renal proximal tubule epithelial cells (RPTEC).

[0038] Fig. 11 : Full circular visualization (A) and chemical structure (B) of oligonucleotide candidate CDR076L-21 showing the different linkage modifications and chemical nucleotide modifications specific to the oligonucleotide.

[0039] Fig. 12: In vitro exploratory immunotoxicity study in hPBMC. (A) Flow chart on treatment scheme used for cytokine ELISA and gene expression analysis. hPBMC derived from three different healthy donors were used. (B) Functional level of miR-21- 5p normalized to the reference gene U6 snRNA and the Placebo group in hPBMC from three different donors 24 h post administration of CDR076L-21 or CDR076L-02 (0.1 , 1 or 10 pM) or Placebo. Two experiments were performed for donor 1 and one experiment for donor 2 and 3, respectively. Data are mean ± SD (n=2-6). (C) IL-6 and (D) TNF-a concentrations assed by ELISA from cell culture supernatant of hPBMC from three different donors 24 h post administration of CDR076L-21 or CDR076L-02 (0.1 , 1 , or 10 pM), Placebo control or positive control (10 ng / ml LPS). Data are mean ± SD (n=2-3).

[0040] Fig. 13: In vitro exploratory cytotoxicity study in hPBMC. (A) Flow chart on treatment scheme used for cell viability analysis (MTT assay). hPBMC derived from three different healthy donors were used. (B) Cell viability of hPBMC from three different donors assessed by MTT assay shown as corrected absorbance relative to Placebo after 24 h of treatment with CDR076L-21 or CDR076L-02 at different dose levels (0.1 , 1 or 10 pM). The dashed lines indicate mean corrected absorbance relative to Placebo for the Placebo group and for the positive control group with 5 pM Doxorubicin. Data are mean ± SD (n=3).

[0041] Fig. 14: RNA sequencing after CDR076L-02 treatment in a human in vitro model of (cardiac) inflammation. (A) Flow chart on treatment scheme used to study transcriptomic changes after administration of 100 nM CDR076L-02 in the human monocyte / macrophage cell line (THP-1) polarized into M1 macrophage by stimulation with 100 ng / ml LPS + 20 ng / ml IFN-y for 48h. (B) Volcano plot displaying differentially expressed genes in M1 macrophages treated with CDR076L-02 vs. Placebo which are significantly reciprocally regulated compared to M1 -polarization of cell (M1 Placebo vs. M0 Placebo) with a |FC| > 1.5 and p value < 0.05. (C) Gene set enrichment analysis of reciprocally regulated genes (using enrichment analysis, Chen et al., 2013; Huang et al., 2019) including the gene-set library from the pathway database NCATS BioPlanet; input: 402 reciprocal regulated genes (|FC| > 1.5 and p value < 0.05 for both comparisons; displayed are pathways with p value <5x10-7).

[0042] Fig. 15: RNA sequencing after CDR076L-02 treatment in a human in vitro model of cardiac fibrosis. (A) Flow chart on treatment scheme used to study transcriptomic changes after administration of 100 nM CDR076L-02 in human ventricular cardiac fibroblasts (NHCF-V) after pro- fibrotic stimulation with 10 ng / ml TGF- P for 72 h. (B) Volcano plot displaying differentially expressed genes in TGF-p stimulated NHCF-V treated with CDR076L-02 or Placebo which are significantly reciprocally regulated between Placebo treatment and unstimulated condition with a |FC| > 1.5 and p value < 0.05. (B) Gene set enrichment analysis of reciprocally deregulated genes (using enrichment analysis, Chen et al., 2013; Huang et al., 2019) including the gene-set library from the pathway database NCATS BioPlanet; input: 317 reciprocally regulated genes (|FC| > 1.5 and p value < 0.05 for Placebo vs. unstimulated and p value < 0.05 for CDR076L-02 vs. Placebo; displayed are pathways with p<0.0001).

[0043] Fig. 16: RNA sequencing after CDR076L-21 treatment in a human in vitro model of (cardiac) inflammation. (A) Flow chart on treatment scheme used to study transcriptomic changes after administration of 100 nM CDR076L-21 in the human monocyte / macrophage cell line (THP-1) polarized into M1 macrophage by stimulation with 100 ng / ml LPS + 20 ng / ml IFN-y for 48h. (B) Volcano plot displaying differentially expressed genes in M1 macrophages treated with CDR076L-21 vs. Placebo which are significantly reciprocally regulated compared to M1 -polarization of cell (M1 Placebo vs. M0 Placebo) with a |FC| > 1.5 and p value < 0.05. (C) Gene set enrichment analysis of reciprocally regulated genes (using enrichment analysis, Chen et al., 2013; Huang et al., 2019) including the gene-set library from the pathway database NCATS BioPlanet; input: 108 reciprocal regulated genes (|FC| > 1.5 and p value < 0.05 for both comparisons; displayed are pathways with p value <0.05).

[0044] Fig. 17: RNA sequencing after CDR076L-21 treatment in a human in vitro model of cardiac fibrosis. (A) Flow chart on treatment scheme used to study transcriptomic changes after administration of 100 nM CDR076L-21 in human ventricular cardiac fibroblasts (NHCF-V) after pro- fibrotic stimulation with 10 ng / ml TGF- P for 72 h. (B) Volcano plot displaying differentially expressed genes in TGF-p stimulated NHCF-V treated with CDR076L-21 or Placebo which are significantly reciprocally regulated between Placebo treatment and unstimulated condition with a |FC| > 1.5 and p value < 0.05. (B) Gene set enrichment analysis of reciprocally deregulated genes (using enrichment analysis Chen et al., 2013; Huang et al., 2019)including the gene-set library from the pathway database NCATS BioPlanet; input: 28 reciprocally regulated genes (|FC| > 1.5 and p value < 0.05 for Placebo vs. unstimulated and p value < 0.05 for CDR076L-21 vs. Placebo; displayed are pathways with p<0.02).

[0045] Fig. 18: In vivo biodistribution study with CDR076L-02 in mice. (A) (i) Flow chart of a single intraperitoneal (i.p.) administration of 20 mg / kg CDR076L-02 and (ii) functional levels of miR-21-5p normalized to the reference gene U6 snRNA in murine tissue or to cel-miR-39 in murine plasma samples at day 2, day 3 or day 7 post injection of 20 mg / kg CDR076L-02 or day 7 post administration of Placebo. Data are mean ± SD (n=4-6). P values are derived from Mann-Whitney II test (**: p < 0.01). (B) (i) Flow chart of a single intraperitoneal (i.p.) administration of 10 mg / kg CDR076L-02 and (ii) functional levels of miR-21-5p normalized to the reference gene U6 snRNA in murine tissue or to cel-miR-39 in murine plasma samples at day 7 post injection of 10 mg / kg CDR076L-02 or Placebo. Data are mean ± SD (n=4-6). P-values are derived from Mann- Whitney II test (**: p < 0.01).

[0046] Fig. 19: In vivo biodistribution study with CDR076L-21 in mice. (A) Flow chart of a single intraperitoneal (i.p.) administration of 2.5 mg / kg CDR076L-21 and (B) functional levels of miR-21-5p normalized to the reference gene U6 snRNA in murine tissue or to cel-miR-39 in murine plasma samples at day 7 post injection of 2.5 mg / kg CDR076L-21 or Placebo. Data are mean ± SD (n=4-6). P-values are derived from Mann-Whitney II test (**: p < 0.01).

[0047] Fig. 20: Functional level of miR-21-5p in a mouse model of Angiotensin Il- induced cardiac fibrosis. (A) Experimental set up of the Angiotensin II (Angll) study: To induce fibrosis, Angll was infused using subcutaneously implanted osmotic minipumps at a dose of 3 mg / kg / day for 14 days. (B) Functional levels of miR-21-5p in tissue were normalized to the reference gene U6 snRNA, while plasma miR-21-5p were used without reference gene normalization and calculated as fold change to the Placebo group at study endpoint 14 days post infusion of Angll or Placebo. Data are mean ± SD (n=6-11). P-values are derived from Mann-Whitney II test (ns: p>0.05, *: p < 0.05, **: p < 0.01 ; ***: p < 0.001).

[0048] Fig. 21 : Functional level of miR-21-5p in a mouse model of Angiotensin Il- induced cardiac fibrosis after treatment with 2.5 mg / kg CDR076L-21 . (A) Experimental set up of the Angiotensin II (Angll) study: To induce fibrosis, Angll was infused using subcutaneously implanted osmotic minipumps at a dose of 3 mg / kg / day for 14 days. Animals treated with CDR076L-21 received two intraperitoneal (i.p.) administration at adose of 2.5 mg / kg. (B) Functional levels of miR-21-5p in heart tissue normalized to the reference gene U6 snRNA (Left), and Plasma miR-21-5p levels were used without reference gene normalization and calculated as fold change to the control group at study endpoint 14 days post infusion of Angll (Right). Data are mean ± SD (n=6-13). P- values are derived from Mann-Whitney II test (ns: p>0.05, *: p < 0.05, **: p < 0.01 ; ***: p < 0.001 ; ****: p= 0.0001). (C) Correlation between cardiac miR-21-5p and plasma miR-21-5p using Pearson product-moment correlation and Spearman rank-order correlation.DETAILED DESCRIPTIONTerminology

[0049] In order that the present invention may be more readily understood, certain terms are first defined.

[0050] Articles "a" and "an" used herein refer to one or to more than one ( / .e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element, e.g., a plurality of elements.

[0051] The term “including” is used herein to mean, and is used interchangeably with, the phrase “including, but not limited to”. Likewise, the term “comprising” is used herein to mean, and is used interchangeably with, the phrase “comprising, but not limited to".

[0052] The terms “about” and “approximately” may be understood to permit standard variation as would be understood by those of ordinary skill in the art; where ranges are provided, endpoints are included.

[0053] As used herein, a “nucleotide” refers to a monomeric unit of an oligonucleotide or polynucleotide that includes a nucleoside and an internucleosidic linkage.

[0054] As used herein, the term "nucleic acid" is intended to include any DNA molecules (e.g., cDNA or genomic DNA) and any RNA molecules (e.g., mRNA) and analogues of the DNA or RNA generated using nucleotide analogues. The nucleic acid can be single-stranded or double-stranded. Each component of the DNA or RNA structure can be modified and be categorized by modification of (1) the internucleoside linkage, (2) the deoxyribose / ribose, and / or (3) the nucleobase.

[0055] The term “oligonucleotide" or “oligonucleotides” as used herein are defined as it is generally understood by the skilled person as a molecule including two or more covalently linked nucleosides (e.g., short nucleic acid polymer(s) linked byinternucleosidic linkage(s)). They can comprise DNA and / or RNA. The oligonucleotides provided herein have a backbone comprising deoxyribonucleotides.

[0056] The term “nucleobase” refers to nitrogen-containing biological building blocks that form nucleosides, which, in turn, are components of nucleotides. The naturally occurring bases [guanine, (G), adenine, (A), cytosine, (C), thymine, (T), and uracil (II)] are derivatives of purine or pyrimidine, though it should be understood that naturally and non-naturally occurring base analogues are also included and that the term “nucleobase” also includes “modified nucleobases”.

[0057] Within the context of this invention, the term "modified nucleobase" and "modified base" may be used interchangeably with the term “nucleobase”. Nucleobases may be modified or unmodified. Hence, in some embodiments, a modified nucleobase is a nucleobase which comprises a modification. In some embodiments, a modified nucleobase is capable of at least one function of a nucleobase, e.g., forming a moiety in a polymer capable of base-pairing to a nucleic acid comprising an at least complementary sequence of bases. In one embodiment, the modified nucleobase is capable of increasing hydrogen bonding, base pair stacking interactions and / or stabilizing a nucleic acid complex. In some embodiments, a modified nucleobase is substituted A, T, C, G, or II, or a substituted tautomer of A, T, C, G, or II. In some embodiments, a modified nucleobase in the context of oligonucleotides refer to a nucleobase that is not A, T, C, G or II. Modifications include but are not limited to nonstandard nucleobases 5-methyl-2’-deoxycytidine (d5m), pseudouridine (pll), dihydrouridine, inosine (I), and 7-methylguanosine.

[0058] The term "nucleoside(s)" refers to a moiety wherein a nucleobase or a modified nucleobase is covalently bound to a sugar or a modified sugar. In some embodiments, a “nucleoside” refers to a nucleoside unit in an oligonucleotide or a nucleic acid. The term "nucleoside(s)" encompasses all modified versions and derivatives “modified nucleobases”.

[0059] As used herein, the term “internucleosidic linkage(s)” refers to a linkage between adjacent nucleosides. “Internucleoside linkage” and “linkage” may be used interchangeably. Linkages comprise, but are not limited to, phosphate (PO), phosphorodiamidate or phosphorothioate (PS) linkages. Internucleosidic linkages may include any modified internucleosidic linkage. Unless otherwise specified, description of oligonucleotides and elements thereof (e.g., base sequence, sugar modifications, internucleosidic linkages, linkage phosphorus stereochemistry, patterns thereof, etc.) isfrom 5' to 3'. As those skilled in the art will appreciate, in some embodiments, oligonucleotides may be provided and / or utilized as salt forms, particularly pharmaceutically acceptable salt forms, e.g., sodium salts.

[0060] As used herein the term “antisense oligonucleotide” or “ASO” refers to a short strand of nucleotide analogue that hybridizes with the complementary RNA (microRNA, mRNA, ncRNA, etc.) or any other target RNA in a sequence-specific manner via Watson-Crick base pairing. The ASO can comprise DNA and RNA. The ASO may be chemically modified. As used herein the terms “antisense oligonucleotide” (ASO) and “oligonucleotide” may be used interchangeably.

[0061] The term “hydroxy”, as used herein, represents an -OH group.

[0062] The term "modified sugar" refers to a moiety that can replace a sugar. A modified sugar mimics the spatial arrangement, electronic properties, or some other physicochemical property of a sugar. A modified sugar may be substituted ribose or deoxyribose. In some embodiments, a modified sugar comprises a 2'-modification. Nucleotide modifications include LNA-modification(s) and / or 5-methyl-2’-deoxycytidine modification(s). The terms “locked nucleic acid” (LNA) or “locked nucleic acids” (LNAs) are known as bridged nucleic acid (BNA) and refer to the modification of the 2’-sugar modification of one or more nucleotides with an extra bridge (connecting the 2' oxygen and 4' carbon of the sugar). Within each oligonucleotide there can be one or more nucleotides having the same modification. In one embodiment, the oligonucleotide comprises LNA(s). In one embodiment, the oligonucleotide comprises DNA and / or RNA and / or LNA(s).

[0063] As used herein, the term “complementary” or “partially complementary” or “substantially complementary” refer to nucleic acid sequences, which due to their complementary nucleotides are capable of specific intermolecular base-pairing. For example, the oligonucleotide may comprise a nucleic acid sequence complementary to a target sequence. Hence, the complementarity of the ASOs of the invention may be 100%. In one embodiment, the complementarity is at least 80%, 85%, 90%, 95%. In one embodiment, the complementarity is 85%-99%.

[0064] As used herein the term “common base sequence” generally refers to a sequence of nucleotides (bases) that is commonly found within or shared among different DNA or RNA molecules, e.g., different oligonucleotides. As used herein the common base sequence is shared between the different oligonucleotides of theinvention. According to the invention, the common base sequence is 5'- T C A G T C T G A TAA G C T -3' (SEQ ID NO: 52).

[0065] As used herein, the terms “disease” or “disorder” are used interchangeably to refer to a condition in a subject. In certain embodiments, the condition is a disease in a subject, the severity of which is decreased by inducing an immune response through the administration of a pharmaceutical composition.

[0066] As used herein, the term “effective amount” in the context of administering a therapy to a subject refers to the amount of a therapy which has a prophylactic and / or therapeutic effect(s).

[0067] As used herein, the term “in combination” in the context of the administration of two or more therapies, refers to the use of more than one therapy (e.g., more than one prophylactic agent and / or therapeutic agent). The use of the term "in combination" does not restrict the order in which therapies are administered.

[0068] As used herein, the terms “prevent”, “preventing” and “prevention” in the context of the present invention and the administration of a therapy(ies) to a subject refers to the inhibition of the development or onset of a disease or a symptom thereof. In one embodiment, it relates to the administration of the compound to a patient who is known to have an increased risk of developing a certain disorder.

[0069] As used herein, the terms “treat”, “treating” and “treatment” refer to the administration of the compound to a patient which has already developed signs and / or symptoms of a certain disorder. Treating covers treatment of an existing condition, inhibiting the progress or development of the condition, ameliorating the condition, and providing palliation of the condition.

[0070] The terms “subject” and “patient” are used interchangeably and relate to an animal (e.g., mammals) in need of administration of the oligonucleotide or composition of the invention. In specific embodiments, the subject is a human.

[0071] As used herein, the term “pharmaceutically acceptable” means that which is useful in preparing a pharmaceutical composition and is generally safe, non-toxic, and neither biologically nor otherwise undesirable and includes that which is acceptable for veterinary use as well as human pharmaceutical use. Pharmaceutically acceptable substances are those that are generally approved by a regulatory agency.

[0072] The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the oligonucleotide or pharmaceutical composition comprising the same of theinvention is administered. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatine, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The formulation should suit the mode of administration. As those skilled in the art will appreciate, methods and compositions described herein relating to provided oligonucleotides generally also apply to pharmaceutically acceptable salts of such compounds.

[0073] Within the context of this application, the expression “standard of care” (SoC) refers to any given regimen involved in the prevention and / or treatment of a cardiac and / or fibrotic condition, disease or disorder. This may include informal or formal guidelines generally accepted in the medical community for the treatment of the particular condition, disease or disorder. SoC may be a medical treatment guideline and / or it may be developed by a specialist society or organization. For instance, renin-angiotensin system (RAS) inhibitors are currently used as standard therapy for heart failure (HF).

[0074] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present disclosure; other, suitable methods and materials known in the art can also be used.(Antisense) Oligonucleotides

[0075] Oligonucleotides are useful in various therapeutic, diagnostic, and research applications. Use of naturally occurring nucleic acids is limited, for example, by their susceptibility to endo- and exonucleases. As such, various synthetic counterparts have been developed to circumvent these shortcomings and / or to further improve various properties and activities. These include synthetic oligonucleotides that contain chemical modifications, e.g., base modifications, sugar modifications, backbone modifications, etc., which, among other things, render these molecules less susceptible to degradation and improve other properties and / or activities.

[0076] Therefore, among other things, the present disclosure utilizes technologies for controlling various structural elements, e.g., sugar modifications and patterns thereof, nucleobase modifications and patterns thereof, modified internucleosidic linkages andpatterns thereof, linkage phosphorus stereochemistry and patterns thereof, additional chemical moieties (moieties that are not typically in an oligonucleotide chain) and patterns thereof, etc.. With the capability to fully control structural elements of oligonucleotides, the present disclosure provides oligonucleotides with improved and / or new properties and / or activities for various applications, e.g., as therapeutic and / or diagnostic agents.

[0077] Provided herein are, inter alia, chemically modified antisense oligonucleotides (ASOs), comprising varying designs of nucleobase modifications and patterns thereof, sugars and patterns thereof, internucleosidic linkages and patterns thereof, and / or additional chemical moieties and patterns thereof. While not intending to be bound by any particular theory of operation, it is believed that particular nucleobase and backbone linkage modifications of said ASOs are useful in stabilising and improving the effect of the oligonucleotide.

[0078] Specifically, provided herein are oligonucleotides (ASOs) that target and / or inhibit miR-21. The oligonucleotides provided herein are oligonucleotides that comprise or consist essentially of a sequence complementary to the target miR-21. Hence, they provide the advantage of effectively modulating endogenous miR-21 function. Therefore, it is believed that the oligonucleotides of the invention and compositions comprising the same may be generally useful for therapeutic use and for treating and preventing cardiac and / or pulmonary inflammation and / or fibrotic disorders.

[0079] Provided herein are modified oligonucleotides and compositions comprising said oligonucleotides as well as oligonucleotides and compositions comprising the same for therapeutic use and for use in the prophylaxis and / or treatment of inflammatory diseases, cardiopulmonary disorders and / or fibrotic disorders in a subject. Since miRNA-21 plays an important role in regulating inflammatory and immunomodulatory responses, its regulation might be controlled through target-specific oligonucleotides.

[0080] Provided herein are modified oligonucleotides and compositions comprising said oligonucleotides as well as oligonucleotides and compositions comprising the same for therapeutic use and for use in the treatment of Heart Failure, more particularly Heart Failure with reduced ejection fraction (HFrEF).

[0081] As is known to those of skill in the art, ASOs can be used as diagnostic agents, therapeutic agents, probes, etc.. It is believed that the claimed oligonucleotides, having a particular sequence, nucleotide modification(s), and backbone modification(s),provide highly specific and stable oligonucleotides that target miR-21. It is believed that these synthetic anti-sense oligonucleotides (ASOs) are effective in controlling miR-21 compared to unmodified ASOs with less modified backbones that are less stable and less effective in targeting miR-21.

[0082] Advantageously, the oligonucleotides provided herein are useful in the specific targeting of miR-21 to modulate an immune response and thereby provide means against inflammatory diseases, cardiopulmonary disorders and / or fibrotic disorders. Thus, the oligonucleotides of this invention and compositions comprising the same are useful as agents in medicine, particularly in the prevention and / or treatment of inflammatory diseases and / or fibrotic disorders, specifically in the prevention and / or treatment of cardiac and / or pulmonary inflammatory diseases and / or cardiac fibrotic disorders. Even more specifically, for the prevention and / or treatment of Heart Failure with reduced ejection fraction (HFrEF).

[0083] The inventors have realised that providing oligonucleotides in the context of non-coding RNA-based therapeutics, it is beneficial to incorporate certain features into the oligonucleotides in order to provide effective ASOs for the therapeutic targeting of non-coding RNAs. Moreover, the inventors submit that the ASOs of the invention have the ability to act on several key disease pathways simultaneously, triggering a concerted therapeutic effect against key hallmarks of heart disease including, cardiac hypertrophy, fibrosis, impaired contractility, and reduced vascularization.

[0084] Accordingly, provided herein are oligonucleotides comprising one of the sequences selected from: (i) 5’- +T C A +G T C +T G A +T A +A G +C +T -3’ (formula iy, or (ii) 5’- +T C +A G T +C T G +A +T A +A G C +T -3’ (formula If); wherein A, T, G and C are deoxyribonucleotide building blocks, or C is 5-methyl-2’-deoxycytidine; and wherein +T, +A, +G, and +C are bridged nucleotide building blocks and / or morpholino building blocks. In one embodiment, +G, +T, +A, and +C are locked nucleic acid (LNA) building blocks.

[0085] The oligonucleotide of the invention may comprise or consist of an oligonucleotide listed in Table 1. In one embodiment, the oligonucleotide comprises the sequence of formula I (i.e., CDR076L-21 U; see, Table 1). In one embodiment, the oligonucleotide comprises or consists of SEQ ID NO: 21 (CDR076L-21 U). In one embodiment, the oligonucleotide comprises of SEQ ID NO: 21 (CDR076L-21 II). In one embodiment, the oligonucleotide consists of SEQ ID NO: 21 (CDR076L-21 U). In one embodiment, the oligonucleotide comprises or consists of the sequence of formula la(i.e., CDR076L-21 ; see Table 1). In one embodiment, the oligonucleotide comprises or consists of SEQ ID NO: 46 (CDR076L-21). In one embodiment, the oligonucleotide comprises SEQ ID NO: 46 (CDR076L-21). In one embodiment, the oligonucleotide consists of SEQ ID NO: 46 (CDR076L-21). In one embodiment, the oligonucleotide comprises or consists of the formula as shown in Fig. 11(A). In one embodiment, the oligonucleotide comprises or consists of the formula as shown in Fig. 11(B).

[0086] In one embodiment, the oligonucleotide comprises or consists of the sequence of formula II, i.e., CDR076L-02B. In one embodiment, the oligonucleotide comprises or consists of SEQ ID NO: 2 (CDR076L-02B). In one embodiment, the oligonucleotide comprises or consists of the sequence of formula Ila, i.e., CDR076L-02. In one embodiment, the oligonucleotide comprises or consists of SEQ ID NO: 27 (CDR076L- 02). In one embodiment, the oligonucleotide comprises SEQ ID NO: 27 (CDR076L- 02). In one embodiment, the oligonucleotide consists of SEQ ID NO: 27 (CDR076L- 02). In one embodiment, the oligonucleotide comprises bridged nucleotide building blocks. In one embodiment, the oligonucleotide comprises morpholino building blocks.

[0087] Sugars can be bonded to internucleosidic linkages at various positions. As non-limiting examples, internucleosidic linkages can be bonded to the 2', 3', 4' or 5' positions of sugars. In some embodiments, as most commonly in natural nucleic acids, an internucleosidic linkage connects with one sugar at the 5' position and another sugar at the 3' position unless otherwise indicated. In one embodiment, the oligonucleotide comprises at least one modified internucleosidic linkage. In one embodiment, at least 1 , 2, 3, 4, 5, 6, or 7 internucleosidic linkages are modified. In one embodiment, the oligonucleotide comprises one or more phosphate (PO) linkages. In one embodiment, the modified internucleosidic linkages are phosphorothioate or phosphorodiamidate linkages. In one preferred embodiment, the oligonucleotide comprises one or more phosphorothioate (PS) linkages. In one embodiment, the oligonucleotide comprises one or more phosphorodiamidate linkages. In one embodiment the oligonucleotide comprises phosphorothioate, phosphate, and / or phosphorodiamidate linkages. In one embodiment, all internucleosidic linkages are modified. In one preferred embodiment, all internucleosidic linkages are phosphorothioate (PS) linkages.

[0088] Various nucleobases may be utilized in provided oligonucleotides in accordance with the present disclosure. Suitable technologies for nucleobase modification in oligonucleotide synthesis may be utilized in accordance with the presentdisclosure. Nucleobases may be modified. In some embodiments, modified nucleobases improve properties and / or activities of oligonucleotides. For example, in many cases, 5mC (5-methyl-cytidine) may be utilized in place of C to modulate certain undesired biological effects, e.g., immune responses. In some embodiments, a nucleobase is a natural nucleobase, the most commonly occurring ones being A, T, C, and G. In some embodiments, a nucleobase is a modified nucleobase in that it is not A, T, C, or G. In one embodiment, a nucleobase is a substituted purine base. In one embodiment, a nucleobase is a substituted pyrimidine base. In a preferred embodiment, each C is 5-methyl-2’-deoxycytidine or 5-methyl-cytidine. In a preferred embodiment, each C carries a 5-methyl modification. In one embodiment, the oligonucleotide comprises 5-methyl-2’-cytidine having an LNA modification.

[0089] The oligonucleotides of the invention may comprise various combinations of unmodified and modified nucleobases. In some embodiments, a nucleobase is a natural nucleobase, or a modified nucleobase derived from a natural nucleobase. In some embodiments, the present disclosure provides oligonucleotides comprising one or more modified nucleobases. In some embodiments, the oligonucleotide comprises one or more modified nucleobases and / or modified sugars. In some embodiments, the oligonucleotide comprises one or more modified nucleobases, one or more modified sugars and / or one or more modified internucleosidic linkages.

[0090] In some embodiments, oligonucleotides are of suitable lengths and sequence complementarity to specifically hybridize with target nucleic acids. In one embodiment, an oligonucleotide is 80% complementary to the target RNA, i.e., miR-21. In one embodiment, an oligonucleotide is 85% complementary to the target RNA, i.e., miR-21. In one embodiment, an oligonucleotide is 92% complementary to the target RNA, i.e., miR-21. In one preferred embodiment, an oligonucleotide is 100% complementary to the target RNA, i.e., miR-21.

[0091] Oligonucleotides may have varying lengths. In one embodiment, the oligonucleotide has a length of at least 12 or 15 nucleotides. In some embodiments, the oligonucleotide has a length of 10-100, 12-80, 12-70, 12-60, or 12-50 nucleotides. In some embodiment, the oligonucleotide has a length of 15-60, 15-50, 12-40, 12-20, or 15 to 20 nucleotides. In embodiment, the oligonucleotide has a length of 10, 11, 12, 13 ,14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, or 60 nucleotides. In one preferred embodiment, an oligonucleotide has a length of15 nucleotides. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.

[0092] In one embodiment, the oligonucleotide is an inhibitor. In some embodiments, the oligonucleotide hybridizes to a target RNA sequence. In one embodiment, the target is miR-21. In one embodiment, the oligonucleotide is an inhibitor of miR-21. In one embodiment, the oligonucleotide decreases or blocks activity of miR-21. In one embodiment, the miR-21 is endogenous miR-21. In some embodiments, an oligonucleotide of the invention hybridizes to one or more variants of miR-21.

[0093] Table 1 identifies LNA / DNA chimeric oligonucleotides of the invention with a phosphorothioate backbone and their specific nucleobase modifications. Length’. 15mer. These sequences are useful in the oligonucleotides, compositions, methods and uses described herein. miRCURY LNA-21 served as negative control and is an oligonucleotide that is commercially available with an unknown LNA pattern (Qiagen). miRCURY LNA is designed to simulate naturally occurring mature miRNAs. Introducing a miRNA mimic into cells increases the proportion of RNA- induced silencing complexes (RISC). A total of 25 candidate oligonucleotides with identical base sequences were generated, whereby each oligonucleotide carries different backbone and nucleobase modifications. Two candidate oligonucleotides are listed in Table 1. The different backbone and nucleobase modifications are identified as follows: +: bridged nucleotide building block or morpholino building block; Ina: locked nucleic acid (link between 2’ oxygen and 4’ carbon); d5mC: 5-methyl-2’- deoxycytidine; lna5mC: Ina modified 5-methyl-2’-cytidine; dG: deoxyguanosine; dT: deoxythymidine; dA: deoxyadenosine; +A: bridged or morpholino linked adenosine; +G: bridged or morpholino linked guanosine; +C: bridged or morpholino linked cytosine; +T : bridged or morpholino linked thymine; *: phosphorothioate (PS) linkage.Table 1. Exemplary miR-21 targeting oligonucleotide sequences and their modifications. miRCURY LNA-21 serves as negative control. For candidates CDR076L- 01 A to CDR076L-25Y & miRCURY LNA-21 : A, C, T G = nucleotide bases; + = bridged nucleotide building blocks and / or morpholino building blocks; and * = phosphorothioate (PS) linkage. For candidates CDR076L-01 to CDR076L-25: Ina = locked nucleic acids; dN = 2’-H (deoxyribose; DNA), wherein N is nucleotide base; dA = 2’-deoxyadenosine; dT = 2’- deoxythymidine; dC = 2’-deoxycytosine; dG = 2’-deoxyguanosine; d5mC = 5-methyl-2’- deoxycytidine; lna5mC = Ina modified 5-methyl-2’-cytidine; * = phosphorothioate (PS) linkage

[0094] The sequences disclosed herein are also shown in the enclosed sequence listing. The relevant modifications associated with the sequences are disclosed in Table 1 of this application. The oligonucleotide may comprise any one of the sequences selected from SEQ ID NO: 1 to SEQ ID NO: 50 (see, Table 1).

[0095] An oligonucleotide of the invention may comprise or consist of formula I, formula la, SEQ ID NO: 21 , or SEQ ID NO: 46. Hence, in one embodiment, an oligonucleotide comprises the sequence of formula I: 5’- +T C A +G T C +T G A +T A +A G +C +T -3’ , wherein A, T, G and C are deoxyribonucleotide building blocks, or C is 5-methyl-2’-deoxycytidine; and wherein +T, +A, +G, and +C are bridged nucleotide building blocks and / or morpholino building blocks. In one preferred embodiment, an oligonucleotide comprises the sequence 5'- +T C A +G T C +T G A +TA +A G +C +T -3' (CDR076L-21 U) (SEQ ID NO: 21), wherein A, T, G and C are deoxyribonucleotide building blocks, or C is 5-methyl-2’-deoxycytidine; and wherein +T, +A, +G, and +C are bridged nucleotide building blocks and / or morpholino building blocks. In one embodiment, an oligonucleotide comprises the sequence of formula la’. 5‘- +T*d5mC*dA*+G*dT*d5mC*+T*dG*dA*+T*dA*+A*dG*+5mC*+T -3‘; wherein dA is 2’-deoxyadenosine, dG is 2’-deoxyguanosine, dT is 2’-deoxythymidine; wherein +T is an LNA-T building block, +A is an LNA-A building block, +G is anLNA-G building block and +5mC is an LNA-5-methyl-2’-cytidine building block; wherein d5mC is 5-methyl-2’-deoxycytidine; and wherein * is a phosphorothioate linkage. In one preferred embodiment, an oligonucleotide comprises the sequence 5'-lnaT*d5mC*dA*lnaG*dT*d5mC*lnaT*dG*dA*lnaT*dA*lnaA*dG*lna5mC*lnaT -3' (CDR076L-21) (SEQ ID NO: 46), wherein InaT is an LNA-T building block; InaA is an LNA-A building block; dA is 2’-deoxyadenosine, dG is 2’-deoxyguanosine, dT is 2’-deoxythymidine; wherein lna5mC is an LNA-5-methyl-2’-cytidine building block; wherein d5mC is 5-methyl-2’-deoxycytidine; and wherein * is a phosphorothioate linkage. In one preferred embodiment, an oligonucleotide comprises SEQ ID NO: 46. In one preferred embodiment, an oligonucleotide consists of SEQ ID NO: 46.

[0096] Alternatively, an oligonucleotide of the invention may comprise or consist of formula II, formula Ila, SEQ ID NO: 2, or SEQ ID NO: 27. Hence, in one embodiment, an oligonucleotide comprises the sequence of formula II: 5'- +T C +A G T +C T G +A+T A +A G C +T- 3', wherein A, T, G and C are deoxyribonucleotide building blocks, or C is 5-methyl-2’-deoxycytidine; and wherein +T, +A, +G, and +C are bridged nucleotide building blocks and / or morpholino building blocks. In one embodiment, an oligonucleotide comprises the sequence of 5'- +T C +A G T +C T G +A+T A +A G C +T- 3' (CDR076L-02B) (SEQ ID NO: 2), wherein A, T, G and C are deoxyribonucleotide building blocks, or C is 5-methyl-2’-deoxycytidine; and wherein +T, +A, +G, and +C are bridged nucleotide building blocks and / or morpholino building blocks. In one embodiment, the oligonucleotide comprises the sequence of formula Ha 5’- +T*d5mC*+A*dG*dT*+5mC*dT*dG*+A*+T*dA*+A*dG*d5mC*+T-3'; wherein dA is 2’-deoxyadenosine, dG is 2’-deoxyguanosine, dT is 2’-deoxythymidine; wherein +T is an LNA-T building block, +A is an LNA-A building block, and +5mC is an LNA-5-methyl-2’-cytidine building block; wherein d5mC is 5-methyl-2’- deoxycytidine; and wherein * is a phosphorothioate linkage. In preferred one embodiment, an oligonucleotide comprises the sequence 5'-lnaT*d5mC*lnaA*dG*dT*lna5mC*dT*dG*lnaA*lnaT*dA*lnaA*dG*d5mC*lnaT-3' (CDR076L-02) (SEQ ID NO: 27), wherein InaT is an LNA-T building block; InaA is an LNA-A building block; dA is 2’-deoxyadenosine, dG is 2’-deoxyguanosine, dT is 2’-deoxythymidine; wherein lna5mC is an LNA-5-methyl-2’-cytidine building block; wherein d5mC is 5-methyl-2’-deoxycytidine; and wherein * is a phosphorothioate linkage. In one preferred embodiment, an oligonucleotide comprises SEQ ID NO: 27. In one preferred embodiment, an oligonucleotide consists of SEQ ID NO: 27.

[0097] A number of heterologous moieties or carriers, ranging from small molecules to macromolecules and supramolecular assemblies, may be attached to the oligonucleotides of the invention. Specifically, the oligonucleotides of the invention may be modified at their 3’ and and / or 5’ end to enhance cellular update and improve target organ delivery. In one embodiment, the oligonucleotide is conjugated to a heterologous moiety. In one embodiment, the oligonucleotide is linked to one or more heterologous moieties. In one embodiment, the oligonucleotide is linked to the heterologous moiety via a covalent bond. In one embodiment, conjugation of the oligonucleotide to a heterologous moiety (or carrier) promotes cellular penetration and / or uptake. The heterologous moiety can be a carrier. Specifically, in one embodiment, the carrier is a lipid or a polymer. In some embodiments, the carrier is a cell-penetrating peptide (CPP). In one embodiment, the carrier is cholesterol. In one embodiment, a marrier is a nanoparticle. In one embodiment, the oligonucleotide comprises a covalent attachment of poly(ethylene glycol) (PEG). In some cases, / V-acetylgalactosamine (GalNac) may be used as a delivery moiety for oligonucleotides. In one embodiment, the oligonucleotide comprises a GalNAc modification.

[0098] In one embodiment, the oligonucleotide is to be administered as such, or the oligonucleotide is to be administered conjugated to a heterologous moiety.

[0099] In one embodiment, one or more oligonucleotides of the invention or a combination thereof is used in the treatment of an inflammatory disease, a cardiopulmonary disorder and / or a fibrotic disorder or combination thereof in a subject. In one embodiment, one or more oligonucleotides of the invention or a combination thereof is used in the treatment of Heart Failure with Reduced Ejection Fraction (HFrEF).Compositions

[0100] The nucleic acids or oligonucleotides (or ASOs) provided herein may be incorporated into compositions of the invention.

[0101] Provided herein is a composition (e.g., oligonucleotide composition, pharmaceutical composition) containing one or more oligonucleotides of the invention. The present disclosure provides oligonucleotide compositions of antisense oligonucleotides described herein (see, Table 1). In some embodiments, the compositions are pharmaceutical compositions. As used herein, pharmaceuticalcomposition means a mixture of substances suitable for administering to an individual. For example, a pharmaceutical composition may comprise one or more active pharmaceutical agents (such as an antisense oligonucleotide) and a sterile aqueous solution. In one embodiment, the composition contains one or more oligonucleotides of the invention. In one embodiment, provided herein is a pharmaceutical composition or a pharmaceutically acceptable salt thereof comprising an antisense oligonucleotide of the invention, or a vector of the invention. In some embodiments, a composition comprises a plurality of oligonucleotides sharing a common base sequence. In some embodiments, the nucleotides of the common base sequence are chemically modified. In some embodiments, a plurality of oligonucleotides share the same common base sequence, and the same base and sugar modification(s). In some embodiments, a plurality of oligonucleotides share the same common base sequence, and the same base, sugar and internucleosidic linkage modification(s). In one embodiment, the composition contains one or more oligonucleotides of the invention. For instance, the composition may include oligonucleotide CDR076L-21 and one or more other oligonucleotides.

[0102] In one embodiment, a composition comprises an oligonucleotide of the invention in an admixture with a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier can simply be a saline solution. This can be isotonic or hypotonic. In one embodiment, a (pharmaceutical) composition comprises an oligonucleotide of the invention, optionally in an admixture with a pharmaceutically acceptable carrier. In one embodiment, a composition of the invention comprises at least one of the oligonucleotides listed in Table 1. In one preferred embodiment, a composition comprises CDR076L-21. In one embodiment, a composition comprises CDR076L-02. In one embodiment, the composition comprises CDR076L-02 and CDR076L-21.

[0103] In some embodiments, a composition is characterized in that when it is contacted with a target nucleic acid, levels of the target nucleic acid and / or a product encoded thereby is reduced. In some embodiment, a composition of the invention inhibits the target nucleic acid. According to the invention, a composition of the invention target is miR-21.

[0104] A composition comprising an oligonucleotide of the invention may be administered as a monotherapy or in combination with one or more further (different) medicaments, particularly a medicament suitable for the prevention or treatment ofcardiac or pulmonary inflammation, or fibrotic disorders. In some embodiments, a pharmaceutical composition may comprise one or more other therapies in addition to an oligonucleotide of the invention. In certain embodiments, an oligonucleotide or composition is administered to a subject once as a single dose. In some embodiments, an oligonucleotide or composition is administered to a subject in combination with one or more therapies. The one or more other therapies may be beneficial in the treatment or prevention of inflammatory diseases and / or fibrotic disorders or may ameliorate a symptom or condition associated with the same.

[0105] For instance, further hear failure drugs with antifibrotic (cardiac) effects include, for example, Angiotensin-converting enzyme (ACE) inhibitors, Angiotensin II receptor blockers (ARB), Angiotensin Receptor-Neprilysin Inhibitor (ARNI), Aldosterone receptor antagonists (MRA), Beta-blockers and sodium-glucose cotransporter 2 (SGLT2) inhibitors. Specifically, ACE inhibitors may include Captopril, Enalapril, Benazepril, Ramipril, Fosinopril, Trandolapril, Perindopril. ARB inhibitors may include, for example, Losartan, Candesartan and Valsartan. An ARNI may be Sacubitril / Valsartan (e.g., 1 :1 ratio). MRAs may include Spironolactone, Eplerenone and Finerenone. Beta-blockers may include Bisoprolol, Carvedilol, and Metoprolol. SGLT2 inhibitors (SGLT2i) may include Empagliflozin, Dapagliflozin, Sotagliflozin, and Canagliflozin.

[0106] In other cases, further drugs may include antifibrotic drugs (e.g., pulmonary fibrosis (IFF)) such as TK inhibitors (e.g., Nintedanib and Imatinib), Antifibrotic drugs (e.g., Pirfenidone), Endothelin R (e.g., Macitentan, Bosentan, Ambrisentan), TNF inhibitors (e.g., Etanercept), immunmodulators (e.g., Cyclophosphamide), and antiinflammatory drugs (e.g., Colchicine).

[0107] Other drugs with antifibrotic effects may include Galectin-3 inhibitors, NLRP3 protein inhibitors, TGF-beta Inhibitors and CTGF Inhibitors.

[0108] The composition of the invention comprises the oligonucleotide of the invention. According to a further aspect, the invention relates to a kit or kit of parts comprising an oligonucleotide of the invention and / or the (pharmaceutical) composition according to the invention. That is, the pharmaceutical compositions described herein can be included in a container, pack, or dispenser together with instructions for administration. In one embodiment, the kit comprises an oligonucleotide or composition of the invention. In one embodiment, the kit additionally comprises instructions for use.

[0109] In some embodiments, there is provided pharmaceutical compositions comprising an oligonucleotide as disclosed in the instant application, and at least one pharmaceutically acceptable excipient. Such pharmaceutical compositions may be prepared as is known in the art. The excipient may serve various purposes, e.g. as a carrier, vehicle, diluent, and / or to improve administration, and / or absorption of the active substance. The formulation of pharmaceutically active ingredients with various excipients is known in the art, see, e.g., Remington: The Science and Practice of Pharmacy, 19th edition (1995), and any later editions. Non-limiting examples of excipients are: solvents, diluents, buffers, preservatives, tonicity regulating agents, chelating agents, and stabilisers.

[0110] Examples of formulations include liquid formulations and solid formulations. Examples of a liquid formulation are aqueous formulations, i.e. formulations that comprise water. A liquid formulation may be a solution. An aqueous formulation typically comprises at least 50% (w / w) water, or at least 60%, 70%, 80% or even at least 90% (w / w) water.

[0111] In some embodiment, the formulation may comprise a buffer. In a further embodiment of the invention the formulation further comprises a pharmaceutically acceptable preservative. In a further embodiment of the invention the formulation further comprises an isotonic agent. In some embodiments, the pharmaceutical composition may comprise a chelating agent. In some embodiments, the pharmaceutical composition may comprise a stabiliser.

[0112] The oligonucleotides of the present invention may be administered in the form of a pharmaceutical composition. Parenteral administration may be performed by subcutaneous, intramuscular, intraperitoneal, or intravenous injection. In some embodiments, the administration may be performed by means of a syringe, optionally a pen-like syringe. Alternatively, parenteral administration can be performed by means of an infusion pump.Prophylactic and Therapeutic Uses

[0113] The invention generally describes the use of the chemically modified oligonucleotide and / or composition comprising the same in the medical setting. Hence, a chemically modified oligonucleotide or composition comprising the same may be used in the treatment and / or prevention of a medical condition. In one aspect provided herein is an oligonucleotide or a composition of the invention for therapeutic use in a subject.

[0114] In another aspect provided herein is an oligonucleotide or a composition for use in the prophylaxis and / or treatment of an inflammatory disease in a subject. In one particular embodiment, the inflammatory disease is cardiac or pulmonary inflammation. In one embodiment, the inflammatory disease is cardiac inflammation. In one embodiment, the inflammatory disease is pulmonary inflammation. In one embodiment, the disease is endocarditis. In one embodiment, the disease is myocarditis. In one embodiment, the disease is pericarditis. Cardiac inflammation and / or pulmonary inflammation may be caused by various factors. In one embodiment, cardiac inflammation is caused by viral, bacterial, fungal, and / or parasitic infections. In one embodiment, cardiac inflammation is caused by an autoimmune disease, e.g., rheumatoid arthritis or lupus. In one embodiment, cardiac inflammation is caused by medicines, such as, e.g., heart medicines, antibiotics, antidepressants, diuretics, benzodiazepines, weight loss medicines etc.. In one embodiment, the pulmonary inflammation is associated with the disease asthma.

[0115] In one aspect provided herein is an oligonucleotide or a composition for use in the prophylaxis and / or treatment of cardiopulmonary disorders in a subject. In one embodiment, the cardiopulmonary disorder is cardiopulmonary inflammation-related remodelling.

[0116] In one aspect provided herein is an oligonucleotide or a composition thereof for use in the prophylaxis and / or treatment of fibrosis or fibrotic disorders in a subject. In one embodiment, the fibrotic disorder includes cardiac fibrotic disorders. In one embodiment, the fibrotic disorder includes cardiac fibrotic disorders, particularly atrial fibrosis, endomyocardial fibrosis or fibrosis resulting from a previous myocardial infarction. In one embodiment, cardiac fibrosis includes left and / or right ventricular fibrosis. In one embodiment, fibrosis results from a previous myocardial infarction (Ml), high blood pressure or myocarditis. In one embodiment, the oligonucleotide or composition thereof for use in the in the prophylaxis and / or treatment of fibrosis related or fibrotic disease related disorders in a subject, such as Heart Failure with Reduced Ejections Fraction (HFrEF).

[0117] Exemplary types of cardiac fibrosis include atrial fibrosis, endomyocardial fibrosis or fibrosis. In some embodiments, fibrosis is myocardial fibrosis. In some embodiments, myocardial fibrosis is interstitial fibrosis, subepicardial fibrosis or replacement fibrosis. In some embodiments, fibrosis is reactive interstitial fibrosis. In other embodiments, cardiac fibrosis results from hypertensive heart disease, diabetichypertrophic cardiomyopathy and / or idiopathic dilated cardiomyopathy or hypertrophic cardiomyopathy. In some embodiments, myocardial fibrosis is associated with myocardial infarction (heart attack).

[0118] In another aspect provided herein, is an oligonucleotide or a composition thereof for use in the treatment or prevention of Heart Failure with Reduced Ejection Fraction (HFrEF). Heart Failure with Reduced Ejection Fraction (HFrEF) is a subtype of heart failure characterized by a diminished ability of the heart's left ventricle to contract effectively, leading to a reduced ejection fraction. This impairs the heart's capacity to pump blood adequately to meet the body's needs, resulting in a range of symptoms and clinical challenges. It is understood that, in certain embodiments, cardiac fibrosis and / or chronic inflammation may be related to the development and progression of HFrEF. In certain embodiments, cardiac fibrosis and / or chronic inflammation may contribute to the development and progression of HFrEF.

[0119] In one embodiment, the fibrotic disorder includes pulmonary fibrotic disorders, particularly pulmonary fibrosis caused by occupational or environmental factors, radiation treatment and / or treatment with medicaments. In one embodiment, the fibrotic disorder includes idiopathic pulmonary fibrosis. In one embodiment, the fibrotic disorder includes hepatic fibrotic disorders, particularly alcoholic liver disease or non-alcoholic fattyliver disease (NAFLD).

[0120] In a specific embodiment, the fibrosis is a pulmonary fibrotic disorder, e.g., a condition involving pathological fibrosis in the lung. In one embodiment, types of pulmonary fibrosis include fibrotic disorders caused by occupational, genetic or environmental factors, for example by exposure to toxins and pollutants such as silica dust, asbestos fibres, metal dust, coal dust, grain dust, bird and animal droppings. In other embodiments, pulmonary fibrotic disorders are caused by radiation treatment and / or treatment with medicaments such as chemotherapeutic drugs, cardiac drugs, antibiotics or anti-inflammatory drugs. In some embodiments, pulmonary fibrosis is a secondary effect of other diseases. In some embodiments, pulmonary fibrotic disorders are caused by disorders including idiopathic pulmonary fibrosis, dermatitis, polymyositis, mixed connective tissue disease, an autoimmune disease such as rheumatoid arthritis, scleroderma, Sjogren's syndrome or systemic lupus erythematosus, sarcoidosis, pneumonia, a viral infection or gastroesophagealreflux disease (GERO). Accordingly, in one embodiment, fibrosis is pulmonary fibrosis. In one embodiment, fibrosis is idiopathic pulmonary fibrosis.

[0121] In one embodiment, the fibrosis is a hepatic fibrosis, e.g., a condition involving pathological fibrosis in the liver. Exemplary types of hepatic fibrosis are caused by a viral infection, e.g., by hepatitis B and / or C virus, hereditary metabolic disorders, autoimmune hepatitis, biliary obstruction, iron overload, non-alcoholic fatty liver disease, including non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH) and alcohol liver disease. In still further embodiments, the fibrosis is a vascular fibrosis, e.g., arterial stiffness, a cutaneous fibrosis, e.g., keloid formation or nephrogenic systemic fibrosis, an arthrofibrosis, some forms of adhesive capsulitis, soft tissue fibrosis such as mediastinal fibrosis or retroperitoneal fibrosis or bone marrow fibrosis such as myelofibrosis.

[0122] Renal fibrosis is a direct consequence of the kidney's limited capacity to regenerate after injury. Renal scarring results in a progressive loss of renal function, ultimately leading to end-stage renal failure and a requirement for dialysis or kidney transplantation. In one embodiment, the fibrotic disorder includes renal fibrosis. Hence, in one embodiment, the fibrosis is a renal fibrosis. In one embodiment, the fibrotic disorder includes dermal fibrosis. In one embodiment, the fibrotic disorder includes bone marrow fibrosis. In one embodiment, the fibrotic disorder includes gut fibrosis.

[0123] In one embodiment, the fibrosis is a dermal fibrosis. In one embodiment, the fibrosis is a bone marrow fibrosis. In one embodiment, the fibrosis is a gut fibrosis. In one embodiment, the fibrosis is any of the fibrotic disorders mentioned above and / or combination thereof.

[0124] In some embodiments, the subject suffers from different types of fibrosis, i.e., the subject may be suffering from one or more different types of fibrosis.

[0125] The oligonucleotide or the pharmaceutical composition of the invention may be administered to a subject in need thereof. In one embodiment, a subject has an increased risk for developing an inflammatory disease. In one embodiment, a subject comprises patients suffering from inflammatory disease and / or patients having an increased risk of inflammatory disease progression. In one embodiment, patients have an increased risk for developing cardiac, kidney or pulmonary disorders. In one embodiment, patients suffer from cardiac disorders and / or patients having an increased risk of cardiac disorder progression. In one embodiment, patients have anincreased risk for developing fibrotic disorders. In one embodiment, patients suffer from fibrotic disorders and / or patients having an increased risk of fibrotic disorder progression. In a particular embodiment, the oligonucleotide or the pharmaceutical composition is administered to the subject selected from: (i) patients having an increased risk for developing inflammatory disease, particularly wherein the inflammatory disease is cardiac inflammation; (ii) patients suffering from inflammatory disease and / or patients having an increased risk of inflammatory disease progression; (iii) patients having an increased risk for developing cardiac, kidney, liver or pulmonary disorders; (iv) patients suffering from cardiac disorders and / or patients having an increased risk of cardiac disorder progression; (v) patients having an increased risk for developing fibrotic disorders; and / or (vi) patients suffering from fibrotic disorders and / or patients having an increased risk of fibrotic disorder progression.

[0126] A number of different cytokine and growth factor pathways play a role in the pathogenesis of fibrotic disorders. Common genes implicated in fibrosis include, e.g., Transforming Growth Factor-Beta (TGF- ), Connective Tissue Growth Factor (CTGF), Epidermal Growth Factor Receptor (EGFR), Interleukin-13 (IL-13), Platelet-Derived Growth Factor (PDGF), and theWnt / beta-catenin signalling pathway (Ma et al., (2022)).

[0127] In some embodiments, fibrosis is associated with one or more inflammatory marker genes. In one embodiment, fibrosis is associated with TNFa, IL-6, IL-i p, STAT3, NOTCH2, PTEN, and / or NOS2. In one embodiment, fibrosis is associated with SPRY1 , FAP, COL1A1 , CTGF and / or CDH2.

[0128] The oligonucleotide or the pharmaceutical composition of the invention may be administered to a subject in need thereof. In one embodiment, the oligonucleotide or the pharmaceutical composition is administered to the subject selected from: (i) patients having an increased risk for developing inflammatory disease, particularly wherein the inflammatory disease is cardiac inflammation; (ii) patients suffering from inflammatory disease and / or patients having an increased risk of inflammatory disease progression; (iii) patients having an increased risk for developing cardiac, kidney, liver or pulmonary disorders; (iv) patients suffering from cardiac disorders and / or patients having an increased risk of cardiac disorder progression; (v) patients having an increased risk for developing fibrotic disorders; and / or (vi) patients suffering from fibrotic disorders and / or patients having an increased risk of fibrotic disorder progression.

[0129] In certain embodiments, an oligonucleotide or composition is administered to a subject, i.e., a subject that does not have a disease or disorder. In one embodiment, an oligonucleotide or composition comprising the same is administered to a subject that is at risk of developing a disease or disorder. In certain embodiments, an oligonucleotide or a composition provided herein is administered to a subject or patient who has been diagnosed with a disease or disorder. In one embodiment, an oligonucleotide or a composition provided herein is administered to a subject who has been diagnosed with a method according to the invention.

[0130] In some embodiments, an oligonucleotide or composition containing an oligonucleotide described herein is administered to a subject before symptoms manifest or symptoms become severe. In one embodiment, the subject is a patient. In some embodiments, a subject to be administered an oligonucleotide or composition is an animal, preferably a mammal. In specific embodiments, a subject to be administered an oligonucleotide or composition is human. In certain embodiments, a subject is a human adult.

[0131] In some embodiments, the subject (e.g., a human) to be administered an oligonucleotide or composition containing an oligonucleotide is any individual at risk of cardiac or pulmonary inflammatory disease or disorder and / or fibrotic diseases. In one embodiment, the patient suffers from or is at risk of developing cardiomyocyte hypertrophy. In one embodiment, the patient suffers from or is at risk of developing cardiomyocyte apoptosis. In one embodiment, the patient suffers from or is at risk of developing myocardial fibrosis. In one embodiment, the patient suffers from or has an increased risk for developing heart failure. In one embodiment, the patient suffers from or is at risk of developing heart failure with reduced ejection fraction (HFrEF).

[0132] In some embodiments, the subject (e.g., a human) to be administered an oligonucleotide or composition containing an oligonucleotide of the invention is an individual affected by any condition that increases susceptibility to cardiac inflammation, cardiomyocyte hypertrophy, cardiomyocyte apoptosis, and / or myocardial fibrosis. In one embodiment, the individual has an increased risk for developing cardiac, kidney, liver or pulmonary disorders.

[0133] Provided herein is a use of an oligonucleotide of the invention in therapy. Also, provided herein is a use of an oligonucleotide of the invention in the manufacture of a medicament for treating a cardiac or pulmonary inflammation. Also provided herein is a use of an oligonucleotide of the invention in the manufacture of a medicament fortreating cardiopulmonary disorders. Also provided herein is a use of an oligonucleotide of the invention in the manufacture of a medicament for treating cardiac fibrotic disorders. Also provided herein is a use of an oligonucleotide of the invention in the manufacture of a medicament for treating kidney and / or liver disorders.

[0134] Further provided herein is a method of preventing and / or treating an inflammatory disease in a subject. In one embodiment, the inflammatory disease is cardiac, kidney, liver or pulmonary inflammation. In one embodiment, the disease is heart failure with reduced ejection fraction (HFrEF).

[0135] Also provided herein is a method of preventing and / or treating cardiopulmonary disorders in a subject. In one embodiment, the disorder is cardiopulmonary inflammation-related remodelling.

[0136] Provided herein is a method of preventing and / or treating fibrotic disorders in a subject. In one embodiment, the fibrotic disorder is cardiac fibrotic disorder. In one embodiment, the fibrotic disorder is left and / or right ventricular fibrosis, atrial fibrosis, endomyocardial fibrosis or fibrosis resulting from a previous myocardial infarction, high blood pressure or myocarditis. In one embodiment, the fibrotic disorder is pulmonary fibrotic disorders. In one embodiment, the fibrotic disorder is pulmonary fibrosis caused by occupational, genetic or environmental factors, radiation treatment and / or treatment with medicaments. In one embodiment, the fibrotic disorder is idiopathic pulmonary fibrosis. In one embodiment, fibrotic disorder is hepatic fibrosis. In one embodiment, the fibrotic disorder is alcoholic liver disease or non-alcoholic fatty liver disease (NAFLD). In one embodiment, the fibrotic disorder is renal fibrosis. In one embodiment, the fibrotic disorder is dermal fibrosis. In one embodiment, the fibrotic disorder is bone marrow fibrosis and / or gut fibrosis. In one embodiment, the fibrotic disorder has contributed to development or progression of heart failure with reduced ejection fraction (HFrEF). Further provided herein is a method of preventing and / or treating heart failure with reduced ejection fraction (HFrEF) in a subject.

[0137] The pharmaceutical compositions provided herein can be in any form that allows for the composition to be administered to a subject. The chemically modified oligonucleotide of the invention or the (pharmaceutical) composition may be administered, for example, orally in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions, or solutions, or parenterally, e.g., by parenteral injection. In some embodiments, formulations suitable for parenteraladministration comprise sterile aqueous preparations of at least one embodiment of the present disclosure, which are approximately isotonic with the blood of the intended recipient. The amount of oligonucleotide or composition to be administered, the dosage and the dosing regimen can vary from cell type to cell type, the disease to be treated, the target population, the mode of administration (e.g., systemic versus local), the severity of disease and the acceptable level of side activity. In some embodiments, the amount of oligonucleotides administered in a pharmaceutical composition is dependent on the subject being treated, the subject's weight, the manner of administration, and / or disease to be treated.

[0138] An oligonucleotide of the invention may be delivered as is ( / .e., naked and / or in isolated form). Hence, in a preferred embodiment, an oligonucleotide of the invention is administered and delivered ‘as is’, also referred to as ‘naked’ or ‘isolated from’. In one embodiment, an oligonucleotide is to be administered as such.

[0139] The key problem for oligonucleotide-based therapeutics is to efficiently deliver the oligonucleotide to the site of action. Hence, oligonucleotides of the invention may be modified at their 3’- and / or 5’-terminal ends to promote cellular uptake and / or in vivo tissue delivery. In one embodiment, the oligonucleotide is conjugated to a heterologous moiety. In one embodiment, the oligonucleotide is linked to one or more heterologous moieties. In one embodiment, the oligonucleotide is to be administered as such, or the oligonucleotide is to be administered conjugated to a heterologous moiety. In one embodiment, the oligonucleotide is linked to the heterologous moiety via a covalent bond. In one embodiment, conjugation of the oligonucleotide to a heterologous moiety (or carrier) promotes cellular penetration and / or uptake. The heterologous moiety can be a carrier. Specifically, in one embodiment, the carrier is a lipid or a polymer. In some embodiments, the carrier is a cell-penetrating peptide (CPP). In one embodiment, the carrier is cholesterol. In one embodiment, a marrier is a nanoparticle. In one embodiment, the oligonucleotide comprises a covalent attachment of poly(ethylene glycol) (PEG). In some cases, N- acetylgalactosamine (GalNac) may be used as a delivery moiety for oligonucleotides. In one embodiment, the oligonucleotide comprises a GalNAc modification.

[0140] The art contains multiple ways of delivering oligonucleotides to cells, tissues or organs, either in vitro, ex vivo or in vivo. For instance, polycationic polymers, nanoparticles, microparticles or liposomal formulations that may be used for in vivo oligonucleotide delivery are well known in the art. In one embodiment, polycationicpolymers are used for delivery of the oligonucleotide. In one embodiment, microparticles are used for delivery of the oligonucleotide. In one embodiment, nanoparticles are used for delivery of the oligonucleotide. In some embodiments, oligonucleotides are delivered using lipid nanoparticles (LNPs). In one embodiment, liposomal formulations are used to deliver the oligonucleotides of the invention.

[0141] Combination therapy has become critical in developing prevention and treatment strategies across many medical disciplines, as drug combinations have the potential to improve treatment response, minimize development of resistance, allow lower doses of component therapies, or reduce adverse events. For instance, heart failure (HF) with reduced ejection fraction has reached a critical juncture with quadruple therapy (a combination of angiotensin receptor-neprilysin inhibitor (ARNI), p-blockers, mineralocorticoid receptor antagonists (MRA), and sodiumglucose co-transporter 2 inhibitor (SGLT2i)) (Heidenreich et al., (2022)).

[0142] The oligonucleotide or composition of the invention may be administered as a monotherapy or in combination with a further different medicament. In some instances, an oligonucleotide or composition of the invention is administered in combination with other oligonucleotides. In one embodiment, the oligonucleotide or composition of the invention is administered in combination with a further different medicament, particularly a medicament suitable for the treatment or prevention of any of the diseases mentioned above. Hence, provided herein is an oligonucleotide or a pharmaceutical composition for use of the invention, wherein the oligonucleotide or the pharmaceutical composition is administered to a subject in combination with one or more other therapies. In one embodiment, the one or more other therapies comprises a standard of care (SoC).

[0143] Examples of further medicaments suitable for the prevention or treatment of cardiac disorders are angiotensin-modulating agents, beta-blockers, diuretics, aldosterone antagonists, vasodilators, ionotrophic agents, statins, neprilysin-inhibitors, or SGL T-2 inhibitors or combinations thereof, e.g., a combination of a neprilysin- inhibitor, e.g., sacubitril, with an angiotensin-ll-receptor blocker, e.g. valsartan.

[0144] In one embodiment, the oligonucleotide is administered in combination with at least one diuretic. In one embodiment, the oligonucleotide is administered in combination with at least one angiotensin-converting enzyme inhibitor. In one embodiment, the oligonucleotide is administered in combination with at least one beta-blocker. In one embodiment, the oligonucleotide is administered in combinationwith an angiotensin-ll-receptor blocker. In one embodiment, the oligonucleotide is administered in combination with an If-channel inhibitor (such as ivabradine). In one embodiment, the oligonucleotide is administered in combination with an angiotensin-receptor-neprilysin-inhibitor. In one embodiment, the oligonucleotide is administered in combination with a glucose Co-transporter 2 inhibitor (such as empagliflozin and dapagliflozin). In one embodiment, the oligonucleotide is administered in combination with stem cell therapeutics. In one embodiment, the oligonucleotide is administered in combination with anti-miRNAs targeting different pathways. In one embodiment, the oligonucleotide is administered in combination with a SGLT-2 inhibitor. In one embodiment, the oligonucleotide is administered in combination with a combination of the above. In one embodiment, the oligonucleotide is to be administered in combination with (i) at least one diuretic, (ii) at least one angiotensin-converting enzyme inhibitor, (iii) at least one beta-blocker, and optionally (iv) an angiotensin-ll-receptor blocker and (v) optionally an If-channel inhibitor such as ivabradine and optionally (vi) an angiotensin-receptor-neprilysin-inhibitor, and optionally (vii) a glucose Co- transporter 2 inhibitor such as empagliflozin and dapagliflozin, and optionally (viii) stem cell therapeutics, and optionally (ix) anti- miRNAs targeting different pathways, and / or optionally (x) a SGLT-2 inhibitor.

[0145] Specifically, in some embodiments, the oligonucleotide or composition of the invention is used in combination therapy with another medicament for treating or preventing cardiac inflammation, cardiomyocyte hypertrophy, cardiomyocyte apoptosis and / or myocardial fibrosis, and / or pulmonary inflammation. In one embodiment, the oligonucleotide or composition of the invention is used in combination therapy with another medicament for treating or preventing an inflammatory disease. In one embodiment, the oligonucleotide or composition of the invention is used in combination therapy with another medicament for treating or preventing cardiopulmonary disorders. In one embodiment, the oligonucleotide or composition of the invention is used in combination therapy with another medicament for treating or preventing fibrosis or a fibrotic disorder.Diagnostic Methods and Uses

[0146] A chemically modified oligonucleotide of the invention or a (pharmaceutical) composition may be used in the diagnosis of a genetic condition, disease, or disorder. Hence, also provided herein is an oligonucleotide for use in the diagnosis of aninflammatory disease, a cardiopulmonary disorder or a fibrotic disorder or a combination thereof.

[0147] Also provided herein is a method of diagnosing an inflammatory disease, a cardiopulmonary, kidney or liver disorder or a fibrotic disorder or combination thereof, wherein the method comprises an oligonucleotide of the invention. In one embodiment, the method is for diagnosing an inflammatory disease. In one embodiment, the method is for diagnosing a cardiopulmonary disorder. In one embodiment, the method is for diagnosing a kidney disorder. In one embodiment, the method is for diagnosing a liver disorder. In one embodiment, the method is for diagnosing a fibrotic disorder (e.g., cardiac, pulmonary, hepatic, renal or dermal fibrotic disorders). In one embodiment, the method comprises one or more oligonucleotides of the invention.

[0148] Also provided herein is the use of an oligonucleotide of the invention for the diagnosis of an inflammatory disease, a cardiopulmonary disorder or a fibrotic disorder or combination thereof. In one embodiment, the use of an oligonucleotide of the invention for the diagnosis of an inflammatory disease. In one embodiment, the use of an oligonucleotide of the invention for the diagnosis of a cardiopulmonary disorder. In one embodiment, the use of an oligonucleotide of the invention for the diagnosis of a fibrotic disorder.

[0149] In some embodiments, the marker gene is one or more inflammatory marker genes. In one embodiment, the marker gene is selected from those listed in Table 6. In one embodiment, the marker gene is TNFa, IL-6, IL-1 p, STAT3, NOTCH2, PTEN, and / or NOS2. In one embodiment, the marker gene is SPRY1 , FAP, COL1A1 , CTGF and / or CDH2.LIST OF FURTHER EMBODIMENTS OF THE INVENTION

[0150] The invention is further described by the following non-limiting embodiments:

[0151] 1 . An oligonucleotide comprising one of the sequences selected from:(i) 5’- +T C A +G T C +T G A +TA +A G +C +T -3’ (formula / ); or(ii) 5’- +T C +A G T +C T G +A +T A +A G C +T -3’ (formula If); wherein A, T, G and C are deoxyribonucleotide building blocks, or C is 5- methyl-2’-deoxycytidine; and wherein +T, +A, +G, and +C are bridged nucleotide building blocks and / or morpholino building blocks.

[0152] 2. An oligonucleotide comprising the sequence 5’- +T C A +G T C +T G A +T A +A G +C +T -3’ (formula / ); wherein A, T, G and C are deoxyribonucleotide building blocks, or C is 5- methyl-2’-deoxycytidine; and wherein +T, +A, +G, and +C are bridged nucleotide building blocks and / or morpholino building blocks.

[0153] 3. An oligonucleotide comprising the sequence 5’- +T C +A G T +C T G +A +T A +A G C +T -3’ (formula / / ); wherein A, T, G and C are deoxyribonucleotide building blocks, or C is 5- methyl-2’-deoxycytidine; and wherein +T, +A, +G, and +C are bridged nucleotide building blocks and / or morpholino building blocks.

[0154] 4. An oligonucleotide comprising CDR076L-21U.

[0155] 5. The oligonucleotide according to embodiment 4, wherein the oligonucleotide consists of CDR076L-21 II.

[0156] 6. An oligonucleotide comprising the sequence and all modifications according to CDR076L-21U.

[0157] 7. The oligonucleotide according to embodiment 6, consisting of the sequence and all modifications according to CDR076L-21 U.

[0158] 8. An oligonucleotide comprising a sequence and all modifications according to SEQ ID NO: 21.

[0159] 9. The oligonucleotide according to embodiment 8, wherein the oligonucleotide consists of a sequence and all modifications according to SEQ ID NO: 21.

[0160] 10. The oligonucleotide according to any one of the previous embodiments, wherein +T, +A, +G, and +C are bridged nucleotide building blocks.

[0161] 11. The oligonucleotide according to any one of the previous embodiments, wherein A, T, G and C are deoxyribonucleotide building blocks.

[0162] 12. The oligonucleotide according to any one of the previous embodiments, wherein at least a C comprises a 5-methyl modification.

[0163] 13. The oligonucleotide according to any one of the previous embodiments, wherein each C comprises a 5-methyl modification.

[0164] 14. The oligonucleotide according to any one of the previous embodiments, wherein at least a C is 5-methyl-2’-deoxycytidine.

[0165] 15. The oligonucleotide according to any one of the previous embodiments, wherein each C is 5-methyl-2’-deoxycytidine.

[0166] 16. An oligonucleotide according to any one of the previous embodiments, wherein +G, +T, +A, and +C are locked nucleic acid (LNA) building blocks.

[0167] 17. The oligonucleotide according to any one of the previous embodiments, comprising at least one modified internucleosidic linkage, optionally wherein all internucleosidic linkages are modified.

[0168] 18. The oligonucleotide according to any one of the previous embodiments, wherein all internucleosidic linkages are modified.

[0169] 19. The oligonucleotide according to any one of the embodiments 17-18, wherein the internucleosidic linkage is a phosphorothioate or phosphorodiamidate linkage, preferably phosphorothioate linkage.

[0170] 20. The oligonucleotide according to any one of the embodiments 17-19, wherein the internucleosidic linkage is a phosphorothioate linkage.

[0171] 21. The oligonucleotide according to any one of the previous embodiments, wherein the oligonucleotide consists of the sequence 5’- +T C A +G T C +T G A +T A +A G +C +T -3’ (formula I).

[0172] 22. An oligonucleotide comprising CDR076L-21.

[0173] 23. The oligonucleotide according to embodiment 22, consisting of CDR076L- 21.

[0174] 24. An oligonucleotide comprising the sequence and all modifications according to CDR076L-21.

[0175] 25. The oligonucleotide according to embodiment 24, wherein the oligonucleotide consists of the sequence and all modifications according to CDR076L- 21.

[0176] 26. An oligonucleotide comprising SEQ ID NO: 46.

[0177] 27. The oligonucleotide according to embodiment 26, consisting of SEQ ID NO: 46.

[0178] 28. An oligonucleotide comprising the sequence and all modifications according to the full length of SEQ ID NO: 46.

[0179] 29. The oligonucleotide according to embodiment 28, wherein the oligonucleotide consists of the sequence and all modifications according to the full length of SEQ ID NO: 46.

[0180] 30. An oligonucleotide comprising CDR076L-02.

[0181] 31. The oligonucleotide according to embodiment 30, consisting of CDR076L- 02.

[0182] 32. An oligonucleotide comprising the sequence and all modifications according to CDR076L-02.

[0183] 33. The oligonucleotide according to embodiment 32, wherein the oligonucleotide consists of the sequence and all modifications according to CDR076L- 02.

[0184] 34. An oligonucleotide comprising SEQ ID NO: 27.

[0185] 35. The oligonucleotide according to embodiment 34, consisting of SEQ ID NO: 27.

[0186] 36. An oligonucleotide comprising the sequence and all modifications according to the full length of SEQ ID NO: 27.

[0187] 37. The oligonucleotide according to embodiment 36, wherein the oligonucleotide consists of the sequence and all modifications according to the full length of SEQ ID NO: 27.

[0188] 38. An oligonucleotide comprising the sequence of formula la:5‘- +T*d5mC*dA*+G*dT*d5mC*+T*dG*dA*+T*dA*+A*dG*+5mC*+T -3‘ ; wherein dA is 2’-deoxyadenosine, dG is 2’-deoxyguanosine, dT is 2’- deoxythymidine; wherein +T is an LNA-T building block, +A is an LNA-A building block, +G is an LNA-G building block and +5mC is an LNA-5-methyl-2’-cytidine building block; wherein d5mC is 5-methyl-2’-deoxycytidine; and wherein * is a phosphorothioate linkage.

[0189] 39. An oligonucleotide comprising the sequence of formula la:5‘- +T*d5mC*dA*+G*dT*d5mC*+T*dG*dA*+T*dA*+A*dG*+5mC*+T -3‘; wherein dA is 2’-deoxyadenosine; dG is 2’-deoxyguanosine; dT is 2’-deoxythymidine;+T is an LNA-T building block;+A is an LNA-A building block;+G is an LNA-G building block;+5mC is an LNA-5-methyl-2’-cytidine building block; d5mC is 5-methyl-2’-deoxycytidine; and* is a phosphorothioate linkage.

[0190] 40. An oligonucleotide comprising the sequence of formula Ila:5’ +T*d5mC*+A*dG*dT*+5mC*dT*dG*+A*+T*dA*+A*dG*d5mC*+T -3' ; wherein dA is 2’-deoxyadenosine, dG is 2’-deoxyguanosine, dT is 2’- deoxythymidine; wherein +T is an LNA-T building block, +A is an LNA-A building block, and +5mC is a LNA-5-methyl-2’-cytidine building block; wherein d5mC is 5-methyl-2’-deoxycytidine; and wherein * is a phosphorothioate linkage.

[0191] 41. An oligonucleotide according to Fig. 11(A).

[0192] 42. An oligonucleotide according to Fig. 11(B).

[0193] 43. The oligonucleotide according to any one of the previous embodiments, wherein the oligonucleotide is conjugated to a heterologous moiety.

[0194] 44. A composition comprising the oligonucleotide according to any one of the previous embodiments, and optionally a pharmaceutically acceptable carrier, delivery agent or excipient.

[0195] 45. The composition of embodiment 44, further comprising at least one of a pharmaceutically acceptable carrier, delivery agent or excipient.

[0196] 46. The composition of embodiment 45, further comprising an acceptable carrier or excipient.

[0197] 47. The composition of embodiment 46, further comprising an acceptable carrier.

[0198] 48. An oligonucleotide according to any one of embodiments 1-43 or a composition of any one of embodiments 44-47 for therapeutic use in a subject.

[0199] 49. An oligonucleotide of any one of embodiments 1-43 or a pharmaceutical a composition of any one of embodiments 44-47, for use in the treatment or prevention of heart failure with reduced ejection fraction (HFrEF).

[0200] 50. An oligonucleotide of any one of embodiments 1-43 or a composition of any one of embodiments 44-47 for use in the prophylaxis and / or treatment of: a) an inflammatory disease in a subject, particularly wherein the inflammatory disease is cardiac or pulmonary inflammation; b) cardiopulmonary disorders in a subject, particularly cardiopulmonary inflammation-related remodelling; and / or c) fibrotic disorders in a subject.

[0201] 51. The oligonucleotide or the pharmaceutical composition for use of embodiment 50, for use in the treatment or prevention of heart failure with reduced ejection fraction (HFrEF).

[0202] 52. The oligonucleotide or the pharmaceutical composition for use of embodiment 50, wherein the fibrotic disorders include cardiac fibrotic disorders and cardia fibrotic related disorder, particularly left and / or right ventricular fibrosis, atrial fibrosis, endomyocardial fibrosis, Heart Failure with Reduced Ejection Fraction (HFrEF), or fibrosis resulting from a previous myocardial infarction, high blood pressure or myocarditis; pulmonary fibrotic disorders, particularly pulmonary fibrosis caused by occupational, genetic or environmental factors, radiation treatment and / or treatment with medicaments; idiopathic pulmonary fibrosis; hepatic fibrotic disorders, particularly alcoholic liver disease or non-alcoholic fatty liver disease (NAFLD); renal fibrosis; dermal fibrosis; bone marrow fibrosis; and / or gut fibrosis.

[0203] 53. The oligonucleotide or the pharmaceutical composition for use of embodiment 50, wherein the fibrotic disorders include cardiac fibrotic disorders, particularly left and / or right ventricular fibrosis, atrial fibrosis, endomyocardial fibrosis, or fibrosis resulting from a previous myocardial infarction, high blood pressure or myocarditis; pulmonary fibrotic disorders, particularly pulmonary fibrosis caused by occupational, genetic or environmental factors, radiation treatment and / or treatment with medicaments; idiopathic pulmonary fibrosis; hepatic fibrotic disorders, particularly alcoholic liver disease or non-alcoholic fatty liver disease (NAFLD); renal fibrosis; dermal fibrosis; bone marrow fibrosis; and / or gut fibrosis.

[0204] 54. The oligonucleotide or the pharmaceutical composition for use of embodiment 50, wherein the fibrotic related disorder is heart failure with reduced ejection fraction (HFrEF).

[0205] 55. The oligonucleotide or the pharmaceutical composition for use of any one of embodiments 48-54, wherein the oligonucleotide or the pharmaceutical composition is administered to the subject selected from:(i) patients having an increased risk for developing inflammatory disease, particularly wherein the inflammatory disease is cardiac inflammation;(ii) patients suffering from inflammatory disease and / or patients having an increased risk of inflammatory disease progression;(iii) patients having an increased risk for developing cardiac, kidney, liver or pulmonary disorders;(iv) patients suffering from cardiac disorders and / or patients having an increased risk of cardiac disorder progression;(v) patients having an increased risk for developing fibrotic disorders; and / or(vi) patients suffering from fibrotic disorders and / or patients having an increased risk of fibrotic disorder progression.

[0206] 56. The oligonucleotide or the pharmaceutical composition for use of any one of embodiments 48-54, wherein the oligonucleotide or the pharmaceutical composition is administered to the subject in combination with one or more other therapies, optionally wherein the one or more other therapies comprises a standard of care (SoC).

[0207] 57. An oligonucleotide of any one of embodiments 1-43 or a composition of any one of embodiments 44-47 for use in the diagnosis of an inflammatory disease, a cardiopulmonary, kidney or liver disorder, or a fibrotic disorder, or a combination thereof in a sample obtained from a subject.

[0208] 58. A method of treating or preventing an inflammatory disease in a subject, wherein the method comprises administering to the subject an effective amount of one of the oligonucleotides selected from Table 1 .

[0209] 59. The method of embodiment 58, wherein the inflammatory disease is cardiac or pulmonary inflammation.

[0210] 60. A method for treating or preventing cardiopulmonary disorders in a subject, wherein the method comprises administering to the subject an effective amount of one of the oligonucleotides selected from Table 1.61. The method of embodiment 60, wherein the cardiopulmonary disorder is cardiopulmonary inflammation-related remodelling.

[0211] 62. A method for treating or preventing fibrotic disorders in a subject, wherein the method comprises administering to the subject an effective amount of one of the oligonucleotides selected from Table 1.

[0212] 63. The method of embodiment 62, wherein the oligonucleotide selected from CDR076L-02B, CDR076L-21 U, CDR076L-02 and CDR076-21.

[0213] 64. The method of embodiment 63, wherein the oligonucleotide is CDR076- 21.

[0214] 65. A method of treating or preventing an inflammatory disease in a subject, wherein the method comprises administering to the subject an effective amount of the oligonucleotide of any one of embodiments 1-43.

[0215] 66. A method for treating or preventing cardiopulmonary disorders in a subject, wherein the method comprises administering to the subject an effective amount of the oligonucleotide of any one of embodiments 1-43.

[0216] 67. A method for treating or preventing fibrotic disorders in a subject, wherein the method comprises administering to the subject an effective amount of the oligonucleotide of any one of embodiments 1-43.

[0217] 68. The oligonucleotide according to embodiments 1-43, for use in the manufacture of a medicament.

[0218] 69. The oligonucleotide according to embodiments 1-43, for use in the manufacture of a medicament for the treatment of a) an inflammatory disease in a subject, particularly wherein the inflammatory disease is cardiac or pulmonary inflammation; b) cardiopulmonary disorders in a subject, particularly cardiopulmonary inflammation-related remodelling; and / or c) fibrotic disorders in a subject.

[0219] 70. An oligonucleotide for use in the manufacture of a medicament for the treatment of a) an inflammatory disease in a subject, particularly wherein the inflammatory disease is cardiac or pulmonary inflammation; b) cardiopulmonary disorders in a subject, particularly cardiopulmonary inflammation-related remodelling; and / or c) fibrotic disorders in a subject, wherein the oligonucleotide comprises one of the sequences selected from: (i) 5’- +T C A +G T C +T G A +T A +A G +C +T -3’ (formula / ); or (ii) 5’- +T C +A G T +C T G +A +T A +A G C +T -3’ (formula If); wherein A, T, G and C are deoxyribonucleotide building blocks, or C is 5-methyl-2’-deoxycytidine; and wherein +T, +A, +G, and +C are bridged nucleotide building blocks and / or morpholino building blocks.

[0220] 71. An oligonucleotide for use in the manufacture of a medicament for the treatment of a) an inflammatory disease in a subject, particularly wherein the inflammatory disease is cardiac or pulmonary inflammation; b) cardiopulmonary disorders in a subject, particularly cardiopulmonary inflammation-related remodelling; and / or c) fibrotic disorders in a subject, wherein the oligonucleotide comprises the sequence of formula la: 5‘-+T*d5mC*dA*+G*dT*d5mC*+T*dG*dA*+T*dA*+A*dG*+5mC*+T -3‘ ; wherein dA is 2’- deoxyadenosine, dG is 2’-deoxyguanosine, dT is 2’-deoxythymidine; wherein +T isan LNA-T building block, +A is an LNA-A building block, +G is an LNA-G building block and +5mC is an LNA-5-methyl-2’-cytidine building block; wherein d5mC is 5- methyl-2’-deoxycytidine; and wherein * is a phosphorothioate linkage.

[0221] 72. The oligonucleotide of any one of embodiments 1-43 or composition of embodiments 44-47 for use in diagnosing a) an inflammatory disease in a subject, particularly wherein the inflammatory disease is cardiac or pulmonary inflammation; b) cardiopulmonary disorders in a subject, particularly cardiopulmonary inflammation- related remodelling; and / or c) fibrotic disorders in a subject.EXAMPLES

[0222] Assays for testing the anti-fibrotic and anti-inflammatory effects of CDR076L oligonucleotides may be conducted using any assay known in the art. Further, any assays known to those skilled in the art can be used to evaluate the prophylactic and / or therapeutic utility of the oligonucleotides and compositions described herein, for example, by measuring a condition or symptoms associated with cardiac inflammation and / or cardiac fibrosis. Oligonucleotides and / or compositions thereof can be tested for in vitro efficacy and / or toxicity by standard experimental procedures in cell culture and / or experimental animals.

[0223] The different cells used in the Examples of the application as filed are listed in Table 2 as indicated below.Table 2: Overview on cells used in Examples 1 to 5 of the application.

[0224] The different cell culture media used in the different Examples are listed in Table 3 as indicated below.Table 3: Overview of cell media and their compositions.

[0225] The different cell culture reagents used in the different Examples are listed in Table 4 as indicated below.Table 4: Overview on cell culture reagents usedExample 1. In vitro efficacy screening in murine RAW 264.7 and human THP- 1 macrophage cell lines.

[0226] CDR076L candidate oligonucleotides were selected and tested to determine their in vitro efficacy and toxicity in inflammatory cells.

[0227] Primary Efficacy Screening in RAW 264.7 cells

[0228] A quantitative in vitro assay for assessing the inhibitory activity of 26 structural analogue compounds derived from an anti-miR-21 library was carried out in RAW 264.7 macrophages (CLS, cat. no. 400319) (Fig. 1A and B).

[0229] Cells were maintained, seeded and treated as described in the following sections.

[0230] Maintenance: RAW264.7 cells were grown in RAW264.7-culture medium (see, Table 3) and maintained in a 5% CO2 humidified incubator at 37°C. Additional RAW264.7-culture medium was added two days after seeding and every 2-3 days thereafter. During weekends, the added volume of medium was doubled. For inflammatory stimulation and treatment experiments, the medium was exchanged for fresh RAW264.7-culture medium containing respective test items and stimuli.

[0231] Seeding: RAW 264.7 macrophages were taken into cultures on day “d-1”, one day prior to polarisation. That is, following scraping, cells were centrifuged at 300 x g for 3 min at room temperature and resuspended in RAW264.7-culture medium. The concentration of cells in suspension was determined and cells were seeded into96 well cell culture plates on day -1 with a density of 4 x 104cells / well. Cells were incubated in a 5% CO2 humidified incubator at 37°C for 24 h before stimulation.

[0232] Treatment with test item and stimulation: On day 0, cells were stimulated and treated in RAW264.7-culture medium supplemented with 10 ng / mL LPS and 100 nM of the corresponding miR-21 inhibitors as indicated. Reference cells were treated with 0.9% NaCI solution. Incubation was performed in a 5% CO2 humidified incubator at 37°C for 48 h (Fig. 1(B)) in DMEM (Dulbecco’s Modified Eagle’s Medium) supplemented with 2 mM L glutamine, 100 ll / rnl penicillin and 100 pg / ml streptomycin, and 10% heat inactivated FBS.

[0233] Cell lysis: At the experimental endpoint, RAW264.7 were washed once with 1 x DPBS (Dulbecco’s phosphate buffered saline), and cell lysis was performed using the Cells-to-Ct Bulk Lysis Reagents (Thermo Fisher Scientific, 4391851 C) according to the manufacturer’s instructions. Briefly, cells were lysed using Lysis Solution (containing DNase I). The lysate reaction was stopped by adding Stop Solution. Cell lysates were stored at -20 °C until reverse transcription.

[0234] cDNA synthesis: To determine functional levels of miR-21-5p in RAW264.7 derived samples, 2.5 pl of cell lysate was reverse transcribed into cDNA using the TaqMan MicroRNA Cells-to-Ct Kit (Thermo Fisher Scientific, 4391848). Appropriate and validated TaqMan MicroRNA Assays (Thermo Fisher Scientific, Germany) were used. miRNA specific TaqMan Assays used in this study are listed below:Table 5: TaqMan MicroRNA Assays used in the Examples. Purchased from Thermo Fisher Scientific. miR = microRNA; snRNA = non-coding small nuclear RNA; cel = C. elegans.

[0235] To determine the inhibitory efficacy of each candidate oligonucleotide in miR- 21 and marker genes, miR-21, TNF-a, Nos2, II-6, 11-1 p, Stat3 and Notch2 expression was quantified by TaqMan® assays and quantitative real-time PCR (qPCR). Placebo and miRCURY LNA-21 were used as control. Data were plotted for individual samples and represented as mean + / - standard deviation (SD). Totest significant differences between groups, a two-tailed unpaired Student’s t-test was used.

[0236] For analysis of gene expression of marker genes, 10 pl of cell lysate from RAW264.7 derived samples was reverse transcribed into cDNA using the TaqMan Gene expression Cells-to-Ct Kit (Thermo Fisher Scientific, 4399002). In samples derived from TH P-1 and NHCF-V, 350 ng and 125 ng RNA was reverse transcribed into cDNA using the Verso cDNA Synthesis Kit (Thermo Fisher Scientific, AB1453A), respectively. cDNA was stored at -20°C until qRT-PCR analysis.

[0237] For quality control, a non-template RT control (RT-NTC; containing water instead of RNA) was included in each reverse transcription reaction. Additionally, a lysis buffer control was included in each reverse transcription reaction in RAW264.7 sample sets. For qRT-PCR analysis, samples were analysed in duplicates. Heatmap was generated using GraphPad PRISM.

[0238] The following validated TaqMan Assays (Thermo Fisher Scientific, Germany) were used in this study.Table 6: TaqMan assays used in this study. GOI = gene of interest; RG = reference gene; miR = microRNA; snRNA = non-coding small nuclear RNA; cel = C. elegans.

[0239] All qRT-PCR data were recorded and saved with QuantStudio Real-Time PCR Software (Thermo Fisher Scientific Inc.). The according plate layout was defined prior each run. Further, the threshold, which defines the threshold of fluorescence detection and thereby determining the Cq values, was manually corrected. It is critical to set the threshold within the early exponential range of amplification.

[0240] The method of choice to analyse the qRT-PCR data was the AACq-Method. This method assumes the optimal qRT-PCR efficiency of 100 % with a doubling of qRT- PCR product with each cycle. The following measurements were applied:ACqSample—CqGene of interest CqReference geneFold difference=2"^^=2"^^ (treatment group) I 2-ACq (placebo)

[0241] Functional levels of miR-21 -5p were normalized to U6 snRNA. Target gene expression and inflammation-associated gene expression or fibrosis-associated gene expression data were normalized to HPRT1 (for RAW246.7 and NHCF-V (see, Example 3)) and TBP (for TH P-1).

[0242] In total, the arithmetical formula 2’AACc> provides the relative difference of expression level between control and treatment groups, both normalized to a reference gene. This mathematical operation was performed for all samples / genes. The 2’AACc> values were plotted and visualized in GraphPad Prism version for Windows (GraphPad Software, La Jolla California USA).

[0243] Statistics: qRT-PCR data were plotted for individual samples and represented as mean + / - standard deviation (SD). To test significant differences between groups, a two-tailed unpaired Student’s t-test was used.

[0244] As shown in Fig. 1(B), the testing of 26 candidates resulted in the identification of six most promising CDR076L candidates that identified based on their in vitro efficacy to target miR-21. Candidates CDR076L-02, -13, -15, -18, - 21 , and -23 (see, e.g., Table 1) were most effective in downregulating miR-21 expression in vitro. Based on the results obtained further secondary efficacyscreenings were conducted in TH P-1 monocyte / macrophage cells (human leukaemia monocytic cell line; CLS, cat. No. 300356).

[0245] Secondary Efficacy Screening in TH P-1 cells

[0246] Secondary efficacy screening was set up in TH P-1 cells as shown in Fig. 2(A) and (B). Placebo-treated cells were used as negative control. Cells were maintained, seeded and treated as described below.

[0247] Maintenance: THP-1 cells were grown in THP-1-culture medium (see, Table 3) and maintained in a 5% CO2 humidified incubator at 37°C. After seeding, THP-1 were cultured in TH P-1 -culture medium. Additional TH P-1 -culture medium was added two days after seeding and every 2-3 days thereafter. During weekends, the added volume of medium was doubled. For inflammatory stimulation and treatment experiments, the medium was exchanged for fresh TH P-1 -culture medium containing respective test items and stimuli.

[0248] Seeding: Collected cells were centrifuged at 300 x g for 3 min at room temperature and resuspended in THP-1-culture medium supplemented with 100 ng / ml M-CSF. The concentration of cells in suspension was determined and cells were seeded into 12-well cell culture plates on day -4 with a density of 3 x 105cells / well (Fig. 2B(i)). Cells were incubated in a 5% CO2 humidified incubator at 37 °C for 4 days. At day -2 additional THP-1-culture medium (no M-CSF) was added to the wells.

[0249] Treatment with test item and stimulation: After 4 days (on “dO”), cells were stimulated and treated in THP-1-culture medium enriched with 100 ng / mL LPS + 20 ng / ml IFN-y and 100 nM of the corresponding miR-21 inhibitors (CDR076L-02, -13, -15, -18, -21 , -23) or placebo as indicated (see, Fig. 2(B)(i)). Reference cells were treated with 0.9% NaCI solution. Incubation was performed in a 5% CO2 humidified incubator at 37°C for 48 h. Cells were harvested 48 hrs post treatment and expression levels assessed using Reverse Transcription (see below) (Fig. 2B(i) and (ii)). The efficacy screening data shown in Fig. 2(B)(ii) are summarised in Table 7.Table 7: Efficacy screening in human THP-1 monocytes / macrophages.

[0250] RNA extraction and quantification: Since stimulation with LPS and IFN-y leads to the development of an adherent TH P-1 subtype, not attached cells were removed, washed once with DPBS and lysed. The adherent cells remaining in the well were washed once with DPBS and lysed as well. Both subtypes were combined afterward. Cell lysis and RNA extraction was performed using the miRNeasy Tissue / Cells Advanced Mini Kit (QIAGEN, 217604) according to the manufacturer’s instruction. Directly after isolation, the RNA concentration was assessed by photometric measurement. Then, RNA was stored at -80 °C until further processing.

[0251] cDNA synthesis and qRT-PCR analysis: The same protocol was used as described for RAW264.7 cells above, with the exception that in samples derived from THP-1 , 20 ng RNA was reverse transcribed into cDNA using the TaqMan MicroRNA Reverse Transcription Kit (Thermo Fisher Scientific, 4366596). Furthermore, for analysis of gene expression of marker genes, in samples derived from THP-1 , 350 ng RNA was reverse transcribed into cDNA using the Verso cDNA Synthesis Kit (Thermo Fisher Scientific, AB1453A).

[0252] Based on the in vitro efficacy data, CDR076L-02 and -21 were identified as lead compounds (Fig. 2(B)). Hence, without being bound by any particular theory, the inventors submit that there must be an optimal balance between the particular modification pattern of the lead compounds and their ability to suppress functional miR-21.Example 2. Candidates CDR076L-02 and -21 reduce (cardiac) inflammation marker genes in macrophages stimulated with inflammatory agents.

[0253] To assess the efficacy of lead candidate compounds CDR076L-02 and -21 in reducing expression of functional miR-21 and modulate expression of (cardiac) inflammation marker genes, murine RAW 264.7 macrophages (Fig. 3) and human THP-1 cells (Fig. 4) were treated with the lead compounds (100 nm) in the presence of 10 ng / ml LPS. In these figures, results that are overlayed with a gray background indicate the presence of LPS. miRCURY LNA-21 (100 nM) served as control. Datarepresent mean ± SD. P values: t-test. ns: p>0.05; *: p<0.05; **: p<0.01; ***: p<0.001 ; ****: p<0.0001.

[0254] The inventors were able to show that CDR076L-02 and -21 significantly downregulate expression of functional miR-21 and (cardiac) inflammation in RAW 264.7 cells when compared to control (placebo + LPS) (Fig. 3(A)). Moreover, the lead candidates showed a significant downregulation in relation to miRCURY LNA- 21 (Fig. 3(A)). The data shown in Fig. 3(A) are summarised in Table 8 below. Moreover, lead compounds CDR076L-02 and -21 showed a significant downregulation of Stat3, Notch2, and Nos2 expression when compared to control (Fig. 3(C)-3(E)).Table 8: Efficacy Screening in Murine RAW 264.7 macrophages.

[0255] Similarly, in THP-1 cells (Fig. 4), lead compounds CDR076L-02 and -21 mediated a significant decrease in functional miR-21 (Fig. 4(A)). Furthermore, there was a significant downregulation in inflammatory markers IL-6 and Notch2 expression levels in the presence of 100 nM CDR076L-21 when compared to control (Fig. 4(B) and 4(D)). At the same, CDR076L-21 significantly repressed expression of inflammatory markers STAT3 and NOTCH2 when compared to control (Fig. 4(C) and 4(D)).

[0256] These results show that CDR076L-02 and -21 significantly downregulate expression of functional miR-21 in murine RAW 264.7 macrophages and human THP-1 monocytes / macrophages. Moreover, these data suggest and confirm the significance of having certain backbone modifications or a certain building block pattern, / .e., a mixture of LNA and DNA with a phosphorothioate (PS) backbone, included in the oligonucleotide in order to provide effective miR-21 targeting and downregulation of functional miR-21.Example 3. In vitro efficacy screening in human cardiac fibroblasts (NHCF-V) of CDR076L candidates and identification of lead compounds.

[0257] Drug efficacy assessment without using animals is important for development of cardiac fibrosis treatment. Human Cardiac Fibroblasts are derived from normal human heart tissues and have been used to study the effects of candidate drugs on fibroblast function. Inhibition of miR-21 with a synthetic oligonucleotide effectively inhibits miR-21 activity. Accordingly, the inventors went on to carry out a quantitative in vitro assay for miRNA inhibitory activity of 26 structural analogue compounds (see Table 1) derived form an anti-miR-21 library was carried out together with in vitro dose optimization in Normal Human Ventricular Cardiac Fibroblasts (NHCF-V) (Lonza, cat. no. CC-2904). The experimental set up and results are shown in Fig. 5.

[0258] Maintenance: NHCF-V cells were cultured in NHCF-V growth medium containing 10% FBS (see, Table 3) in a humid incubator providing at 5% CO2 environment at 37°C. Medium was changed one day after seeding and every 1-3 days thereafter. For treatment experiments, the medium was exchanged for fresh NHCF-V growth medium containing respective test items and stimuli.

[0259] Seeding: Following trypsinization, cells were centrifuged at 200 x g for 6 min at room temperature and resuspended in NHCF-V growth medium containing 10% FBS. The concentration of cells in suspension was determined, cells were seeded into 12 well cell culture well plates on day -1 [on day “d-1”, one day prior to fibrotic stimulation (on “d0”)] with a density of 2.5x104cells / well. Cells were incubated in a 5% CO2 humidified incubator at 37°C for 24 h (± 4 h) before stimulation with 10 ng / ml TGF-p and treatment with the test items (Fig. 5(B)).

[0260] Treatment with test item and stimulation: Before treatment on day 0, cells were washed once with prewarmed DPBS. Cells were treated with the test items (CDR076L-01 to -26 or miRCURY LNA-21 (a customized LNA oligonucleotide provided by Qiagen as listed in Table 1) or Placebo (0.9% NaCI)) supplementing either 100 nM (efficacy screening with 26 candidates) or 50 nM, 100 nM or 200 nM (dose optimization with 9 candidates) of each miR-21 inhibitor variant as indicated to the NHCF-V growth medium containing 1% FBS and 10 ng / ml TGF-p. Incubation was performed in a 5% CO2 humidified incubator at 37°C for 72 h. Reference / placebo cells were treated with 0.9% NaCI solution (Fig. 5(B)).

[0261] RNA extraction and quantification: After 72 h of TGF-p stimulation, NHCF- V cells were washed once with DPBS. Cell lysis and RNA extraction wasperformed using the miRNeasy Tissue / Cells Advanced Mini Kit (QIAGEN, 217604) according to the manufacturer’s instruction. After cell lysis, samples were frozen and stored at -80°C until further processing. RNA was extracted from all samples. Directly after isolation, the RNA concentration was assessed by photometric measurement. Then, RNA was stored at -80°C until further processing.

[0262] cDNA synthesis and qRT-PCR analysis: The same protocol was used as described for RAW264.7 cells above (see, Example 1), with the exception that in samples derived from NHCF-V, 20 ng RNA was reverse transcribed into cDNA using the TaqMan MicroRNA Reverse Transcription Kit (Thermo Fisher Scientific, 4366596). Furthermore, for analysis of gene expression of marker genes, in samples derived from NHCF-V, 125 ng RNA was reverse transcribed into cDNA using the Verso cDNA Synthesis Kit (Thermo Fisher Scientific, AB1453A).

[0263] As shown in Fig. 5(B)(iii), each structural analogue compound was assigned a scoring rank (1 (highest score) to 5 (lowest score)) based on the output of the qRT-PCR of functional miR-21 and fibrotic marker gene expression including SPRY1 , FAP, CDH2, COL1a1 , and CTGF as described above. For example, the inventors were able to show that targeting of miR-21 by lead candidate compound CDR076L-02, -15, -19, and -21 resulted in a high change in, e.g., FAP, a key regulator of fibrosis and CDH2 (Fig. 5(B)(iii)). Based on the efficacy screening in NHCF-V cardiac fibroblasts the inventors were able to identify nine lead compounds (CDR076L-02, -04, -05, -06, -11 , -15, -19, and -21) which were selected for further dose optimization.

[0264] As shown in Fig. 5(B)(iv), NHCF-V cells were taken into culture on day “d-1”, one day prior to fibrotic stimulation (which took place on “dO”). On “dO”, NHCF-V cells were stimulated with 100ng / ml TGF-p in the presence of 50 nM, 100 nM, or 200 nM of either CDR076L-02, -04, -05, -06, -11 , -15, -19, -21 or miRCURY LNA-21 (a customized LNA oligonucleotide provided by Qiagen, the sequence and modification of which is listed in Table 1) or Placebo (0.9% NaCI) in FGM-3 medium supplemented with 1% FBS. Treated cells were incubated for 72 hrs before harvesting (endpoint). As described above, each structural analogue compound was subsequently assigned a scoring rank based on the output of the qRT-PCR of functional miR-21 and fibrotic marker gene expression including SPRY1 , FAP, CDH2, COL1a1 , and CTGF after treatment with 100 nM of each oligonucleotide (Fig. 5(B)(iv)). As shown in Fig. 5(B)(iv), theinventors were able to further narrow down the candidate compounds to four lead candidate compounds (-02, -15, -19, and 21) based on their scoring (1 = highest score, 5 = lowest score). The data are summarised in Table 9.Table 9: Efficacy Screening in human cardiac fibroblasts.

[0265] The inventors subsequently went on to show that efficacy screening of lead candidates CDR076L-02 and -21 reduced fibrotic marker genes in TGF-p stimulated cardiac fibroblasts (Fig. 6). Placebo treated cells were used as control. In these figures, results that are overlayed with a gray background indicate the presence of TGF-p. Data are mean ± SD. P values: t-test; ns: p>0.05; *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001. Both lead candidates (CDR076L-02 and - 21) significantly downregulate expression of functional miR-21 in human cardiac fibroblasts when compared to control (see, Fig. 6(A); placebo and miRCURY LNA- 21). The data depicted in Fig. 6(A) is summarised in Table 10. Further, the oligonucleotides were shown to have a significant effect on the expression of CDH2, COL1a1 , CTGF and FAP (see, Fig. 6(B)-(E)).Table 10: Efficacy Screening in human cardiac fibroblasts.

[0266] Dose Optimization

[0267] In view of the lead candidates identified during the efficacy screening, the inventors went on to conduct dose optimisation experiments in human cardiac fibroblasts. Placebo and miRCURY LNA-21 treated cells were used as control.Data are mean ± SD. P values: t-test; ns: p>0.05; *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001. As shown in Fig. 7, both lead candidate compounds (CDR076L-02 and -21) were effective at a dose of 50 nM when compared to miRCURY LNA-21.

[0268] These data confirm the results observed in murine RAW 264.7 and human TH P-1 macrophage cell lines, in that certain backbone modifications or a certain building block pattern, / .e., a mixture of LNA and DNA with a phosphorothioate (PS) backbone, should be included in the oligonucleotide to provide effective targeting and suppression of functional miR-21 and reduction in fibrotic marker gene expression.Example 4. Candidates CDR076L-02 and -21 Reduce Fibrotic Marker Genes in Lung Fibroblasts.

[0269] Inhibition of miR-21 with synthetic oligonucleotides effectively inhibits miR-21 activity and reduces (cardiac) inflammation marker genes in macrophages (see, Example 2). It is known that miR-21 is a pro-fibrotic microRNA and a molecular master-switch driving heart failure (Thum etal., 2008). Therefore, the inventors went on to conduct a proof of concept study to determine the efficacy of lead candidate compounds CDR076L-02 and -21 to regulate miR-21 expression in an in vitro model of fibrosis. To assess their efficacy in reducing expression of functional miR-21 and modulate expression of fibrotic marker gene COL1a1 , normal human lung fibroblasts (NHLF) were treated with each of the lead compounds (100 nm) at day 0 (dO) in the presence of 10 ng / ml TGF-p. Marker expression was detected as described below. Gene expression analysis (cell lysis, RNA extraction and quantification, cDNA synthesis, and qRT-PCR) took place as described under Example 1 above.

[0270] Normal Human Lung Fibroblasts (NHLF)

[0271] Maintenance: NHLF cells were cultured in NHLF growth medium containing 2% FBS (see, Table 3) in a humid incubator providing a 5% CO2 environment at 37°C. Medium was changed one day after seeding and every 1-3 days thereafter. For treatment experiments, the medium was exchanged for fresh NHLF growth medium containing respective test items and stimuli.

[0272] Seeding: Following trypsinization, cells were centrifuged at 200 x g for 6 min at room temperature and resuspended in NHLF growth medium containing 2% FBS. The concentration of cells in suspension was determined and cells were seeded into12-well cell culture plates on day -1 with a density of 3.5x104cells / well. Cells were incubated in a 5% CO2 humidified incubator at 37°C for 24 h (± 4 h) before stimulation with 10 ng / ml TGF-p and treatment with the candidate compounds.

[0273] Treatment with test item and stimulation: On day 0, cells were stimulated and treated in NHLF growth medium containing 0.2% FBS, 10 ng / ml TGF-p and 100 nM of the corresponding miR-21 inhibitor as indicated. Reference cells were treated with 0.9% NaCI solution. Incubation was performed in a 5% CO2 humidified incubator at 37°C for 48 h (Fig. 8(A)).

[0274] cDNA synthesis and qRT-PCR analysis: In samples derived from NHLF, 250 ng RNA was reverse transcribed into cDNA using the Verso cDNA Synthesis Kit (Thermo Fisher Scientific, AB1453A), respectively. cDNA was stored at -20°C until qRT-PCR analysis.

[0275] RNA extraction and quantification: After 72 h of TGF-p stimulation, NHCF- V cells were washed once with DPBS. Cell lysis and RNA extraction was performed using the miRNeasy Tissue / Cells Advanced Mini Kit (QIAGEN, 217604) according to the manufacturer’s instruction. After cell lysis, samples were frozen and stored at -80°C until further processing. RNA was extracted from all samples. Directly after isolation, the RNA concentration was assessed by photometric measurement. Then, RNA was stored at -80°C until further processing.

[0276] Placebo treated cells were used as control. Data are mean ± SD. P values: t-test; ns: p>0.05; *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001. Results are shown in Fig. 8(B) and 8(C). In these figures, results that are overlayed with a gray background indicate the presence of TGF-p. The data shown in Fig. 8(B) are further summarised in Table 11.Table 11 : Efficacy Screening in human lung fibroblasts.

[0277] As shown in Fig. 8(B), addition of candidate compounds CDR076L-02 and -21 led to a significant reduction in functional miR-21 expression in lung fibroblasts when compared to placebo (with or without TGF-p). Specifically, there was about a 100-fold reduction in the level of miR-21 for both candidates CDR076L-02 and -21. Likewise, as shown in Fig. 8(C), candidate compounds CDR076L-02 and -21 significantly recued expression of COL1A1.

[0278] These data not only show that CDR076L-02 and -21 efficiently downregulate expression of functional miR-21 and fibrotic marker gene in lung fibroblasts but also provide a proof of concept that chemically modified oligonucleotide compounds comprising particular backbone modifications and a certain building block pattern, / .e., a mixture of LNA and DNA with a phosphorothioate (PS) backbone, are efficient in targeting and downregulation of functional miR-21. Accordingly, such compounds may provide a useful approach to treating fibrosis.Example 5. In vitro Toxicity Screening in human hepatocytes (HepaRG) and renal proximal tubule epithelial cells (RPTEC) cells.

[0279] It has been shown that that relatively minor modifications to the base pair sequence or backbone of an oligonucleotide can have drastic consequences for toxicity. Consequently, cytotoxicity has a major implication on oligonucleotide use and patient safety. The inventors went on to assess the toxicity of candidate compounds CDR076L-02 and -21 in vitro.

[0280] HepaRG are the most reliable hepatocyte-like cells for studying liver functions or disorders. At the same time, RPTEC are commonly used to determine human renal cell responses to drugs or environmental toxicants. Hence, the inventors went on to assess the effect of lead compounds on cell apoptosis (Caspase assay), metabolic activity (MTT assay) and cell / membrane damage (LDH assay) of HepaRG and RPTEC (Fig. 9(A)-(C)). As will be appreciated by the skilled person, many assays well-known in the art can be used to assess cellular viability and metabolic activity following exposure to an oligonucleotide, active compound or a composition thereof. The cell culture conditions and assays used in Example 4 are outlined below.

[0281] HepaRG

[0282] Maintenance: HepaRG cells were cultivated in a humid incubator providing a 5% CO2 environment at 37°C.

[0283] Seeding: HepaRG were thawed and seeded in HepaRG Thaw, Plate & General Purpose Medium (see, Table 3) into 96 well cell culture well plates on day -6 with a density of 7.2x104cells / well. For caspase assay, collagen I coated white-walled 96-well plates with clear bottom were used. For MTT and LDH assay, collagen Icoated clear 96-well plates were used. One day after thawing, medium was replaced to HepaRG Maintenance / Metabolism Medium (see, Table 3) and renewed every 2-3 days thereafter.

[0284] Treatment with test item: On day 0, cells were treated in HepaRG Maintenance / Metabolism Medium enriched with 1 nM, 10 nM, 25 nM, 50 nM, 100 nM, 200 nM of the corresponding miR-21 inhibitors as indicated. Reference cells were treated with 0.9% NaCI solution. Incubation was performed in a 5% CO2 humidified incubator at 37°C. As a positive control for the caspase assay, Staurosporine treatment (0 pM, 0.3 pM, 1 pM, 3 pM, 10 pM, 30 pM Staurosporine in HepaRG Maintenance / Metabolism Medium) of the cells was performed 44 h post administration of the miR-21 inhibitors as indicated (Fig. 9(A)). A colorimetric commercial MTT assay was used (CellTiter 96® Non-Radioactive Cell Proliferation Assay, Promega) to assess cytotoxicity. For the MTT assay (Fig. 9(B)), doxorubicin treatment (0.25 pM, 0.5 pM, 1 pM, 2 pM, 5 pM, 10 pM Doxorubicin in HepaRG Maintenance / Metabolism Medium) of the cells served as positive control and was performed for 48 h. Therefore, treatment with doxorubicin was completed at the same time as the treatment with the miR-21 inhibitors as indicated. Since the LDH dilution series, which served as positive control for the LDH cytotoxicity assay (LDH-Glo Cytotoxicity Assay Kit, Promega) (Fig. 9(C)), was prepared immediately before assay performance, no additional cell treatment beforehand was necessary. After a total incubation time of 48 h, incubation at 37°C was stopped and cell toxicity assays were performed.

[0285] RPTEC

[0286] Maintenance: RPTEC were cultivated in Renal epithelial growth medium (see, Table 3) in a humid incubator providing a 5% CO2 environment at 37°C. Medium was changed one day after seeding and every 1-3 days thereafter.

[0287] Seeding: Following trypsinization, cells were centrifuged at 200 x g for 6 min at room temperature and resuspended in Renal epithelial growth medium. The concentration of cells in suspension was determined, cells were seeded into 96 well cell culture well plates on day -6 with a density of 1x104cells / well. For caspase assay, white-walled 96-well plates with clear bottom were used. For MTT and LDH assay, clear 96-well plates were used.

[0288] Treatment with test item and stimulation: On day 0, cells were treated in renal epithelial growth medium enriched with 1 nM, 10 nM, 25 nM, 50 nM, 100 nM,200 nM of the corresponding miR-21 inhibitors as indicated. Reference cells were treated with 0.9% NaCI solution. Incubation was performed in a 5% CO2 humidified incubator at 37°C. As a positive control for the caspase assay, Staurosporine treatment (0 M, 0.3 M, 1 pM, 3 pM, 10 pM, 30 pM Staurosporine in Renal epithelial growth medium) of the cells was performed 44 h post administration of the miR-21 inhibitors as indicated (Fig. 9(A)). As described above, a colorimetric commercial MTT assay was used (CellTiter 96® Non-Radioactive Cell Proliferation Assay, Promega) to assess cytotoxicity. For the MTT assay (Fig. 9(B)), doxorubicin treatment (0.25 pM, 0.5 pM, 1 pM, 2 pM, 5 pM, 10 pM Doxorubicin in Renal epithelial growth medium) of the cells served as positive control and was performed for 48 h. Therefore, treatment with doxorubicin was completed at the same time as the treatment with the miR-21 inhibitors as indicated. Since the LDH dilution series, which served as positive control for the LDH cytotoxicity assay (LDH-Glo Cytotoxicity Assay Kit, Promega) (Fig. 9(C)), was prepared immediately before assay performance, no additional cell treatment beforehand was necessary. After a total incubation time of 48 h, incubation at 37°C was stopped and cell toxicity assays were performed.

[0289] Caspase assay: Specifically, 48 h after treatment with the oligonucleotides, incubation at 37°C was stopped and cell culture plates harbouring HepaRG and RPTEC were removed from the incubator. Apoptosis was determined by measuring the activation of the enzyme’s caspase-3 and -7 using the luminescence caspase-Glo 3 / 7 assay kit (Promega, G8090). Samples were analysed in singlets. Caspase assay data was recorded and saved via the BioTek Gen5 software. The luminescence measurements were background corrected. Therefore, the average luminescence of the blank controls was subtracted from the luminescence value of each sample.

[0290] To determine the caspase activity relative to the negative control (cells treated with vehicle), the following formula was used:Corrected Luminescence sample)Caspase activity (rel. to vehicle) = - - - - - - - —Corrected Luminescence (mean of negative controls)Data were visualized in GraphPad Prism for Windows (GraphPad Software, La Jolla California USA).

[0291] MTT assay: Cell viability can be assessed by using MTT Cell Viability Assay as shown in Fig. 9(B), which utilizes the well-established and widely used MTT reagent to determine mammalian cell viability. The redox potential in viable mammalian cells causes the water soluble MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to convert to an insoluble formazan product. Cell culture plates harbouring HepaRG and RPTEC were taken from the 37°C incubator at 48 h after treatment with the miR-21 inhibitors as indicated. Cell viability was performed by measuring the metabolic activity of cellular enzymes via the conversion of a tetrazolium salt into a formazan product at a wavelength of 570 nm using CellTiter96 NonRadioactive Cell Proliferation Assay (Promega, G4000 / G4100). Samples were analysed in singlets. MTT assay data was recorded and saved via the BioTek Gen5 software. Absorbance measurements in MTT assay were background corrected. The background correction was conducted for background derived from the measurement procedure itself and from the test system. For the correction of the measurement background, the absorbance values of the reference wavelength at 650 nm were subtracted from absorbance values measured at a wavelength of 570 nm. Afterwards, the background signal derived from the treatment medium was subtracted for correction. Therefore, the average blank absorbance was subtracted from the “background-corrected absorbance value” of each sample:Corrected absorbance sample = (sample OD at 570 nm — sample OD at 650 nm) — (blank OD at 570 nm — blank OD at 650 nm)For the calculation of the absorbance relative to the negative control (cells treated with vehicle), the following formula was used:Corrected absorbance (rel. to vehicle)Corrected absorbance (sample) Corrected absorbance (mean of negative controls)Data were and visualized in GraphPad Prism for Windows (GraphPad Software, La Jolla California USA).

[0292] LDH assay: As shown in Fig. 9(C), cell damage can be monitored by Lactate dehydrogenase (LDH) release. LDH is a soluble cytosolic enzyme present in many cell types that is rapidly released into the cell culture medium upon disruption of the plasma membrane. Incubation of HepaRG and RPTEC was stopped 48 h after treatment with the miR-21 inhibitors as indicated. The quantification of lactate dehydrogenase in cell culture media was determined by measuring the bioluminescent signals generated by enzymatic reactions using the LDH-Glo Cytotoxicity Assay Kit (Promega, J2380 / J2381). Samples were analyzed in singlets. LDH assay data was recorded and saved via the BioTek Gen5 software. The luminescencemeasurements were background corrected. Therefore, the average luminescence of the blank controls was subtracted from the luminescence value of each sample. Specifically, to calculate the LDH release relative to the negative control (cells treated with vehicle), the following formula was used:Corrected Luminescence sample)LDH release (rel. to vehicle) = - - - - - - - —Corrected Luminescence (mean of negative controls)Data were and visualized in GraphPad Prism for Windows (GraphPad Software, La Jolla California USA).

[0293] Statistics: Toxicity assay data is presented as mean + / - standard deviation (SD) and were plotted qualitatively using GraphPad Prism for Windows (GraphPad Software, La Jolla California USA). No statistical tests were applied.

[0294] Overall, the data demonstrate low cytotoxicity for the different lead oligonucleotides tested (see, Fig. 9 and 10). The summary heatmap data for oligonucleotides CDR076L-02, -15, -19, and -21 as shown in Fig. 9(D) are summarised in Table 12.Table 12: In v / fro toxicity assays in human renal (RPTEC) and liver (HepaRG) cells.For ranking sequential numbers from 1 to 5 were assigned. Where 1 indicates a low cytotoxicity score and 5 a high cytotoxicity score.

[0295] Notably, analogues CDR076L-02 and CDR076L-21 showed low in vitro cytotoxicity in HepaRG and RPTEC (Fig. 10(A) and (B)). That is, both compounds resulted in low caspase activity (relative to Staurosporine), even at a concentration of 200 nM (Fig. 10(A)(i) and 10(B)(i)). Furthermore, the MTT Cell Viability Assay resulted in good absorbance and metabolic activity similar to the miRCURY LNA-21 compound when compared to doxorubicin (Fig. 10(A)(ii) and 10(B)(ii)). Finally, theinventors were able to show that both lead compounds led to low LDH release even at a concentration of 200 nM when compared to LDH, which indicates to membrane damage (Fig. 10(A)(iii) and 10(B)(iii)).

[0296] Overall, the inventors have shown that a mixture of backbone modifications of the oligonucleotides can have a significant impact on their ability to target and effectively suppress expression of functional miR-21. In particular, the inventors have shown that a specific combination of precisely placed LNA, DNA and phosphorothioate (PS) backbone modifications provides for effective miR-21 targeting. That way lead compounds CDR076L-02 and -21 were identified that demonstrate a significant anti-inflammatory and anti-fibrotic effect, while having very low cellular toxicity (cellular apoptosis and cell / membrane damage). The inventors also showed that administration of these lead compounds did not result in any decrease in metabolic activity of the cell.Example 6. In vitro immunotoxicity assays in human peripheral blood mononuclear cells (hPBMCs).

[0297] It has been shown that that minor modifications to the base pair sequence or backbone of an oligonucleotide can have potential adverse immunological effects. The inventors went on to assess the immunological effects of candidate compounds CDR076L-02 and -21 in vitro.

[0298] hPBMCs were used as an in vitro model. hPBMCs are a mixed population of single nucleus cells containing individual cell types such as natural killer cells, T cells, and B cells. This wide-spread in vitro model can be used for the early investigation of immunological effects induced by oligonucleotides in blood. The cell culture conditions and assays used in Example 6 are outlined below. hPBMC

[0299] Seeding: hPBMCs were thawed and seeded in hPBMC cell culture medium into 96-well cell culture plates on day -1 with a density of 0.75x106cells / ml (200 pl / well) for cytokine ELISA and RNA isolation or a density of 2x106cells / ml (100 pl / well) for cell viability assays. Cells were incubated in a 5% CO2 humidified incubator at 37°C.

[0300] Treatment with test item: On day 0, cells were treated by addition of the test item to the cell culture medium resulting in a final concentration of 10 pM, 1 pM or 0.1 pM of the corresponding miR-21 inhibitors as indicated. Reference cells were treated with an equivalent volume of 0.9% NaCI. As positive control for cytokine ELISA,treatment with lipopolysaccharide (LPS; final concentration 10 ng / ml) was performed, while Doxorubicin (final concentration 5 pM) served as positive control for cell viability assays (Fig. 12(A), Fig. 13(A)). Cells were incubated in a 5% CO2 humidified incubator at 37°C.

[0301] RNA extraction: At the experimental endpoint (24 h post administration), RNA extraction was performed using the miRNeasy Tissue / Cells Advanced Micro Kit (QIAGEN; Cat. No. 217684). RNA was eluted in 20 pl in RNase-free water. Isolated RNA was used directly or stored at -80°C until further processing.

[0302] cDNA synthesis and qRT-PCR analysis: To determine functional level of miR- 21 -5p (Fig. 12(B)) in hPBMC-derived samples the same protocol was used as described for RAW264.7 cells above, with the exception that 2.5 pl of RNA was reverse transcribed into cDNA using the TaqMan MicroRNA Reverse Transcription Kit (Thermo Fisher Scientific, 4366596) and miRNA specific TaqMan Assays (see, Table 5). For qRT-PCR analysis validated TaqMan Assays for miR-21-5p and U6 snRNA (see, Table 6) and the ABsolute Blue qPCR Mix (low ROX, Thermo Fisher Scientific, AB4319A) were used. qRT-PCR analysis was performed as described for RAW264.7 cells above. The results shown in Fig. 12(B) are summarized in Table 13.Table 13: In vitro immuntoxicity assays in human peripheral blood mononuclear cells.

[0303] ELISA: Release of the cytokines TNF-a and IL-6 from hPBMCs after treatment with the miR-21 inhibitors or controls was determined by ELISA (Fig. 12(C) and (D)). Therefore, cell culture supernatant was collected at experimental endpoint (24 h post administration). A 1 :10 pre-dilution of sample cell culture supernatant andthe human IL-6 ELISA Kit (Thermo Fisher Scientific, KHC0061) or the human TNF-a ELISA Kit (Thermo Fisher Scientific, KHC3011) were used.

[0304] MTT Assay: Cell culture plates harboring hPBMCs were taken from the 37°C incubator 24 h after treatment with the miR-21 inhibitors or controls as indicated. Cell viability can be assessed by using MTT Cell Viability Assay as shown in Fig. 13(B), which utilizes the well-established and widely used MTT reagent to determine mammalian cell viability. The redox potential in viable mammalian cells causes the water soluble MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to convert to an insoluble formazan product. Cell culture plates harbouring hPMBC were taken from the 37°C incubator at 24 h after treatment with the miR-21 inhibitors as indicated. Cell viability was performed by measuring the metabolic activity of cellular enzymes via the conversion of a tetrazolium salt into a formazan product at a wavelength of 570 nm using CellTiter96 Non-Radioactive Cell Proliferation Assay (Promega, G4000 / G4100). Samples were analysed in singlets. MTT assay data was recorded and saved via the BioTek Gen5 software. Absorbance measurements in MTT assay were background corrected. The background correction was conducted for background derived from the measurement procedure itself and from the test system. For the correction of the measurement background, the absorbance values of the reference wavelength at 650 nm were subtracted from absorbance values measured at a wavelength of 570 nm. Afterwards, the background signal derived from the treatment medium was subtracted for correction. Therefore, the average blank absorbance was subtracted from the “background-corrected absorbance value” of each sample:Corrected absorbance sample = (sample OD at 570 nm — sample OD at 650 nm) — blank OD at 570 nm — blank OD at 650 nm)For the calculation of the absorbance relative to the negative control (cells treated with vehicle), the following formula was used:Corrected absorbance (rel. to vehicle)Corrected absorbance (sample)Corrected absorbance (mean of negative controls)

[0305] Data were and visualized in GraphPad Prism for Windows (GraphPad Software, La Jolla California USA).

[0306] Statistics: The data represent mean ± SD. Results are shown in Fig. 12 and 13.

[0307] Overall, the results show that cytokines and gene expression analysis could be successfully determined in hPBMCs (Fig. 12). As shown in Fig. 12(B), there was a decrease in miR-21 in the presence of CDR076L-21 and -02 for each of the three donors tested, even at the lowest concentration of 0.1 pM (also see, Table 13). Further, no cytokines (IL-6 and TNF-a) were detectable (Fig. 12(C) and (D)). These observations suggest effective downregulation of miR-21, with no evidence of immunotoxic effects.Example 7. RNA sequencing in human macrophages and cardiac fibroblasts

[0308] RNA sequencing (RNAseq) is a method that uses next-generation sequencing to reveal the presence and quantity of RNA molecules in a biological sample, providing transcriptome-wide analysis of differential gene expression. To identify novel pathways and responsive target genes by CDR076L-02 and CDR076L- 21 RNA sequencing the inventors performed RNAseq using the in vitro model of cardiac inflammation and cardiac fibrosis described above.

[0309] THP-1:

[0310] Maintenance: THP-1 cells were grown in THP-1-culture medium (see, Table 3) and maintained in a 5% CO2 humidified incubator at 37°C. After seeding, THP-1 were cultured in TH P-1 -culture medium. Additional TH P-1 -culture medium was added two days after seeding and every 2-3 days thereafter. During weekends, the added volume of medium was doubled. For inflammatory stimulation and treatment experiments, the medium was exchanged for fresh TH P-1 -culture medium containing respective test items and stimuli.

[0311] Seeding: Collected cells were centrifuged at 300 x g for 3 min at room temperature and resuspended in THP-1-culture medium supplemented with 100 ng / ml M-CSF. The concentration of cells in suspension was determined and cells were seeded into 12-well cell culture plates on day -4 with a density of 3 x 105cells / well (Fig. 14(A), Fig. 16(A)). Cells were incubated in a 5% CO2 humidified incubator at 37 °C for 4 days. At day -2 additional TH P-1 -culture medium (no M-CSF) was added to the wells.

[0312] Treatment with test item and stimulation: After 4 days (on “dO”), cells were stimulated and treated in THP-1-culture medium enriched with 100 ng / mL LPS +20 ng / ml IFN-y and 100 nM of the corresponding miR-21 inhibitors (see Fig. 14(A) for CDR076L-02; see Fig. 16(A) for CDR076L-21) or placebo as indicated. Reference cells were treated with 0.9% NaCI solution. Incubation was performed in a 5% CO2 humidified incubator at 37°C for 48 h. Cells were harvested 48 hrs post treatment.

[0313] RNA extraction and quantification: Since stimulation with LPS and IFN-y leads to the development of an adherent TH P-1 subtype, not attached cells were removed, washed once with DPBS and lysed. The adherent cells remaining in the well were washed once with DPBS and lysed as well. Both subtypes were combined afterward. Cell lysis and RNA extraction was performed using the miRNeasy Tissue / Cells Advanced Mini Kit (QIAGEN, 217604) according to the manufacturer’s instruction. Directly after isolation, the RNA concentration was assessed by photometric measurement. Then, RNA was stored at -80 °C until further processing.

[0314] NHCF-V:

[0315] Maintenance: NHCF-V cells were cultured in NHCF-V growth medium containing 10% FBS (see, Table 3) in a humid incubator providing at 5% CO2 environment at 37°C. Medium was changed one day after seeding and every 1-3 days thereafter. For treatment experiments, the medium was exchanged for fresh NHCF-V growth medium containing respective test items and stimuli.

[0316] Seeding: Following trypsinization, cells were centrifuged at 200 x g for 6 min at room temperature and resuspended in NHCF-V growth medium containing 10% FBS. The concentration of cells in suspension was determined, cells were seeded into 12 well cell culture well plates on day -1 [on day “d-1”, one day prior to fibrotic stimulation (on “d0”)] with a density of 2.5x104cells / well. Cells were incubated in a 5% CO2 humidified incubator at 37°C for 24 h (± 4 h) before stimulation with 10 ng / ml TGF-p and treatment with the test items (Fig. 15(A), Fig. 17(A)).

[0317] Treatment with test item and stimulation: Before treatment on day 0, cells were washed once with prewarmed DPBS. Cells were treated with the test items (see Fig. 15(A) for CDR076L-02; see Fig. 17(A) for CDR076L-21) or Placebo (0.9% NaCI)) supplementing at a dose of 100 nM supplemented to the NHCF-V growth medium containing 1 % FBS and 10 ng / ml TGF-p. Incubation was performed in a 5% CO2 humidified incubator at 37°C for 72 h. Reference / placebo cells were treated with 0.9% NaCI solution.

[0318] RNA extraction and quantification: After 72 h of TGF-p stimulation, N HCF- V cells were washed once with DPBS. Cell lysis and RNA extraction was performed using the miRNeasy Tissue / Cells Advanced Mini Kit (QIAGEN, 217604) according to the manufacturer’s instruction. After cell lysis, samples were frozen and stored at -80°C until further processing. RNA was extracted from all samples. Directly after isolation, the RNA concentration was assessed by photometric measurement. Then, RNA was stored at -80°C until further processing.

[0319] RNA sequencing: Libraries for RNA sequencing were constructed in a strandspecific manner from 1 pg of total RNA using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB) and index codes were added to attribute sequences to each sample. The enrichment of mRNA was based on poly-adenylation selection. RNA was then fragmented, subjected to two rounds of cDNA synthesis and NEBNext Adaptors were then ligated to ds cDNAto prepare for hybridization. Library quality was assessed on the Agilent Bioanalyzer 2100 system (Agilent).

[0320] The clustering of the index-coded samples was performed on a cBot Cluster Generation System using PE Cluster Kit cBot-HS (Illumina). After cluster generation, the library preparations were sequenced (Illumina Inc.) and paired-end reads were generated.

[0321] For all experiments with CDR076L-02 (see, Fig. 14, Fig. 15) data analysis was performed. Raw paired-end reads were aligned to human reference genome using STAR (version 2.7.9a), and mapped reads were counted using featurecounts (version 2.0.1). Using the software environment for statistical computing and graphics R (version 4.2.1), principal component analysis (PCA) was performed with the ggplot2 package (version 3.4.0) to determine the potential outliers among samples in each batch.

[0322] For all experiments involving CDR076L-21 (see, Fig. 16, Fig. 17), the following workflow was implemented: Raw paired-end sequencing reads were processed using Trim Galore (version 0.6.10) for uniform adapter and quality trimming. Transcript abundances were quantified with kallisto (version 0.51.0) through pseudoalignment to the human reference genome GRCh38.p14, which includes both cDNA and ncRNA sequences. Gene annotation was conducted in the statistical computing environment RStudio (version 2023.12.1 Build 402) using the EnsDb.Hsapiens.v86 package (version 2.99.0). To identify potential outliers withineach batch, principal component analysis (PCA) was performed with the ggplot2 package (version 3.5.1). Differentially expressed genes (DEGs) were identified using the edgeR (version 4.0.16) and limma (version 3.58.1) packages.

[0323] Reciprocal gene analysis for CDR076L-02: To identify reciprocally differentially expressed genes cells in THP-1 cells, the settings |FC| > 1.5 and p value< 0.05 for M1 Placebo vs. MO Placebo in combination with |FC| > 1.5 and p value < 0.05 for M1 CDR076L-02 versus M1 Placebo were used. For NHCF-V, the filtering criteria |FC| > 1.5 and p value < 0.05 comparing the Placebo vs. unstimulated group in combination with a significant reciprocal regulation by CDR076L-02 treatment (p value < 0.05 for CDR076L-02 versus Placebo) were used. Gene set enrichment analysis of reciprocally regulated genes after CDR076L-02 treatment was performed using enrichment analysis Chen et al., 2013; Huang et al., 2019) including the geneset library from the pathway database NCATS BioPlanet (see, Fig. 14, Fig. 15).

[0324] Reciprocal gene analysis for CDR076L-21 : To identify reciprocally differentially expressed genes cells in THP-1 cells, the settings |FC| > 1.5 and p value< 0.05 for M1 Placebo vs. M0 Placebo in combination with |FC| > 1.5 and p value < 0.05 for M1 CDR076L-21 versus M1 Placebo were used. For NHCF-V, the filtering criteria |FC| > 1.5 and p value < 0.05 comparing the Placebo vs. unstimulated group in combination with a significant reciprocal regulation by CDR076L-21 treatment (p value < 0.05 for CDR076L-21 versus Placebo) were used (see, Fig. 16, Fig. 17). Pathway analysis with reciprocally regulated genes was performed using EnrichR (Pathways BioPlanet 2019).

[0325] Overall, the results show that treatment with CDR076L-02 or CDR076L-21 led to the identification of various pathways comprising genes whose expression levels were reciprocally regulated in response to oligonucleotide treatment. For example, those that are involved in the regulation of the immune system, interferon signalling (Fig. 14(C)), TGF-beta regulation (Fig. 15(C)), sulfur amino acid metabolism (Fig. 16(C)), and IL-1 regulation (Fig. 17(C)).Example 8. In vivo biodistribution study.

[0326] Biodistribution and exploratory efficacy assessments in animals form an integral part of the drug development process, providing data to support the design of subsequent preclinical studies and human clinical trial enabling studies. Rodent animals including mice are commonly used since their pharmacological relevance isgenerally demonstrated. Mice express the target and evoke a similar pharmacological response as that expected in humans. Therefore, mice are a well-established model for biodistribution and exploratory efficacy.

[0327] Animal husbandry: The animal husbandry fulfils all recommendations as required by the guidelines of the European Union. All animal experiments were approved by the Local Institutional Animal Care and Research Advisory Committee and approved by the local authority.

[0328] Treatment: Mice (C57BL / 6N mice, male, 6-12 weeks, Charles River, Germany) received a single intraperitoneal (i.p.) injection: 20 mg / kg CDR076L-02 (Fig. 18(A)), 10 mg / kg CDR076L-02 (Fig. 18(B)) or 2.5 mg / kg CDR076L-21 (Fig. 19(A)) in saline solution (0.9% NaCI), or a Placebo (0.9% NaCI). At endpoints (day 2, day 3 or day 7 as indicated), animals were euthanized and dissected. Plasma and selected organs (heart, liver, kidney, lung, skeletal muscle, and spleen) were collected for the assessment of functional miR-21 level.

[0329] RNA extraction and quantification: RNA extraction from tissue samples was performed using the miRNeasy Tissue / Cells Advanced Mini Kit (QIAGEN, 217604) according to the manufacturer’s instruction. Directly after isolation, the RNA concentration was assessed by photometric measurement. Then, RNA was stored at -80 °C until further processing. RNA extraction from plasma samples was performed using the miRNeasy Serum / Plasma Advanced Kit (Qiagen, 217204). As starting material, 75 pl plasma diluted with 75 pl PBS were used and supplemented with an exogenous cel-miR-39 spike-in control (final amount of spike-in control 5.6 x 108copies per sample). RNA from plasma samples was eluted in 14 pl RNase-free water and stored at -80 °C until further processing.

[0330] cDNA synthesis and qRT-PCR analysis: To determine functional levels of miR-21-5p in tissue and circulating levels in plasma the same protocol was used as described for RAW264.7 cells above (see, Example 1), with the exception that 20 ng of RNA from tissue samples or 2.5 pl of RNA from plasma samples were reverse transcribed into cDNA using the TaqMan MicroRNA Reverse Transcription Kit (Thermo Fisher Scientific, 4366596) and miRNA specific TaqMan Assays using U6 snRNA as reference gene for tissue samples and cel-miR-39 for plasma samples (see, Table 5). For qRT-PCR analysis validated TaqMan Assays (see, Table 6) and the ABsolute Blue qPCR Mix (low ROX, Thermo Fisher Scientific, AB4319A) were used. qRT-PCR analysis was performed as described for RAW264.7 cells above.

[0331] Statistics: Data are mean ± SD. P values: Mann-Whitney U test; ns: p>0.05; *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001. Results are shown in Fig. 18(A), Fig. 18(B), and Fig. 19(B).

[0332] Overall, the results show that CDR076L-02 and CDR076L-21 can effectively be taken up and effectively inhibit miR-21 expression in various organs and plasma in vivo. More specifically, the results show that CDR076L-02 and CDR076L-21 are particularly effective in inhibiting miR-21 expression in the heart.Example 9. Functional miR-21 level in a mouse model of (cardiac) fibrosis

[0333] The murine angiotensin II (Angll) model is a highly relevant and accepted model for cardiac remodeling and cardiac fibrosis. This well-established model was used to investigate the effects of Angll infusion on functional miR-21 levels.

[0334] Animal husbandry: The animal husbandry fulfils all recommendations as required by the guidelines of the European Union. All animal experiments were approved by the Local Institutional Animal Care and Research Advisory Committee and approved by the local authority.

[0335] Angll infusion: To induce cardiac fibrosis, Alzet osmotic minipumps (model 1002) were implanted subcutaneously into mice (C57BL / 6N mice, male, 6-8 weeks, Charles River, Germany) in the dorsal region to obtain a delivery rate of 3 mg / kg / day Angll over the course of 2 weeks. Control animals received osmotic mini pumps filled with Placebo (Fig. 20(A)).

[0336] RNA extraction and quantification: RNA extraction from tissue samples was performed using the miRNeasy Tissue / Cells Advanced Mini Kit (QIAGEN, 217604) according to the manufacturer’s instruction. Directly after isolation, the RNA concentration was assessed by photometric measurement. Then, RNA was stored at -80 °C until further processing. RNA extraction from plasma samples was performed using the miRNeasy Serum / Plasma Advanced Kit (Qiagen, 217204). As starting material, 75 pl plasma diluted with 75 pl PBS were used and supplemented with an exogenous cel-mi R-39 spike-in control (final amount of spike-in control 5.6 x 108copies per sample). RNA from plasma samples was eluted in 14 pl RNase-free water and stored at -80 °C until further processing.

[0337] cDNA synthesis and gRT-PCR analysis: To determine functional levels of miR-21-5p in tissue and circulating levels in plasma the same protocol was used as described for RAW264.7 cells above (see, Example 1), with the exception that 20 ngof RNA from tissue samples or 2.5 pl of RNA from plasma samples were reverse transcribed into cDNA using the TaqMan MicroRNA Reverse Transcription Kit (Thermo Fisher Scientific, 4366596) and miRNA specific TaqMan Assays using U6 snRNA as reference gene for tissue samples and cel-miR-39 for plasma samples (see, Table 5). For qRT-PCR analysis validated TaqMan Assays (see, Table 6) and the ABsolute Blue qPCR Mix (low ROX, Thermo Fisher Scientific, AB4319A) were used. qRT-PCR analysis was performed as described for RAW264.7 cells above. Statistics: Data are mean ± SD. P values: Mann-Whitney II test; ns: p>0.05; *: p<0.05; **: p<0.01 ; ***: p<0.001 ; ****: p<0.0001. Results are shown in Fig. 20(B). The data shown in Fig. 20(B) is summarised in Table 14.Table 14. Summary of functional miR-21 in various organs and plasma.

[0338] As shown in Fig. 20(B), in the presence of angiotensin II (3 mg / kg), functional miR-21 levels were upregulated in various organs and plasma relative to placebo. Specifically, there was a significant increase in function miR-21 in the heart, liver, muscle, and plasma.

[0339] Overall, these results show that the murine angiotensin II (Angll) model can be effectively used to investigate the effects of Angll infusion on functional miR-21 levels in vivo.Example 10. Functional miR-21 level in a mouse model of (cardiac) fibrosis after treatment with CDR076L-21

[0340] For this study, the same murine Angll model as described in Example 9 was used and treated with two intraperitoneal (i.p.) administrations of 2.5 mg / kg CDR076L- 21.

[0341] Animal husbandry: As described above, the animal husbandry fulfils all recommendations as required by the guidelines of the European Union. All animal experiments were approved by the Local Institutional Animal Care and Research Advisory Committee and approved by the local authority.

[0342] Anqll infusion: To induce cardiac fibrosis, Alzet osmotic minipumps (model 1002) were implanted subcutaneously into mice (C57BL / 6N mice, male, 6-8 weeks, Charles River, Germany) in the dorsal region to obtain a delivery rate of 3 mg / kg / day Angll over the course of 2 weeks. Control animals received osmotic mini pumps filled with Placebo (Fig. 21). Intraperitoneal (i.p.) treatment of mice exposed to Angll was performed on day 0 and day 7 either with 2.5 mg / kg CDR076L-21 or with Placebo (0.9% NaCI solution). Control animals remained untreated.

[0343] RNA extraction and quantification: RNA extraction from tissue samples was performed using the miRNeasy Tissue / Cells Advanced Mini Kit (QIAGEN, 217604) according to the manufacturer’s instruction. Directly after isolation, the RNA concentration was assessed by photometric measurement. Then, RNA was stored at -80 °C until further processing. RNA extraction from plasma samples was performed using the miRNeasy Serum / Plasma Advanced Kit (Qiagen, 217204). As starting material, 75 pl plasma diluted with 75 pl PBS were used and supplemented with an exogenous cel-mi R-39 spike-in control (final amount of spike-in control 5.6 x 108copies per sample). RNA from plasma samples was eluted in 14 pl RNase-free water and stored at -80 °C until further processing.

[0344] cDNA synthesis and qRT-PCR analysis: To determine functional levels of miR-21-5p in tissue and circulating levels in plasma the same protocol was used as described for RAW264.7 cells above (see, Example 1), with the exception that 20 ng of RNA from tissue samples or 2.5 pl of RNA from plasma samples were reverse transcribed into cDNA using the TaqMan MicroRNA Reverse Transcription Kit (Thermo Fisher Scientific, 4366596) and miRNA specific TaqMan Assays using U6 snRNA as reference gene for tissue samples and cel-miR-39 for plasma samples (see, Table 5). For qRT-PCR analysis validated TaqMan Assays (see, Table 6) and the ABsolute Blue qPCR Mix (low ROX, Thermo Fisher Scientific, AB4319A) were used. qRT-PCR analysis was performed as described for RAW264.7 cells above.

[0345] Statistics: Data are mean ± SD. P values: Mann-Whitney U test; ns: p>0.05; *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001. Correlation between cardiac miR- 21-5p and plasma miR-21-5p was performed using Pearson product-momentcorrelation and Spearman rank-order correlation. Results are shown in Fig. 21(B) & 21(C). In Fig. 21(B), results that are overlayed with a gray background indicate the presence of Angiotensin II. The results shown in Fig. 21(B) (cardiac) and Fig. 21(C) are further summarised in Table 15 and Table 16 respectively.Table 15: Angiotensin II mouse model of (cardiac) fibrosis with CDR076L-21 treatment. Detection of cardiac miR-21.Table 16: Angiotensin II mouse model of (cardiac) fibrosis with CDR076L-21 reatment Detection of plasma miR-21.

[0346] As shown in Fig. 21(B), the levels of cardiac miR-21 were significantly downregulated in the presence of CDR076L-21 when compared to control. Similarly, there was a significant decrease in plasma miR-21 in the presence of CDR076L-21 when compared to control. These results confirm that CDR076L-21 effectively downregulates cardiac and plasma levels of miR-21 in vivo. Further, as shown in Fig. 21(C), in the presence of CDR076L-21, there was a correlation between low levels of cardiac miR-21 and plasma miR-21 when compared to control and placebo. Overall, these results further confirm the ability of CDR076L-21 to efficiently reduce cardiac and plasma miR-21 levels in the murine Angll model.

[0347] Those having ordinary skill in the art will appreciate that the disclosure can be modified in ways not specifically described herein.REFERENCES . Bang C.; Batkai S.; Dangwal S.; Gupta S. K.; Foinquinos A.; Holzmann A., et al. (2014). Cardiac fibroblast-derived microRNA passenger strand-enriched exosomes mediate cardiomyocyte hypertrophy. J. Clin. Invest. 124: 2136-46. . Barnett, R. E.; Conklin, D. J.; Ryan, L.; Keskey, R. C.; Ramjee, V.; Sepulveda, E. A.; Srivastava, S.; Bhatnagar, A.; Cheadle, W. G. (2016). Anti-inflammatory effects of miR-21 in the macrophage response to peritonitis. JLB. 99(2):361-71. . Barry, S.P.; Townsend, P.A. (2010). What causes a broken heart- Molecular insights into heart failure. Int. Rev. Cell Mol. Biol. 284, 113- 179. . Berk, B. C.; Fujiwara, K.; Lehoux, S. (2007). ECM remodeling in hypertensive heart disease. J. Clin. Invest. 117(3): 568-575. . Chen, E.Y.; C. M. Tan; Y. Kou; Q. Duan; Z. Wang; G. V. Meirelles; N. R. Clark; and A. Ma’ayan. (2013). Enrichr: interactive and collaborative HTML5 gene list enrichment analysis tool. BMC Bioinformatics (14): 128. . Chung S. E.; Packer, M.; Lo, K. H.; Fasanmade, A. A.; Willerson, J. T. (2003). Randomized, Double-Blind, Placebo-Controlled, Pilot Trial of Infliximab, a Chimeric Monoclonal Antibody to Tumor Necrosis Factor-a, in Patients with Moderate-to- Severe Heart Failure. Circulation. 107: 3133-3140. . Frantz, S.; Hu, K.; Adamek, A.; et al. (2008). Transforming growth factor beta inhibition increases mortality and left ventricular dilatation after myocardial infarction. Basic Res. Cardiol. 103(5): 485-492. . Guinea-Viniegra, J.; Jimenez, M.; Schonthaler, H. B.; Navarro, R.; Delgado, Y; Concha-Garzon, M. C.; Tschachler, E.; Obad, S.; Dauden, E.; Wagner, E. F. (2014). Targeting miR-21 to treat psoriasis. Sci. Transl. Med. 6(225): 225. . Gullestad, J.; lleland, T; Vinge, L. E.; Finsen, A.; Yndestad, A.; Aukrust, P. (2012). Inflammatory cytokines in heart failure: mediators and markers. Cardiol. 122(1): 23- 25. . Heidenreich P.A., Bozkurt B., Aguilar D., Allen L.A., Byun J. J., Colvin M.M., et al. (2022) AHA / ACC / HFSA Guideline for the Management of Heart Failure: a report of the American College of Cardiology / American Heart Association Joint Committee on Clinical Practice Guidelines. Circulation', 145: e895-e1032. 1. Hill, J. A. and Olson, E. N. (2008). Cardiac plasticity. N. Engl. J. Med. 358: 1370- 1380. . Huang, R.; I. Grishagin; Y. Wang; T. Zhao; J. Greene; J. C. Obenauer; D. Ngan; D. T. Nguyen; R. Guha; A. Jadhav; N. Southall; A. Simeonov, and C. P. Austin. (2019). The NCATS BioPlanet - An Integrated Platform for Exploring the Universe of Cellular Signaling Pathways for Toxicology, Systems Biology, and Chemical Genomics. Front Pharmacol. 10: 445. . Jiang W; Xiong, Y; Li, X.; Yang, Y. (2021). Cardiac Fibrosis: Cellular Effectors, Molecular Pathways, and Exosomal Roles. Frontier Cardio. Med. 8: Article 715258. . Kong, P.; Christia, P.; Frangogiannis, N. G. (2014). The Pathogenesis of Cardiac Fibrosis. Cell. Mol. Life Sci. 71 (4): 549-574. . Leask A. (2010). Potential therapeutic targets for cardiac fibrosis: TGFbeta, angiotensin, endothelin, CCN2, and PDGF, partners in fibroblast activation. Circ. Res.WQ 1675-1680. . Ma, H.; Liu, S.; li, S.; and Xia Y. (2022) Targeting Growth Factor and Cytokine Pathways to Treat Idiopathic Pulmonary Fibrosis. Front Pharmacol. 13: 918771. . Mann, D. L.; McMurray, J. J. V.; Packer, M.; Swedberg, K.; Borer, J. S.; Colucci, W S.; Djian, J.; Drexler, H., et al. (2004). Targeted anticytokine therapy in patients withchronic heart failure: results of the Randomized Etanercept Worldwide Evaluation (RENEWAL). Circulation. 109(13): 1594-1602. Morfino, P.; Aimo, A.; Castiglione, V.; Galvez-Monton, C.; Emdin, M.; and Bayes- Genis, A. (2022). Treatment of cardiac fibrosis: from neuro-hormonal inhibitors to CAR-T cell therapy. Heart Fail. Rev. 11 : 1-15. Piccoli, M.-T.; Gupta, S. K.; Viereck, J.; Foinquinos, A.; Samolovac, S.; Kramer, F. L.; Garg, A.; Remke, J.; Zimmer, K.; Batkai, S. and Thum, T. (2017). Inhibition of the Cardiac Fibroblast-Enriched IncRNA Meg3 Prevents Cardiac Fibrosis and Diastolic Dysfunction. Circulation Research. 121(5): 575-583. Quemener, A. M.; Bachelot, L.; Forestier, A.; Donnou-Fournet, E.; Gilot, D.; Galibert, M.-D. (2019). The powerful world of antisense oligonucleotides: From bench to bedside. Wiley Interdiscip. Rev. RNA. 11 (5): e1594. Rincon M. and Irvin, C. G. (2012). Role of IL-6 in Asthma and Other Inflammatory Pulmonary Diseases. Int. J. Biol. Sci. 8(9): 1281-1290. Roubille F, Busseuil D, Merlet N, et al. (2014) Investigational drugs targeting cardiac fibrosis. Expert Rev. Cardiovasc. The 12: 111-125. Stephenson, M. L.; Zamecnik, P. C. (1978). Inhibition of Rous sarcoma viral RNA translation by a specific oligodeoxyribonucleotide. Proc. Natl. Acad. Sci. USA. 75(1): 285-288. Sheedy, F. J. (2015). Turning 21 : introduction of miR-21 a key switch in the inflammatory response. Frontiers in Immunol. 6: Article 19. Sicard, F.; Gayral, M.; Lulka, H.; Buscail, L.; Cordelier, P. (2013). Targeting miR-21 for the Therapy of Pancreatic Cancer. Mol. The 21 (5): 986-994. Sims, E. K., A. J. Lakhter, E. Anderson-Baucum, T. Kono, X. Tong, and C. Evans- Molina. (2017). MicroRNA 21 targets BCL2 mRNA to increase apoptosis in rat and human beta cells. Diabetologia. 60(6): 1057-1065. Surina, S.; Fontanella, R. A.; Sciscioloa, L.; Marfella, R.; Paolisso, G.; Barbieri, M. (2021). miR-21 in Human Cardiomyopathies. Frontiers in Cardiovascular Medicine. 8: Article 767064. Tanaka, T.; Narazaki, M.; Kishimoto, T. (2014). IL-6 in Inflammation, Immunity, and Disease. Cold Spring Harb Perspect Biol. 6: a016295. Thum, T.; Gross, C.; Fiedler, J.; Fischer, T.; Kissler, S.; Bussen, M.; et al. (2008) MicroRNA-21 contributes to myocardial disease by stimulating MAP kinase signalling in fibroblasts. Nature. 456: 980-984. Wu, H.; Wang, J.; Ma, H.; Xiao, Z.; Dong, X. (2017). MicroRNA-21 inhibits mitochondria-mediated apoptosis in keloid. Oncotarget. 8(54): 92914-92925. Yu M.; Zheng, X.; Witschi, H.; Pinkerton, K. E. (2002). The Role of lnterleukin-6 in Pulmonary Inflammation and Injury Induced by Exposure to Environmental Air Pollutants. Toxicol. Sci. 68: 488-497.

Claims

CLAIMS1 . An oligonucleotide comprising one of the sequences selected from:(i) 5’- +T C A +G T C +T G A +TA +A G +C +T -3’ (formula / ); or(ii) 5’- +T C +A G T +C T G +A +T A +A G C +T -3’ (formula II)- wherein A, T, G and C are deoxyribonucleotide building blocks, or C is 5- methyl-2’-deoxycytidine; and wherein +T, +A, +G, and +0 are bridged nucleotide building blocks and / or morpholino building blocks.

2. The oligonucleotide according to claim 1 , wherein +G, +T, +A, and +C are locked nucleic acid (LNA) building blocks.

3. The oligonucleotide according to claim 1 or claim 2, comprising at least one modified internucleosidic linkage, optionally wherein all internucleosidic linkages are modified.

4. The oligonucleotide according to claim 3, wherein the at least one modified internucleosidic linkage is a phosphorothioate or phosphorodiamidate linkage, preferably phosphorothioate linkage.

5. The oligonucleotide according to any one of claims 1 to 4, wherein each C is 5- methyl-2’-deoxycytidine.

6. An oligonucleotide comprising the sequence of formula la:5‘- +T*d5mC*dA*+G*dT*d5mC*+T*dG*dA*+T*dA*+A*dG*+5mC*+T -3‘ ; wherein dA is 2’-deoxyadenosine, dG is 2’-deoxyguanosine, dT is 2’- deoxythymidine; wherein +T is an LNA-T building block, +A is an LNA-A building block, +G is an LNA-G building block and +5mC is an LNA-5-methyl-2’-cytidine building block; wherein d5mC is 5-methyl-2’-deoxycytidine; and wherein * is a phosphorothioate linkage.

7. The oligonucleotide according to any one of claims 1-6, wherein the oligonucleotide is conjugated to a heterologous moiety.

8. A pharmaceutical composition comprising the oligonucleotide according to any one of claims 1-7, and optionally a pharmaceutically acceptable carrier, delivery agent or excipient.

9. An oligonucleotide according to any one of claims 1-7 or a pharmaceutical composition of claim 8 for therapeutic use in a subject.

10. An oligonucleotide of any one of claims 1-7 or a pharmaceutical a composition of claim 8, for use in the treatment or prevention of heart failure with reduced ejection fraction (HFrEF).

11. An oligonucleotide of any one of claims 1 -7 or a composition of claim 8 for use in the prophylaxis and / or treatment of: a) an inflammatory disease in a subject, particularly wherein the inflammatory disease is cardiac or pulmonary inflammation; b) cardiopulmonary disorders in a subject, particularly cardiopulmonary inflammation-related remodelling; and / or c) fibrotic disorders in a subject.

12. The oligonucleotide or the pharmaceutical composition for use of claim 10, wherein the fibrotic disorders include cardiac fibrotic disorders and cardiac fibrotic related disorders, particularly left and / or right ventricular fibrosis, atrial fibrosis, endomyocardial fibrosis, Heart Failure with Reduced Ejection Fraction (HFrEF), or fibrosis resulting from a previous myocardial infarction, high blood pressure or myocarditis; pulmonary fibrotic disorders, particularly pulmonary fibrosis caused by occupational, genetic or environmental factors, radiation treatment and / or treatment with medicaments; idiopathic pulmonary fibrosis; hepatic fibrotic disorders, particularly alcoholic liver disease or nonalcoholic fatty liver disease (NAFLD); renal fibrosis; dermal fibrosis; bone marrow fibrosis; and / or gut fibrosis.

13. The oligonucleotide or the pharmaceutical composition for use of any one of claims 9-12, wherein the oligonucleotide or the pharmaceutical composition is administered to the subject selected from:(i) patients having an increased risk for developing inflammatory disease, particularly wherein the inflammatory disease is cardiac inflammation;(ii) patients suffering from inflammatory disease and / or patients having an increased risk of inflammatory disease progression;(iii) patients having an increased risk for developing cardiac, kidney, liver or pulmonary disorders;(iv) patients suffering from cardiac disorders and / or patients having an increased risk of cardiac disorder progression;(v) patients having an increased risk for developing fibrotic disorders; and / or(vi) patients suffering from fibrotic disorders and / or patients having an increased risk of fibrotic disorder progression.

14. The oligonucleotide or the pharmaceutical composition for use of any one of claims 9-13, wherein the oligonucleotide or the pharmaceutical composition is administered to the subject in combination with one or more other therapies, optionally wherein the one or more other therapies comprises a standard of care (SoC).

15. An oligonucleotide of any one of claims 1-7 or a pharmaceutical composition of claim 8 for use in the diagnosis of an inflammatory disease, a cardiopulmonary, kidney or liver disorder, or a fibrotic disorder, or a combination thereof in a sample obtained from a subject.

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