WO2021108546A1

WO2021108546A1 - Inhibition of microrna-224 to treat pulmonary hypertension

Info

Publication number: WO2021108546A1
Application number: PCT/US2020/062226
Authority: WO
Inventors: Yassine Sassi; Roger J. Hajjar; Olympia BIKOU
Original assignee: Icahn School Of Medicine At Mount Sinai
Priority date: 2019-11-27
Filing date: 2020-11-25
Publication date: 2021-06-03

Abstract

Methods and compositions for treating, preventing, or reversing pulmonary hypertension. In an aspect, a method of treating pulmonary hypertension comprising administering to the subject an effective amount of a miR-224 inhibitor. In an aspect, the subject with pulmonary hypertension in a subject having a BMPR2 mutation, the treatment method comprising: (a) obtaining a genetic test result on a subject sample to confirm the presence of a BMPR2 mutation; and (b) administering to the subject an effective amount of a miR-224 inhibitor. In an aspect, the method comprising: (a) identifying a subject suitable for treatment, wherein a suitable subject is one who has an increased level of miR-224 in a biological sample; and (c) administering an effective amount of an miR-224 inhibitor to a subject. A pharmaceutical composition comprising an miR-224 inhibitor, and a kit comprising the pharmaceutical composition, and an oligonucleotide capable of being used to detect a BMPR2 mutation.

Description

INHIBITION OF MICRORNA-224 TO TREAT PULMONARY HYPERTENSION

CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit and priority under 35 U.S.C. § 119(e) of the filing date of United States Provisional Patent Application Serial Number 62/941,608, filed November 27, 2019. The entire contents of the aforementioned application are hereby incorporated herein by reference.

SEQUENCE LISTING SUBMITTED AS ASCII TEXT FILE VIA EFS-WEB [0002] Applicant expressly incorporates by reference all of the information and material located in the file designated “10526.70008WO00-SEQ-GJM” which was created on November 18, 2020, and is 1.7 kilobytes in size. By this statement, the Sequence Listing constitutes a part of the instant Specification. The submission contains no new matter.

BACKGROUND OF THE INVENTION

[0003] Pulmonary hypertension (PH) is a rare and life-threatening condition characterized by high blood pressure in the lungs and occurs when the pulmonary arteries become clogged and narrowed. The pulmonary arteries are the blood vessels that are responsible for the transport of blood from the heart to the lungs. As a result of this condition, the heart becomes strained in order to properly pump the blood, which can result in enlargement and weakening of the heart and ultimately death. PH has an estimated prevalence of 15 to 50 cases per million population. The most common symptoms associated with PH include shortness of breath, fatigue, dizziness or fainting spells, pressure and pain in the chest, edema of the ankles, legs or abdomen, bluish color in the lips and skin, and irregular heartbeat. Symptoms tend to worsen over time as the PH is a progressive disease. However, the disease manifests differently according to the patients’ characteristics and disease subtype.

[0004] The appearance of PH depends on numerous factors that include the presence of other conditions of the lung or heart, congenital heart defects, coronary artery disease, connective tissue disease, liver disease, high blood pressure, and blood clots affecting the lungs, as well as the PH subtype. According to the World Health Organization (WHO), PH is classified into five subtypes. Group 1 PH is pulmonary arterial hypertension (PAH) and has no definitive cause. Possible causes are recognized to include genetic mutations that define heritable PAH. Other causes may include use of some prescription drugs or non-prescription drugs, such as methamphetamines, as well as exposure to toxins. Causes of PAH may also relate to congenital heart conditions, or other disease conditions of the lungs, connective tissues, and liver. Group 2 PH is caused by left-sided heart disease. Causes can include left sided heart valve disease, such as mitral valve or aortic valve disease, and also failure of the lower left heart chamber. Group 3 PH is caused by lung disease, including chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, sleep apnea, and high-altitude exposure in people at high risk for PH. Group 4 PH is caused by chronic blood clot disorders. Group 5 PH is caused by other health conditions falling outside of Groups 1-4, and can include blood disorders, inflammatory disorders, metabolic disorders, kidney disease, and tumors impacting pulmonary arteries. Different types of PH arise from a variety of factors that generally develop slowly over a period of months or even years.

[0005] When the cause of PH is not known, it is generally classified as “idiopathic” PH, which occurs in about 40% of the cases. By contrast, “heritable” PH occurs when the condition is associated with underlying genetic familial factors.²

[0006] PH is a disease characterized by the progressive remodeling of the pulmonary arteries, resulting in the loss of vascular cross-sectional area and elevated pulmonary vascular resistance and PH. Pulmonary arterial remodeling is perhaps the chief contributor to elevated pulmonary vascular resistance. All three layers of the arterial wall are involved in vascular remodeling, including the intimal endothelial cells, medial smooth muscle cells (SMC), and adventitial fibroblasts.³ Typical arterial lesions in PH consist of neointima formation and intimal fibrosis, medial hyperplasia of SMC, and adventitial fibrosis accompanied by a variable degree of perivascular inflammation.⁴ PH also involves increased pulmonary venous pressure, which is often a result of other medical conditions that harm the left side of the heart and increase left heart ventricular pressure. Due to the excessive stress, the pulmonary arteries also gain high pressure, which can result in acute injuries in the alveolar capillary wall and subsequent edema. This can also cause irreversible thickening of the walls of the alveolar-capillary membrane, compromising lung function.

Without intervention, PH is progressive, leading to heart failure and eventual death. The management of PH has advanced rapidly in recent years due to improved understanding of the condition’s pathophysiology. Five classes of drugs are available and include phosphodiesterase-5 inhibitors, soluble guanylate cyclase stimulators, prostacycline analogues, prostacyclin receptor agonists, and endothelin receptor antagonists. Despite the availability of these therapies and intensive research in the last decades, PH is still associated with significant morbidity and mortality, with 1-year and 5-year rates at 15% and 45%, respectively. Many of the current targeted therapies also have significant limitations, including adverse effects. New strategies and therapies for treating PH, including PAH, are desperately needed and would significantly advance the art.

SUMMARY OF THE INVENTION

[0007] The present disclosure provides a new therapeutic strategy to prevent and inhibit PH based on microRNA-224 (miR-224) inhibition. It was found that miR-224 levels are significantly increased in subjects with clinical pulmonary arterial hypertension PAH (a subtype of PH), as well as in different PH- animal models. In vitro , miR-224 overexpression induces an increase in human pulmonary artery smooth muscle cell (hPASMC) proliferation, whereas miR-224 inhibition decreases hPASMC proliferation. The overexpression of miR- 224 in vivo exacerbates the PH phenotype of mice exposed to Sugen/Hypoxia (referring to mice exposed to a combination of a vascular endothelial growth factor receptor antagonist, Sugen 5416 (SU5416), and chronic hypoxia, which is to cause pronounced pulmonary hypertension (PH) in mice - see Vitali et ah, “The Sugen 5416/hypoxia mouse model of pulmonary hypertension revisted: long-term follow-up,” Pulm Cric. 2014, Dec; 4(4): 619- 629, the entire contents of which are incorporated herein by reference). The mice may also be referred to as the “SuHx mouse model” of PH, which are also described in Crucian L, Bonneau O, Hussey M, et al., “A novel murine model of severe pulmonary arterial hypertension,” Am J Respir Crit Care Med 2011;184:1171-1182, the contents of which are incorporated by reference. The findings described in the instant Specification and Examples indicate that miR-224 inhibition, using chemically modified oligonucleotides or AAV1- Tough decoy miRNA, reverses pulmonary vascular remodeling and Sugen/Hypoxia-induced PH.

[0008] MicroRNAs (miRNAs) are small non-coding ribonucleic acids (RNAs) that control expression of complementary target messenger RNAs (mRNAs). miRNAs interact specifically with mRNAs by repressing their translation or inducing their degradation. They are thus, by their impact on gene expression, key factors in the development and maintenance of tissue, both in healthy and disease states. A growing number of miRNAs have been implicated in the pathogenesis of PH, mostly based on the observation that they are dysregulated in diseased lungs and not on functional data. Consequently, our knowledge regarding a potential pulmonary role of the majority of miRNAs is limited. However, a small number of miRNAs (e.g., miR-21, miR-124, miR-145, miR-204) have been reported to play a critical role in pulmonary hypertension.^{20 22} Several miRNAs have been shown to be dysregulated in lungs of subjects with clinical PH and in mice or rats subjected to in vivo PH models.^{23 25} By performing array analysis, it has been reported that miR-224 is among the most upregulated miRNAs in lungs of subjects with PH.²⁶ In addition, it has been demonstrated that miR-224 level is upregulated in certain tumor types. Indeed, miR-224 has been reported to be significantly upregulated in non- small cell lung cancer tissues and to be associated with tumor size.^{27 29} In addition, increased miR-224 expression was shown to promote carcinoma (notably non- small cell lung cancer) cell proliferation, migration and invasion, while low miR-224 expression was demonstrated to suppress carcinoma cell migration and invasion.^{27 30} Prior to the instant disclosure, the role of miR-224 in PH, including PAH, was not known or contemplated.

[0009] Accordingly, in an aspect, the disclosure relates to a method of treating, preventing, or reversing pulmonary hypertension in subject in need thereof, comprising administering to the subject an effective amount of a miR-224 inhibitor. In some embodiments, the miR-224 inhibitor comprises an oligonucleotide that has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence which is fully complementary to SEQ ID NO: 1.

[0010] In some embodiments, the methods comprise administering an oligonucleotide wherein, the oligonucleotide comprises at least 12 nucleotides, at least 12 +1 nucleotides, at least 12 + 2 nucleotides, at least 12 + 3 nucleotides, at least 12 + 4 nucleotides, at least 12 + 5 nucleotides, at least 12 + 6 nucleotides, at least 12 + 7 nucleotides, at least 12 + 8 nucleotides, at least 12 + 9 nucleotides, at least 12 + 10 nucleotides, at least 12 + 11 nucleotides, at least 12 + 12 nucleotides, or at least 12 + 13 nucleotides.

[0011] In some embodiments, the oligonucleotide comprises an antagomir, antisense molecule, small hairpin RNA molecule, small interfering RNA molecule, microRNA sponge, tiny seed-targeting locked nucleic acid (LNA), or an RNA decoy. In some embodiments, the RNA decoy is a tough decoy (TuD).

[0012] In some embodiments, the oligonucleotide comprises a TuD-224 (SEQ ID NO: 3) or LNA-224 (SEQ ID NO: 4). In some embodiments the oligonucleotide is TuD-224 comprising a nucleic acid sequence which has at least 70% sequence identity to SEQ ID NO: 3. In some embodiments the oligonucleotide is TuD-224 comprising a nucleic acid sequence of SEQ ID NO: 3. In some embodiments the oligonucleotide is LNA-224 comprising a nucleic acid sequence which has at least 70% sequence identity to SEQ ID NO: 4. In some embodiments the oligonucleotide is LNA-224 comprising a nucleic acid sequence of SEQ ID NO: 4.

[0013] In some embodiments, the oligonucleotide comprises a nucleic acid modification to enhance stability. In some embodiments, the nucleic acid modification comprises a 2'-0- methoxyethyl sugar, a 2'-fluoro sugar modification, a 2 '-O-methyl sugar, a bicyclic sugar moiety, a cholesterol, or a phosphorothioate.

[0014] In some embodiments, the subject in need thereof comprises a mutation in the Bone Morphogenetic Protein Receptor 2 (BMPR2) gene, a genetic marker of PAH.

[0015] In some embodiments, administering the miR-224 inhibitor results in a decrease in human pulmonary artery smooth muscle cells (hPASMC) proliferation.

[0016] In some embodiments, the miR-224 inhibitor reverses Sugen/Hypoxia-induced pulmonary hypertension in a mouse model.

[0017] In an aspect, the disclosure relates to a method of treating, preventing, or reversing pulmonary hypertension in a subject in need thereof, comprising administering to the subject an effective amount of an miRNA inhibitor comprising a nucleic acid sequence that has at least 90% sequence identity to a sequence which is fully complementary to an miR-224 sequence.

[0018] In an aspect, the disclosure relates to a method of treating, preventing, or reversing pulmonary hypertension in a subject having a BMPR2 mutation, comprising: (a) obtaining a genetic test result on a subject sample to confirm the presence of a BMPR2 mutation; and (b) administering to the subject an effective amount of a miR-224 inhibitor.

[0019] In some embodiments, the genetic test result is obtained by a polymerase chain reaction (PCR) based method, a sequencing-based method, or a microarray-based method. [0020] In some embodiments, the miR-224 inhibitor comprises an oligonucleotide that has at least 85% sequence identity to a sequence which is fully complementary to SEQ ID NO: 1.

In some embodiments, the miR-224 inhibitor comprises an oligonucleotide that has 100% sequence identity to a sequence which is fully complementary to SEQ ID NO: 1.

[0021] In some embodiments, the methods comprise administering an oligonucleotide wherein, the oligonucleotide comprises at least 12 nucleotides, at least 12 +1 nucleotides, at least 12 + 2 nucleotides, at least 12 + 3 nucleotides, at least 12 + 4 nucleotides, at least 12 + 5 nucleotides, at least 12 + 6 nucleotides, at least 12 + 7 nucleotides, at least 12 + 8 nucleotides, at least 12 + 9 nucleotides, at least 12 + 10 nucleotides, at least 12 + 11 nucleotides, at least 12 + 12 nucleotides, or at least 12 + 13 nucleotides. [0022] In some embodiments, the oligonucleotide comprises an antagomir, antisense molecule, small hairpin RNA molecule, small interfering RNA molecule, microRNA sponge, tiny seed-targeting locked nucleic acid (LNA) oligonucleotide, or an RNA decoy. In some embodiments, the RNA decoy is a tough RNA decoy (TuD).

[0023] In some embodiments, the oligonucleotide comprises a TuD-224 (SEQ ID NO: 3) or LNA-224 (SEQ ID NO: 4). In some embodiments the oligonucleotide comprises a TuD-224 comprising a nucleic acid sequence with at least 70% identity to SEQ ID NO: 3. In some embodiments the oligonucleotide comprises a TuD-224 comprising a nucleic acid sequence of SEQ ID NO: 3. In some embodiments the oligonucleotide comprises an LNA-224 comprising a nucleic acid sequence with at least 70% identity to SEQ ID NO: 4. In some embodiments the oligonucleotide comprises an LNA-224 comprising a nucleic acid sequence of SEQ ID NO: 4.

[0024] In some embodiments, the oligonucleotide comprises a nucleic acid modification to enhance stability. In some embodiments, the nucleic acid modification comprises a 2'-0- methoxyethyl sugar, a 2'-fluoro sugar modification, a 2 '-O-methyl sugar, a bicyclic sugar moiety, a cholesterol, or a phosphorothioate.

[0025] In an aspect, the disclosure relates to a method for treating, preventing, or reversing pulmonary hypertension in a subject comprising: (a) identifying a subject suitable for treatment, wherein a suitable subject is one who has an increased level of miR-224 in a biological sample; (b) optionally obtaining a genetic test result on the biological sample to confirm the presence of a BMPR2 mutation; and (c) administering an effective amount of an miR-224 inhibitor to a subject having an increased level of miR-224, and optionally a BMPR2 mutation.

[0026] In some embodiments, the genetic test result is obtained by a PCR-based method, a sequencing-based method, or a microarray-based method.

[0027] In some embodiments, the miR-224 inhibitor comprises an oligonucleotide that has at least 85% sequence identity to a sequence which is fully complementary to SEQ ID NO: 1.

[0028] In some embodiments, the methods comprise administering an oligonucleotide wherein, the oligonucleotide comprises at least 12 nucleotides, at least 12 +1 nucleotides, at least 12 + 2 nucleotides, at least 12 + 3 nucleotides, at least 12 + 4 nucleotides, at least 12 + 5 nucleotides, at least 12 + 6 nucleotides, at least 12 + 7 nucleotides, at least 12 + 8 nucleotides, at least 12 + 9 nucleotides, at least 12 + 10 nucleotides, at least 12 + 11 nucleotides, at least 12 + 12 nucleotides, or at least 12 + 13 nucleotides.

[0029] In some embodiments, the oligonucleotide comprises an antagomir, antisense molecule, small hairpin RNA molecule, small interfering RNA molecule, microRNA sponge, tiny seed-targeting locked nucleic acid (LNA) oligonucleotide, or RNA decoy. In some embodiments, the RNA decoy is a tough decoy (TuD).

[0030] In some embodiments, the oligonucleotide comprises a TuD-224 (SEQ ID NO: 3) or LNA-224 (SEQ ID NO: 4). In some embodiments the oligonucleotide comprises a TuD-224 comprising a nucleic acid sequence with at least 70% identity to SEQ ID NO: 3. In some embodiments the oligonucleotide comprises a TuD-224 comprising a nucleic acid sequence of SEQ ID NO: 3. In some embodiments the oligonucleotide comprises an LNA-224 comprising a nucleic acid sequence with at least 70% identity to SEQ ID NO: 4. In some embodiments the oligonucleotide comprises an LNA-224 comprising a nucleic acid sequence of SEQ ID NO: 4.

[0031] In some embodiments, the oligonucleotide comprises a nucleic acid modification to enhance stability. In some embodiments, the nucleic acid modification comprises a 2'-0- methoxyethyl sugar, a 2'-fluoro sugar modification, a 2 '-O-methyl sugar, a bicyclic sugar moiety, a cholesterol, or a phosphorothioate.

[0032] These and other aspects and embodiments will be described in greater detail herein. The description of some exemplary embodiments of the disclosure are provided for illustration purposes only and not meant to be limiting. Additional compositions and methods are also embraced by this disclosure.

[0033] The summary above is meant to illustrate, in a non-limiting manner, some of the embodiments, advantages, features, and uses of the technology disclosed herein. Other embodiments, advantages, features, and uses of the technology disclosed herein will be apparent from the Detailed Description, Drawings, Examples, and Claims.

BRIEF DESCRIPTION OF THE DRAWINGS [0034] FIGs. 1A-1B show miR-224 expression in the lung. FIG. 1A: miR-224 expression profile across different rat tissues. Expression of miR-224 was assessed by real time PCR in stomach, right ventricle, left ventricle, liver, kidney, lung, brain, spleen, skeletal muscle and pancreas from 5 different rats. FIG. IB: Endogenous levels of miR-224 in pulmonary fibroblasts (leftmost column), endothelial cells (center column), and smooth muscle cells (rightmost column) (n = 4 experiments performed in duplicate). * P < 0.05.

[0035] FIGs. 2A-2D show miR-224 upregulation in PAH. FIG. 2A: Lung expression of miR-224 was assessed by real time PCR in lungs from non-PAH (n = 5), hPAH (n = 4), and iPAH subjects (n = 5). FIG. 2B: Endogenous levels of miR-224 in lung homogenates from normoxic and 6 weeks hypoxic mice (n = 5-6 mice/group). FIG. 2C: Changes in miR-224 levels determined by real time PCR analysis of lung extracts from control and MCT-injected rats (n = 4-6 rats/group). FIG. 2D: miR-224 expression in lungs from control and PV banding-induced PH pigs (n = 5-6 pigs per group). * P < 0.05.

FIGs. 3A-3D show that miR-224 regulates human pulmonary smooth muscle cells proliferation. FIG. 3A: miR-224 expression in hPASMCs transfected with miR-224 or miR- Ctrl. n = 6 experiments performed in duplicate. FIG. 3B: Effect of miR-224 overexpression on hPASMC proliferation. PASMCs were transfected with miR-Ctrl or miR-224 (50 nM each) and proliferation assessed 48 hours later n = 6-7 experiments in triplicate. FIG. 3C: Quantification of miR-224 expression in cultured hPASMC transfected with Anti-miR-Ctrl or Anti-miR-224 (50 nM each) n = 4 experiments in duplicate. FIG. 3D: Proliferation of hPASMCs transfected with Anti-miR-Ctrl or Anti-miR-224. Proliferation was assessed 48 hours after the transfection n = 3 experiments performed in triplicate. * P < 0.05; ** P < 0.01; *** P < 0.001. FBS: Fetal Bovine Serum.

[0036] FIGs. 4A-4D show that miR-224 exacerbates Sugen/Hypoxia-induced PH. FIG. 4A: Design of the study. Ten-week-old C57B6 mice subcutaneously received 20 mg/kg of SU5416, and were then exposed to three weeks of chronic hypoxia. SU5416 was injected once a week during the next two weeks. Mice were then randomly assigned to receive AAVl-miR- 224 or AAV 1 -Ctrl at week 13 for 3 weeks. The end point for hemodynamic measurements and sacrifice was at week 16. FIG. 4B: miR-224 expression in lung (Left) and RV (Right) homogenates from normoxic, hypoxic AAV 1 -Ctrl-treated mice and hypoxic AAVl-miR-224-treated mice (n = 3-4 mice/group). FIG. 4C: Left ventricular (LV) weight, right ventricular (RV) weight and Fulton index of the indicated groups n = 3-4 mice/group. FIG. 4D: Right ventricular pressure (RVSP) of the indicated groups n = 3-4 mice/group. [0037] FIGs. 5A-5B show the efficacy of LNA-antimiR-224 in vitro and in vivo. FIG. A: miR-224 expression in KEK293 cells transfected with LNA-224 or LNA-Ctrl. n = 3 experiments performed in duplicate. FIG. B: Pulmonary expression of miR-224 in healthy mice injected with anti-miR-224 (LNA-224) or a control molecule (LNA-Ctrl). LNAs were delivered via intraperitoneal injection (IP) or Intratracheal injection (ITT) at 5 or 10 mg/kg. LNA-Ctrl was delivered at 10 mg/kg. Mice were sacrificed 3 weeks after the injection n = 3- 4 mice/ group. * P < 0.05; ** P < 0.01.

[0038] FIGs. 6A-6F show that pharmacological inhibition of miR-224 reverses Sugen/Hypoxia-induced PH. FIG. 6A: Sequences of miR-224 and of the miR-224 inhibitor (LNA-224). miR-224 seed region is shown boxed in. FIG. 6B: Design of the study. Ten- week-old C57B6 mice subcutaneously received 20 mg/kg of SU5416, and were then exposed to three weeks of chronic hypoxia. SU5416 was injected once a week during the next two weeks. Mice were then randomly assigned to receive LNA-Ctrl or LNA-224 at week 13 for 3 weeks. The end point for hemodynamic measurements and sacrifice was at week 16. FIG. 6C: miR- 224 expression in lung homogenates from normoxic, hypoxic LNA-Ctrl-treated mice and hypoxic LNA-224-treated mice (n = 7-9 mice/group). FIG. 6D: Right ventricular (RV) weight, Fulton index and Right ventricular pressure (RVSP) of the indicated groups n = 7-9 mice/group. FIG. 6E: The three panels above the bar-graph show representative WGA- staining of ventricular sections to assess hypertrophy of cardiac myocytes. Scale bar: 50 pm. The bar-graph below the three panels shows quantitative analysis; n =7- 9 mice/group. FIG. 6F: The three panels above the bar-graph show representative H&E-stained sections of small pulmonary arteries from the indicated groups. Scale bar: 50 pm. The bar-graph below the three panels shows the percentage of artery medial thickness in relation to cross-sectional diameter n = 7-9 mice'/group. * P < 0.05; ** P < 0.01; *** P < 0.001.

[0039] FIGs. 7A-7F shows that inhibition of miR-224 using an AAVl-Tud-224 ameliorates pulmonary vascular remodeling and right ventricular function. FIG. 7A: Design of the TuD- miR-224 inhibitor. miR-224 seed region is show boxed in. FIG. 7B: Design of the study. Ten-week-old C57B6 mice subcutaneously received 20 mg/kg of SU5416, and were then exposed to three weeks of chronic hypoxia. SU5416 was injected once a week during the next two weeks. Mice were then randomly assigned to receive AAVl-Ctrl or AAV 1-TuD- 224 at week 13 for 3 weeks. The end point for hemodynamic measurements and sacrifice was at week 16. FIG. 7C: miR-224 expression in lung homogenates from normoxic, hypoxic AAV1- Ctrl-treated mice and hypoxic AAVl-TuD-224-treated mice (n = 4-5 mice/group). FIG. 7D: Right ventricular (RV) weight, Fulton index and Right ventricular pressure (RVSP) of the indicated groups n = 4-5 mice/group. FIG. 7E: The three panels above the bar-graph show representative WGA- staining of ventricular sections to assess hypertrophy of cardiac myocytes. Scale bar: 50 pm. The bar- graph below the three panels shows quantitative analysis; n = 4-5 mice/group. FIG. 7F: The three panels above the bar-graph show representative H&E-stained sections of small pulmonary arteries from the indicated groups. Scale bar: 50 pm. The bar-graph below the three panels shows the percentage of artery medial thickness in relation to cross-sectional diameter n = 4-5 mice/group. * P < 0.05; ** P < 0.01; *** P < 0.001.

[0040] FIGs. 8A-8F shows that miR-224 targets key signaling pathways in PH. FIG. 8A: Experimental scheme to identify miR-224 targets. Human PASMCs were transfected with miR-Ctrl or miR-224. 48 hours later, RNA was extracted and RNA sequencing was performed. FIG. 8B: Venn Diagram illustrating overlap between downregulated genes (assessed by RNA sequencing) and miR-224 predicted targets as predicted by miRDB, Targetscan, and Pictar databases. FIG. 8C: Diseases known to be associated with the 21 identified targets as assessed by the DAVID 6.8 algorithm (GAD: Genetic Association Database). FIG. 8D: Most relevant pathways associated with the 21 identified targets assessed using the Reactome Database. FIG. 8E: Scheme depicting the control of the BMP signaling pathway through miR-224. BMP-11, BMP-14, BMPRlb, BMPR2, Smad4, Smad5, Smad8, Idl, and Id3 are predicted to be targets of miR-224. FIG. 8F: Validation of microarray data by quantitative polymerase chain reaction. Human PASMCs were transfected with miR- 224 or miR-Ctrl. n = 2-5 experiments performed in duplicate.

*P<0.05; ***P<0.001.

[0041] FIG. 9 shows several genes with genetic evidence of mutations associated with PH are targets of miR-224. mRNAs expression levels of the predicted targets were assessed by real-time PCR in human PASMCs transfected with miR-224 or miR-Ctrl. n = 3 experiments performed in duplicate.

[0042] FIGs. 10A-10E show that miR-224 inhibition reverses Sugen/Hypoxia-induced PAH in rats. FIG. 10A: Design of the study. Sprague Dawley rats received 20 mg/kg of SU5416, and were then exposed to 3 weeks of hypoxia. Rats were then randomly assigned to receive FNA-Ctrl or FNA-224 at day 21. The end point for hemodynamic measurements and sacrifice was at day 42. FIG. 10B: miR-224 expression in lung homogenates from the indicated groups n = 8-10 rats/group. FIG. IOC: (Feft) RV hypertrophy reflected by the RV weight over FV plus interventricular septum (S) weight ratio (Fulton index), and (Right) right ventricular systolic pressure (RVSP) of the indicated groups n = 8-12 rats/group. FIG. 10D: Pulmonary artery systolic pressure (PASP), diastolic pressure (PADP), and mean pulmonary pressure (mPAP) measured in the indicated groups n = 8 rats/group. FIG. 10E: (top panel) Representative H&E-stained pulmonary arteries sections from the indicated groups. Scale bar: 10 mih. (bottom panel) Percentage of arteries medial thickness n = 7 rats/group.* P < 0.05; ** P < 0.01; *** P < 0.001.

[0043] FIGs. 11A-11G show that pharmacological inhibition of miR-224 improves survival and reverses MCT-induced PAH in rats. FIG. 11 A: Design of the study. Rats received 60 mg/kg of MCT, and were then randomly assigned to receive LNA-Ctrl or LNA-224 at day 14. The end point for MRI, hemodynamic measurements and sacrifice was at day 28. FIG.

1 IB: miR-224 expression in lung homogenates from control and MCT rats treated with LNA- Ctrl or LNA-224. FIG. 11C: Kaplan-Meier survival analysis of rats treated as in (FIG. 11A). FIG. 11D: Fulton index of the indicated groups. FIG. 11E: Right Ventricular Systolic Pressure (RVSP) of the indicated groups. FIG. 11F: Pulmonary artery systolic pressure (PASP), diastolic pressure (PADP), and mean pulmonary pressure (mPAP) measured in the indicated groups. FIG. 11G: (top panel) Representative H&E-stained pulmonary arteries sections from the indicated groups. Scale bar: 10 pm. (bottom panel) Percentage of arteries medial thickness. * P < 0.05; ** P < 0.01; *** P < 0.001. n = 8-12 rats/group.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

Definitions

Antagomir

[0044] The term “antagomir,” as used herein, refers to a class of oligonucleotides ( e.g ., polymers of nucleotides) that prevent binding by other molecules (e.g., miRNA) from binding to a specific site on an mRNA. Antagomirs are nearly always engineered (e.g., synthetic), often containing nucleic acid modifications (e.g., 2'-methoxy groups, phosphorothioate linkages) nucleic acids which are fully (e.g., perfectly) complementary to the miRNA target site. Many antagomirs however, will have either a mispairing or a nucleic acid medication at the Ago2 cleavage site. In one mechanism, the antagomirs, being complementary to a target miRNA, the antagomirs (miRNA themselves) bind and inhibit the inhibition of the target mRNA, thereby permitting the translation (e.g., expression) of the mRNA of the target miRNA. Additionally, in other instances, the antagomir is complementary to at least a portion of the mRNA which is targeted by the miRNA, and which antagomir upon binding to the mRNA sterically hinders the binding of the miRNA, thereby preventing degradation of the mRNA (“blockmirs”). Antagomirs and blockmirs may be formed of antisense molecules.

Antisense Molecule [0045] The term “antisense molecule,” as used herein, refers to an oligonucleotide ( e.g ., polymer of nucleotides) which is synthesized or contains a sequence of nucleotides complementary to a target nucleic acid sequence. For example, with the respect to RNA (e.g., mRNA, miRNA), a strand may read 5'-AAGGUCCU-3', wherein the antisense molecule will read 3'-UUCCAGGA-5'. In the case of antisense molecules targeting RNA they can modulate expression in a variety of ways. For example, strands may target mRNA (thereby blocking translation and promoting degradation of the mRNA transcript) or in another manner, the strands may target miRNA (thereby inhibiting the blocking miRNA from targeting the mRNA and promoting or restoring translation from the mRNA, and promoting degradation of the blocking miRNA).

Complementary

[0046] The terms “complementary” and “complementarity,” as used herein, refers a property of a nucleotide (e.g., A, C, G, T, U) in a nucleic acid (e.g., RNA, DNA) in a strand (e.g., oligonucleotide) to pair with another particular nucleotide in a nucleic acid strand of the opposite orientation (e.g., strands running parallel, but in the reverse direction (i.e., 5 '-3' aligns with 3'-5', and 3'-5' with 5 '-3 ')) (i-e., Watson-Crick base-pairing rules). With respect to deoxyribonucleic acids (DNA) the base pairings which are complementary are adenine (A) and thymine (T) (e.g., A with T, T with A) and guanine (G) and Cytosine (C) (e.g., G with C, C with G) and with respect to ribonucleic acid (RNA) the base pairings which are complementary are A and uracil (U) (e.g., A with U, U with A) and G and C (e.g., G with C, C with G). This occurs because of the ability of each base pair to form an equivalent number of hydrogen bonds with its complementary base (e.g., A-T/U, T/U-A, C-G, G-C), for example the bond between guanine and cytosine shares three hydrogen bonds compared to the A-T/U bond which always shares two hydrogen bonds.

[0047] When every base in at least one strand of a pair of nucleic acids is found opposite it’s complementary base pair, such strand is considered fully complementary to its sequence in the other strand. When one, or more, bases of such a strand is found in a position where it is opposite any other base excepting its complementary base pair, that base is considered “mis matched” and the strand is considered partially complementary. Accordingly, strands can be varying degrees of partially complementary, until no bases align, at which point they are non complementary.

[0048] Other non-standard nucleotides (e.g., 5-methylcytosine, 5-hydroxymethylcytosine) are known in the art and their properties and complementarity will be readily apparent to the skilled artisan. Effective Amount

[0049] The terms “effective amount” and “therapeutically effective amount,” as may be used interchangeably herein, refer to an amount of a biologically active agent ( e.g ., miR-224 inhibitor) sufficient to elicit a desired biological response. For example, in some embodiments, an effective amount of a miR-224 inhibitor may refer to the amount of the inhibitor sufficient to inhibit the target miRNA (e.g., miR-224). In some embodiments, an effective amount of a miR-224 inhibitor provided herein, may refer to the amount of the miR- 224 inhibitor sufficient to induce inhibition of the miRNA (e.g., miR-224). As will be appreciated by the skilled artisan, the effective amount of an agent (e.g., miR-224 inhibitor) may vary depending on various factors as, for example, on the desired biological response (e.g., on the miRNA to be inhibited, the mRNA transcript to be promoted), on the cell or tissue being targeted, and on the agent being used.

Host Cell

[0050] The term “host cell” as used herein, refers to a cell that can host, replicate, and express a vector described herein, e.g., a vector comprising a nucleic acid molecule encoding an miRNA inhibitor (e.g., miR-224 inhibitor). miR-224

[0051] The term “miR-224,” as used herein, should be understood to mean miR-224 in any form, for example precursor (e.g., pre-miRNA), primary (e.g., pri-miRNA), and/or mature (e.g., miRNA) sequences (e.g. SEQ ID NO: 1). miR-224 is often found to be -22 nucleotides long in its mature form and is encoded on the X-chromosome in mammals. miR-224 inhibitor

[0052] The term “miR-224 inhibitor,” as used herein, refers to an agent (e.g., molecule) capable of inhibiting or preventing the miR-224 from carrying out its functions. The inhibitor may inhibit miR-224 in any of its forms (e.g., precursor, primary, mature).

Generally, inhibition includes direct inhibition in which an agent (e.g., molecule) binds to a target miRNA and directly inhibits its activity. Inhibition also includes indirect inhibition in which, for example, expression of the miRNA molecule is modulated by suitable means including for example use of repressors or siRNA molecules.

[0053] Inhibition of miRNA function should be understood to encompass administering any agent (e.g., molecule) which directly or indirectly inhibits the mature form as well as agents (e.g., molecules) which target the precursor or primary forms of the target miRNA, such molecules, generally will be understood to be miRNA inhibitors. Suitable agents may include, for example, antagomirs (i.e., anti-micro RNAs (anti-miRs) or blockmirs (a class of chemically engineered oligonucleotides that prevent microRNAs (e.g., miR-224)) from binding to a desired site on an mRNA molecule, thereby silencing the effects of microRNAs), antisense molecules, small hairpin RNA molecules (shRNA), small interfering RNA molecules (siRNA), microRNA sponges (miRNA sponges), tiny seed-targeting locked nucleic acids (LNA), decoy oligonucleotides (e.g., RNA decoys, TuDs), aptamers, ribozymes, or antibodies that specifically recognize DNA:RNA hetero-duplexes. As may be used herein, anti-miRs refer to anti-micro RNAs.

[0054] For example, an inhibitor oligonucleotide is typically 7-30 linked nucleosides that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or fully, complementary to the sequence of the target micro RNA in a mature form, which may contain a nucleic acid modification. miRNA inhibitors can be obtained commercially (e.g., MIRvANA™ miRNA inhibitors sold by AMBION™). microRNA

[0055] The terms “microRNA” and “miRNA,” as may be used interchangeably herein, refer to short (e.g., about 20 to about 24 nucleotides in length) non-coding ribonucleic acids (RNAs) that are involved in post-transcriptional regulation of gene expression in multicellular organisms by affecting both the stability and translation of mRNAs. miRNAs are transcribed by RNA polymerase II as part of capped and polyadenylated primary transcripts (pri-miRNAs) that can be either protein-coding or non-coding. The primary transcript is cleaved by the Drosha ribonuclease III enzyme to produce a stem-loop precursor miRNA (pre-miRNA) approximately 70 nucleotides in length, which is further processed in the RNAi pathway. As part of this pathway the pre-miRNA is cleaved by the cytoplasmic Dicer ribonuclease to generate the mature miRNA and antisense miRNA star (miRNA*) products. The mature miRNA is incorporated into an RNA-induced silencing complex (RISC), which recognizes target mRNAs through imperfect base pairing (i.e., partial complementarity) with the miRNA and most commonly results in translational inhibition or destabilization of the target mRNA. This mechanism is most often seen through the binding of the miRNA on the 3' untranslated region (UTR) of the target mRNA, which can decrease gene expression by either inhibiting translation (for example, by blocking the access of ribosomes for translation) or directly causing degradation of the transcript. The term (i.e., miRNA) may be used herein to any form of the subject miRNA (e.g., precursor, primary, and/or mature miRNA). miRNA Spouse [0056] The term “miRNA sponge,” as used herein, refers to RNA molecules (e.g., antagomirs, antisense molecules, RNA decoys, miR-224 inhibitors, etc.) which have repeated miRNA antisense sequences, and accordingly can act to bind (e.g., sequester) multiple target miRNA per miRNA sponge molecule. miRNA sponges can be expressed through the introduction of plasmid constructs either transiently or stably transfected into mammalian cells (e.g., by retroviral vectors) containing multiple miR-binding sites for a chosen miRNA gene. The binding sites are located in tandem along an expressed transcript. The transcripts (e.g., miRNA inhibitors (e.g., miR-224 inhibitors)) are expressed via a strong promoter element and the endogenous, targeted miRNA of interest is soaked up (i.e., bound, sequestered) by the sponge transcript (e.g., miRNA inhibitors (e.g., miR-224 inhibitor)). miRNA sponges mimic the effects of miRNA inhibitors (i.e., the target miRNA’ s function, and the target miRNA’ s target (e.g., mRNA) sequence’s function is promoted or restored).

Nucleic Acid Modifications

[0057] The term “nucleic acid modifications,” as used herein, refers to modifications made to an oligonucleotide, or the constituent portions or linkages thereof (i.e., the nitrogenous base, sugar, or phosphate group). Modifications may be introduced for a variety of reasons, often to increase stability, reduce off-target effects, increase hybridization (i.e., binding) properties, or to reduce toxicity.

[0058] Purine and/or pyrimidine nucleobases may be modified, for example by amination or deamination of the heterocyclic rings. Further, modified sugars, such as a 2'-0-methoxyethyl sugar, a 2'-fluoro sugar modification, a 2 '-O-methyl sugar, a bicyclic sugar moiety, or substitutions such as a 2'-0 moiety with a lower alkyl, an alkenyl, an alkynyl, a methoxyethyl (2'-0-MOE), an -H (as in DNA), or other substituent may be introduced. Other examples may include the addition of a conjugate linked to the oligonucleotide, such as a cholesterol or phosphorothioate, to render the molecule more resistant to degradation.

[0059] Other chemistries and modification are known in the field of oligonucleotides that can be readily used in accordance with the disclosure and are encompassed within the definition of a nucleic acid modification. Linkages between the nucleotides may be modified by means of thioation of the phosphodiester bonds which can be used to yield phosphorothioate esters or phosphorodithioate esters. Further modifications to the linkages include amidation and peptide linkers.

Oligonucleotide

[0060] The term “oligonucleotide” as used herein, refers to a polymer of nucleotides. The polymer may include natural nucleosides (i.e., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine), nucleoside analogs ( e.g ., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C5 bromouridine, C5 fluorouridine, C5 iodouridine, C5 propynyl uridine, C5 propynyl cytidine, C5 methylcytidine, 7 deazaadenosine, 7 deazaguanosine, 8 oxoadenosine, 8 oxoguanosine, 0(6) methylguanine, 4-acetylcytidine, 5- (carboxyhydroxymethyl)uridine, dihydrouridine, methylpseudouridine, 1 -methyl adenosine, 1-methyl guanosine, N6-methyl adenosine, and 2-thiocytidine), chemically modified bases, biologically modified bases (e.g., methylated bases), intercalated bases, modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, 2'-0-methylcytidine, arabinose, and hexose), or modified phosphate groups (e.g., phosphorothioates and 5'-N phosphoramidite linkages).

Percent Identity

[0061] The terms “percent identity,” “sequence identity,” “% identity,” “% sequence identity,” and % identical,” as they may be interchangeably used herein, refer to a quantitative measurement of the similarity between two sequences (e.g., nucleic acid or amino acid). The percent identity of genomic DNA sequence, intron and exon sequence, and amino acid sequence between humans and other species varies by species type, with chimpanzee having the highest percent identity with humans of all species in each category. Percent identity can be determined using the algorithms of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such algorithms is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul et al., J. Mol. Biol. 215:403-10, 1990. BLAST protein searches can be performed with the XBLAST program, score=50, word length=3, to obtain amino acid sequences homologous to the protein molecules of interest. Where gaps exist between two sequences, Gapped BLAST can be utilized as described in Altschul et ah, Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. When a percent identity is stated, or a range thereof (e.g., at least, more than, etc.), unless otherwise specified, the endpoints shall be inclusive and the range (e.g., at least 70% identity) shall include all ranges within the cited range (e.g., at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least

97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% identity) and all increments thereof ( e.g ., tenths of a percent (i.e., 0.1%), hundredths of a percent (i.e., 0.01%), etc.).

RNA Decoys

[0062] The term “RNA Decoy,” as used herein, refers to nucleic acids (e.g., RNA, often single-stranded RNA), useful for, at least, regulating RNAi mechanisms or applications.

They are typically strands of RNA containing at least one antisense miRNA binding site, which when present, or introduced into an environment, containing miRNA coordinating to the miRNA binding site, knock down the miRNA effect on their target mRNA transcripts. In other words RNA decoys act to bind miRNA, thereby inhibiting their activity on mRNA. Since miRNA bind mRNA transcripts to knock down protein expression, RNA decoys act to decrease this inhibition, thereby increasing (e.g., lessening inhibition, restoring) protein expression. A variant of RNA, “Tough RNA Decoys” or “TuDs” are stabilized stem-loop RNA constructs containing two miRNA binding domains (as further described in Haraguchi et al., Nucleic Acids Res., 2009; Xie et al., Nature, 2012). RNA decoys (including TuDs), are form of antisense molecule, and may include antagomirs and blockmirs.

RNA Interference

[0063] The terms “RNA interference” and “RNAi,” as may be used interchangeably herein, refer to a method of gene expression at the step of translation, which is an RNA-dependent gene modulation pathway controlled by the RNA-induced silencing complex (RISC). In RNAi, RNA molecules (e.g., siRNA, miRNA) interact with a target mRNA transcript to alter, modulate, or otherwise effect a change in the target mRNA function in the absence of the “interfering” RNA molecule (e.g., suppress, silence). For example the interfering RNA molecule may direct enzymes to degrade the mRNA (thereby decreasing or eliminating their translation). Examples of interfering RNA molecules include, miRNA and siRNA.

[0064] The RNAi pathway can be initiated by the interaction of short double-stranded RNA (dsRNA) molecules with argonaute (Ago, the a catalytic component of RISC), which dsRNA can be exogenous (e.g., stem- loop dsRNA (e.g., synthetic, viral) introduced into the cytoplasm) and/or endogenous (stem-loop dsRNA originating in the nucleus (e.g., pre- miRNA) and exported to the cytoplasm. Once the dsRNA (dsRNA in plants, and dsRNA in the form of hairpin in humans (i.e., shRNA)) has been introduced into the cytoplasm, it is cleaved by the enzyme Dicer into -20 nucleotide (i.e., base pair) miRNA duplexes (i.e., siRNA). This duplex subsequently binds Ago (the Ago-duplex complex), to form a precessor form of RISC. Aro proteins, a form of endonuclease, then facilitates the cleavage (e.g., separation) of the siRNA strands into single stranded molecules, with one strand being active, referred to as the “guide strand” in the case of siRNA and mature miRNA in the case of miRNA, and the other strand being referred to as the “passenger” strand (which is believed to be largely inactive and will be degraded) in the case of siRNA and antisense miRNA* in the case of miRNA. siRNA and miRNA then interact with a target mRNA to modulate (i.e., silence, reduce, degrade, activate, promote) expression.

Small Hairpin RNA

[0065] The terms “short/small hairpin RNA” and “shRNA,” as may be used interchangeably herein, refer an RNA molecule with a tight turn approximately half-way through the strand, which effects formation in the strand such that it base-pairs with itself forming a dsRNA molecule. In such a shRNA molecule, one end will have two non-connected (i.e., exposed 5' and 3' ends) and one end comprising a closed loop (i.e., hairpin), in other words the dsRNA comprises a single-stranded RNA molecule which has self-base paired.

[0066] shRNA can mimic pri-miRNA, which is processed by Drosha to a form which mimics pre-miRNA, and which is subsequently exported from the nucleus by Exportin 5 where it can be used in the RNAi pathway.

Small Interfering RNA

[0067] The terms “small interfering RNA” and “siRNA,” as may be used interchangeably herein, refer to RNA molecules which present as non-coding double-stranded RNA (dsRNA) molecules of about 20 to about 24 nucleotides (i.e., base pairs) in length (approximately similar to miRNA) which are useful in RNAi. siRNA often are found with phosphorylated 5' ends and hydroxylated 3' ends, which 3' ends typically have a 2 nucleotide overhang beyond the 5' end of the anti-parallel strand (i.e., complementary strand of the dsRNA molecule). [0068] siRNA are most often found interfering with the expression of specific genes through binding of target sequences (e.g., target gene sequences) to which they are complementary and promoting (e.g., facilitating, triggering, initiating) degradation of the mRNA, thereby preventing (e.g., inhibiting, silencing, interfering with) translation.

[0069] After integration and separation into the RISC complex, siRNAs base-pair (i.e., full complementary) to their target mRNA and cleave it, thereby preventing it from being used as a translation template. As discussed herein above, also part of the RNAi pathway, an miRNA-loaded RISC complex scans cytoplasmic mRNAs for potential complementarity (i.e., partial complementarity).

Subject

[0070] The term “subject,” as used herein, refers to any organism in need of treatment or diagnosis using the subject matter herein. For example without limitation, subjects may include mammals and non-mammals which have or are at risk of having PH, including PAH. As used herein, a “mammal,” refers to any animal constituting the class Mammalia (e.g., a human, mouse, rat, cat, dog, sheep, rabbit, horse, cow, goat, pig, guinea pig, hamster, chicken, turkey, or a non-human primate (e.g., Marmoset, Macaque)). In some embodiments, the mammal is a human. Unless specified to the contrary, as may be used herein, a “subject suitable for treatment,” refers to any subject who is in need of treatment using one of the agents disclosed herein or by one of the methods disclosed herein, for example, one who has an increased level of miR-224 in a biological sample.

Tin v Seed-targeting Locked Nucleic Acid

[0071] The terms “Tiny Seed-targeting Locked Nucleic Acid” and “LNA,” as may be used interchangeably herein, refer to oligonucleotides which comprise at least one locked nucleic acid which target the seed region of an miRNA.

[0072] Locked nucleic acids are modified RNA nucleotides in which the ribose sugar is modified by means of a bridge connecting the 2' oxygen and 4' carbon (often seen as a methylene bridge between the 2' oxygen and 4' carbon). This bridge operably "locks" the ribose in the 3'-endo conformation. The locked ribose sugar conformation can enhance base stacking and backbone pre-organization, which can affect (e.g., increase) its hybridization properties (e.g., thermal stability and hybridization specificity). Locked nucleic acids can be inserted into both RNA and DNA oligonucleotides to hybridize with DNA or RNA according to typical Watson-Crick base-pairing rules (i.e., complementarity).

[0073] miRNA seed regions are the regions of the miRNA which primarily affect the ability of the miRNA to recognize (i.e., bind) a target mRNA. These regions are often short stretches of nucleotides, often about 6 to about 8 nucleotides, but in some instances as few as 2 nucleotides, near or at the 5' end of the miRNA which target the 3' UTR of a target mRNA.

Treatment

[0074] The terms “treatment,” “treat,” and “treating,” as may be used interchangeably herein, refer to a clinical intervention aimed to reverse, alleviate, delay the onset of, or inhibit the progress of a, indication (e.g., disease or disorder), or one or more symptoms thereof, as described herein. In some embodiments, treatment may be administered after one or more symptoms have developed and/or after a disease has been diagnosed. In other embodiments, treatment may be administered in the absence of symptoms (e.g., to prevent or delay onset of a symptom or inhibit onset or progression of a disease). For example, treatment may be administered to a susceptible individual (e.g., subject) prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example, to prevent or delay their recurrence.

Vector

[0075] The term “vector,” as used herein, refers to a nucleic acid ( e.g ., RNA, DNA) that can be modified to encode a gene of interest and that is able to enter into a host cell, mutate, and replicate within the host cell, and then transfer a replicated form of the vector into another host cell. Exemplary suitable vectors include viral vectors, such as retroviral vectors or bacteriophages and filamentous phage, and conjugative plasmids. Additional suitable vectors will be apparent to those of skill in the art based on the instant disclosure.

Wild-type

[0076] The term “wild-type” (WT) as used herein, is a term of art understood by skilled persons and means the typical form of an organism, strain, gene, or characteristic as it occurs in nature as distinguished from mutant or variant forms.

Certain Embodiments

[0077] Pulmonary hypertension (PH) is a disease characterized by progressive remodeling of the distal pulmonary arteries, resulting in the loss of vascular cross-sectional area and elevated pulmonary vascular resistance. Without intervention, PH is usually progressive, leading to right heart failure and death. The present disclosure provides a new therapeutic strategy to prevent and inhibit PH based on microRNA-224 (miR-224) inhibition. Through the present disclosure it was found that miR-224 levels are significantly increased in subjects with clinical pulmonary arterial hypertension PAH (a subtype of PH), as well as in different PAH-animal models. In vitro , miR-224 overexpression induces an increase in hPASMC proliferation, whereas miR-224 inhibition decreases hPASMC proliferation. The overexpression of miR-224 in vivo exacerbates the PAH phenotype of mice exposed to Sugen/Hypoxia. These findings indicate that miR-224 inhibition, using chemically modified oligonucleotides (e.g., miRNA inhibitors) or AAVl-Tough decoy miRNA, reverses pulmonary vascular remodeling and Sugen/Hypoxia-induced PAH.

[0078] A growing number of miRNAs have been implicated in the pathogenesis of PAH, mostly based on the observation that they are dysregulated in diseased lungs and not on functional data. Consequently, current knowledge regarding a potential pulmonary role of the majority of miRNAs is limited. However, a small number of miRNAs (e.g., miR-21, miR-124, miR-145, miR-204) have been reported to play a critical role in pulmonary hypertension.^{20 22} Several miRNAs haven been shown to be dysregulated in lungs of subjects with clinical PAH and in mice or rats subjected to In vivo PAH models.^{23 25} By performing array analysis, it has been reported that miR-224 is among the most upregulated miRNAs in lungs of subjects with PAH.²⁶ In addition, it has been demonstrated that miR-224 level is upregulated in certain tumor types. Indeed, miR-224 has been reported to be significantly upregulated in non-small cell lung cancer tissues and to be associated with tumor size.^{27 29} In addition, increased miR-224 expression was shown to promote carcinoma (notably non-small cell lung cancer) cell proliferation, migration and invasion, while low miR-224 expression was demonstrated to suppress carcinoma cell migration and invasion.^{27 30} Accordingly, in an aspect, the disclosure relates to a method of treating, preventing, or reversing pulmonary hypertension in subject in need thereof, comprising administering to the subject an effective amount of a miR-224 inhibitor. By administering an miR-224 inhibitor, the current method will modulate the effects of miR-224 in the body by binding to miR-224 and returning the concentration of miR-224 to levels which may approximate those of healthy subjects, or subjects otherwise not experiencing upregulation ( .<?., overexpression) of miR-224. Such an upregulation may be due to a disease indication (e.g., disease or disorder, e.g., pulmonary hypertension).

[0079] Moreover, the majority of the subjects with familial PAH as well as ll%-40% of the subjects with idiopathic PAH have heterozygous mutations in the Bone Morphogenetic Protein Receptor 2 (BMPR2) gene.^{5 6} Subjects carrying a BMPR2 mutation develop PAH approximately 10 years earlier than non-carriers, with a more severe hemodynamic compromise at diagnosis, and are less likely to respond to acute vasodilator testing.^{7 9} Genetic mutations can either directly inactivate BMPR2 or suppress its function by impairing its trafficking to the cell surface. In addition, mutation or downregulation of BMPR2 induced by RNA interference (RNAi) increases endothelial cell (EC) susceptibility to apoptosis and promotes proliferation of human pulmonary artery smooth muscle cells (PASMCs).^10-11 BMPR2 is a transmembrane serine/threonine kinase receptor, and a member of the transforming growth factor-beta (TGF-b) superfamily. Upon activation through a BMP ligand, BMPR2 builds a heteromeric complex with a type 1 receptor (BMPR1). This leads to phosphorylation of the intracellular part of the type 1 receptor and initiates a Smad protein signaling cascade upon phosphorylation of Smad proteins (Smad) 1, 5, and 8.

Phosphorylation of Smad 1, 5, and 8 leads to their association with the nuclear chaperone Smad4. This signaling complex will translocate to the nucleus, where it acts in combination with transcriptional co-activators and co-repressors to effect control of target gene expression.¹² Major targets of the Smad signaling in the nucleus are the inhibitors of DNA- binding (Id) genes.^{13 14} Id proteins are basic helix-loop-helix transcription factors that lack a DNA binding domain and are the major downstream mediators of BMP signaling. These proteins bind to the ubiquitously expressed E protein family members with high affinity and inhibit their binding to target DNA.^{15 16} This unique function of Id proteins confers a central role in the regulation of gene expression, and hence, cells differentiation and proliferation.

Idl and Id3 are major targets of BMP signaling in PASMCs, and the induction of Idl and Id3 is dependent on intact BMPR2. Several studies have reported that Idl and Id3 promote PASMC growth suppression.^{17 19} Over 300 different BMPR2 mutations have been identified with a prevalence of greater than 75% in families with PAH. Exemplary mutations are described in the art, for example see , Newman JH el al, JACC, 2004; Lane KB el ah, Nature Genetics 2000, both of which are incorporated herein in their entirety.

[0080] Accordingly, in some embodiments, the subject in need of the methods disclosed herein comprises a mutation in the Bone Morphogenetic Protein Receptor 2 (BMPR2) gene. In evaluating subjects for a mutation of BMPR2, the method may more readily identify subjects having, at risk of having, or create suspicion of such subjects having pulmonary hypertension.

[0081] In an aspect, the disclosure relates to a method of treating, preventing, or reversing pulmonary hypertension in a subject having a BMPR2 mutation, comprising: (a) obtaining a genetic test result on a subject sample to confirm the presence of a BMPR2 mutation; and (b) administering to the subject an effective amount of a miR-224 inhibitor. Mutations of BMPR2 can be any known in the art, or those identified by the skilled artisan. As can be appreciated by one of ordinary skill, many mutations are the result of deletions, insertions, frameshifts, and substitutions in the nucleic acid encoding the gene of interest.

[0082] In some embodiments, the genetic test result is obtained by a PCR-based method, a sequencing-based method, or a microarray-based method. Polymerase chain reaction is a well-known technique in the art and has many uses therein. For example, PCR is a well- known technique to the skilled artisan for use in isolating and/or amplifying target sequences ( e.g ., mutations, genes) within a nucleic acid. Techniques of using PCR to confirm the presence of a suspected or identified BMPR2 mutation will be readily apparent to the skilled artisan. In some embodiments, the genetic test result is obtained by a PCR-based method. Generally, primers are designed to bind the target sequence and start the replication. Upon binding, and subsequent synthesis, the target sequence can be quantified. Nucleic acid sequencing is a well-known technique in the art and has many uses therein. For example, nucleic acid sequencing (e.g., next generation sequencing, sanger sequencing) is a well- known technique to the skilled artisan for use in determining the base sequence (e.g., mutations, genes) within a nucleic acid. Techniques of using nucleic acid sequencing to confirm the presence of a suspected or identified BMPR2 mutation will be readily apparent to the skilled artisan. In some embodiments, the genetic test result is obtained by a sequencing- based method. Microarray-based methods for screening for nucleic acid sequences are well known in the art. Generally, the techniques rely on hybridization (i.e., base-pairing) of labeled sequences to a probe for the target sequence which are fixed to a plate or medium (i.e., microarray). Upon binding and subsequent washing, the labeled hybridized nucleic acids can be quantified. In some embodiments, the genetic test result is obtained by a microarray-based method.

[0083] In an aspect, the disclosure relates to methods of treating, preventing, or reversing pulmonary hypertension in a subject in need thereof, comprising administering to the subject an effective amount of an miRNA inhibitor comprising a nucleic acid sequence that has at least 80% sequence identity to a sequence which is fully complementary to an miR-224 sequence. In some embodiments, the methods comprise administering an miRNA inhibitor comprising a nucleic acid sequence that has at least 80% sequence identity to a sequence which is fully complementary to an miR-224 sequence. In some embodiments, the miRNA inhibitor comprises a nucleic acid sequence that has at least 85% sequence identity to a sequence which is fully complementary to an miR-224 sequence. In some embodiments, the miRNA inhibitor comprises a nucleic acid sequence that has at least 90% sequence identity to a sequence which is fully complementary to an miR-224 sequence. In some embodiments, the miRNA inhibitor comprises a nucleic acid sequence that has at least 95% sequence identity to a sequence which is fully complementary to an miR-224 sequence. In some embodiments, the miRNA inhibitor comprises a nucleic acid sequence that has at least 96% sequence identity to a sequence which is fully complementary to an miR-224 sequence. In some embodiments, the miRNA inhibitor comprises a nucleic acid sequence that has at least 97% sequence identity to a sequence which is fully complementary to an miR-224 sequence. In some embodiments, the miRNA inhibitor comprises a nucleic acid sequence that has at least 98% sequence identity to a sequence which is fully complementary to an miR-224 sequence. In some embodiments, the miRNA inhibitor comprises a nucleic acid sequence that has at least 99% sequence identity to a sequence which is fully complementary to an miR-224 sequence. In some embodiments, the miRNA inhibitor comprises a nucleic acid sequence that has at least 99.5% sequence identity to a sequence which is fully complementary to an miR-224 sequence. In some embodiments, the miRNA inhibitor comprises a nucleic acid sequence that has at least 99.9% sequence identity to a sequence which is fully complementary to an miR-224 sequence.

[0084] In some aspects, the disclosure relates to methods of treating, preventing, or reversing pulmonary hypertension in a subject in need thereof the methods administering the miR-224 inhibitor of the methods herein, results in a decrease in human pulmonary artery smooth muscle cells (hPASMC) proliferation. Smooth muscle in the pulmonary artery of subjects with PAH, is characterized by excessive proliferation of hPASMCs. This hyperplasia is primarily the result of the hPASMC of both idiopathic and hereditary PAH proliferating under non-proliferative, non-growth stimulated conditions. Accordingly, as is shown herein, miR-224 inhibitors can attenuate hPASMC hyperplasia.

[0085] In some embodiments, the miR-224 inhibitor reverses Sugen/Hypoxia-induced pulmonary hypertension in a mouse model. A mouse model of PH has been difficult to ascertain. However, a combination of a vascular endothelial growth factor (VEGF) receptor antagonist, Sugen (e.g., Sugen5416 (SU5416)) coupled with 3 weeks of chronic hypoxia has been shown to cause PH in rats.^{41 42} This model is an improvement of previous models, for example, chronic hypoxic and monocrotaline-induced PH rat models. In the SU5416/hypoxia model, the combination causes lesions in the pulmonary arterioles similar those found in human idiopathic pulmonary arterial hypertension. These lesions are not present at the time rats are returned to normoxic conditions (following the 3 weeks of hypoxia), and progressively develop over the following months. The SU5416/hypoxia model exhibits sustained and progressive PH, an improvement over animals exposed to hypoxia alone, which typically revert to a normal phenotype after returning to normoxia ^43-44 This model provides a basis to explore the effect of the agents herein and the ability of the agents herein to facilitate reversion to normal phenotypes.

[0086] In some embodiments, the genetic test result of the method is any of the methods described herein. In some embodiments, the genetic test result is obtained by a polymerase chain reaction (PCR) based method, a sequencing-based method, or a microarray-based method.

[0087] In some embodiments, the disclosure relates methods of treating, preventing, or reversing pulmonary hypertension in a subject in need thereof, by administering an miR-224 inhibitor comprising an oligonucleotide that has at least 80% sequence identity to a sequence which is fully complementary to SEQ ID NO: 1. In some embodiments, the methods comprise administering and miR-224 inhibitor, wherein the miR-224 inhibitor comprises an oligonucleotide that has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence which is fully complementary to SEQ ID NO: 1. In some embodiments, the miR-224 inhibitor comprises an oligonucleotide that has at least 85% sequence identity to a sequence which is fully complementary to SEQ ID NO: 1. In some embodiments, the miR-224 inhibitor comprises an oligonucleotide that has at least 90% sequence identity to a sequence which is fully complementary to SEQ ID NO: 1. In some embodiments, the miR-224 inhibitor comprises an oligonucleotide that has at least 95% sequence identity to a sequence which is fully complementary to SEQ ID NO: 1. In some embodiments, the miR-224 inhibitor comprises an oligonucleotide that has at least 96% sequence identity to a sequence which is fully complementary to SEQ ID NO: 1. In some embodiments, the miR-224 inhibitor comprises an oligonucleotide that has at least 97% sequence identity to a sequence which is fully complementary to SEQ ID NO: 1. In some embodiments, the miR-224 inhibitor comprises an oligonucleotide that has at least 98% sequence identity to a sequence which is fully complementary to SEQ ID NO: 1. In some embodiments, the miR-224 inhibitor comprises an oligonucleotide that has at least 99% sequence identity to a sequence which is fully complementary to SEQ ID NO: 1. In some embodiments, the miR-224 inhibitor comprises an oligonucleotide that has at least 99.5% sequence identity to a sequence which is fully complementary to SEQ ID NO: 1. In some embodiments, the miR-224 inhibitor comprises an oligonucleotide that has at least 99.9% sequence identity to a sequence which is fully complementary to SEQ ID NO: 1. In some embodiments, the miR-224 inhibitor comprises an oligonucleotide which is fully complementary to SEQ ID NO: 1.

[0088] In an aspect, the disclosure relates to a method for treating, preventing, or reversing pulmonary hypertension in a subject comprising: (a) identifying a subject suitable for treatment, wherein a suitable subject is one who has an increased level of miR-224 in a biological sample; (b) optionally obtaining a genetic test result on the biological sample to confirm the presence of a BMPR2 mutation; and (c) administering an effective amount of an miR-224 inhibitor to a subject having an increased level of miR-224, and optionally a BMPR2 mutation. Any tests described herein (e.g., PCR-based methods, nucleic acid sequencing, microarray-based methods) may be used to genetic test result to confirm the presence of a BMPR2 mutation.

[0089] The methods of the disclosure include the administration of at least a nucleic acid (e.g., oligonucleotide, miRNA inhibitor, miR-224 inhibitor), of which a variety are disclosed and contemplated herein. In some embodiments, the methods comprise administering an oligonucleotide wherein, the oligonucleotide comprises at least 12 nucleotides, at least 12 +1 nucleotides, at least 12 + 2 nucleotides, at least 12 + 3 nucleotides, at least 12 + 4 nucleotides, at least 12 + 5 nucleotides, at least 12 + 6 nucleotides, at least 12 + 7 nucleotides, at least 12 + 8 nucleotides, at least 12 + 9 nucleotides, at least 12 + 10 nucleotides, at least 12 + 11 nucleotides, at least 12 + 12 nucleotides, or at least 12 + 13 nucleotides. In some embodiments, the oligonucleotide comprises at least 12 nucleotides. In some embodiments, the oligonucleotide comprises at least at least 12 +1 nucleotides. In some embodiments, the oligonucleotide comprises at least 12 + 2 nucleotides. In some embodiments, the oligonucleotide comprises at least 12 + 3 nucleotides. In some embodiments, the oligonucleotide comprises at least 12 + 4 nucleotides. In some embodiments, the oligonucleotide comprises at least 12 + 5 nucleotides. In some embodiments, the oligonucleotide comprises at least 12 + 6 nucleotides. In some embodiments, the oligonucleotide comprises at least 12 + 7 nucleotides. In some embodiments, the oligonucleotide comprises at least 12 + 8 nucleotides. In some embodiments, the oligonucleotide comprises at least 12 + 9 nucleotides. In some embodiments, the oligonucleotide comprises at least 12 + 10 nucleotides. In some embodiments, the oligonucleotide comprises at least 12 + 11 nucleotides. In some embodiments, the oligonucleotide comprises at least 12 + 12 nucleotides. In some embodiments, the oligonucleotide comprises at least 12 + 13 nucleotides.

[0090] In some embodiments, the methods comprise administering an oligonucleotide, wherein the oligonucleotide comprises an antagomir, antisense molecule, small hairpin RNA molecule, small interfering RNA molecule, microRNA sponge, tiny seed-targeting locked nucleic acid (LNA), or an RNA decoy. In some embodiments, the oligonucleotide comprises an antagomir. In some embodiments, the oligonucleotide comprises an antisense molecule.

In some embodiments, the oligonucleotide comprises a small hairpin RNA molecule. In some embodiments, the oligonucleotide comprises a small interfering RNA molecule. In some embodiments, the oligonucleotide comprises a microRNA sponge. In some embodiments, the oligonucleotide comprises a tiny seed-targeting locked nucleic acid (LNA). In some embodiments, the oligonucleotide comprises an RNA decoy. In some embodiments, the RNA decoy is a tough decoy (TuD).

[0091] In some embodiments, the methods comprise administering an oligonucleotide, wherein the oligonucleotide comprises a TuD-224 (SEQ ID NO: 3) or LNA-224 (SEQ ID NO: 4). In some embodiments the oligonucleotide is TuD-224 comprising a nucleic acid sequence which has at least 70% sequence identity to SEQ ID NO: 3. In some embodiments the oligonucleotide is TuD-224 comprising a nucleic acid sequence which has at least 75% sequence identity to SEQ ID NO: 3. In some embodiments the oligonucleotide is TuD-224 comprising a nucleic acid sequence which has at least 80% sequence identity to SEQ ID NO: 3. In some embodiments the oligonucleotide is TuD-224 comprising a nucleic acid sequence which has at least 85% sequence identity to SEQ ID NO: 3. In some embodiments the oligonucleotide is TuD-224 comprising a nucleic acid sequence which has at least 90% sequence identity to SEQ ID NO: 3. In some embodiments the oligonucleotide is TuD-224 comprising a nucleic acid sequence which has at least 95% sequence identity to SEQ ID NO: 3. In some embodiments the oligonucleotide is TuD-224 comprising a nucleic acid sequence which has at least 96% sequence identity to SEQ ID NO: 3. In some embodiments the oligonucleotide is TuD-224 comprising a nucleic acid sequence which has at least 97% sequence identity to SEQ ID NO: 3. In some embodiments the oligonucleotide is TuD-224 comprising a nucleic acid sequence which has at least 98% sequence identity to SEQ ID NO:

3. In some embodiments the oligonucleotide is TuD-224 comprising a nucleic acid sequence which has at least 99% sequence identity to SEQ ID NO: 3. In some embodiments the oligonucleotide is TuD-224 comprising a nucleic acid sequence which has at least 99.5% sequence identity to SEQ ID NO: 3. In some embodiments the oligonucleotide is TuD-224 comprising a nucleic acid sequence which has at least 99.9% sequence identity to SEQ ID NO: 3. In some embodiments the oligonucleotide is TuD-224 comprising a nucleic acid sequence of SEQ ID NO: 3. In some embodiments the oligonucleotide is LNA-224 comprising a nucleic acid sequence which has at least 70% sequence identity to SEQ ID NO:

4. In some embodiments the oligonucleotide is LNA-224 comprising a nucleic acid sequence which has at least 75% sequence identity to SEQ ID NO: 4. In some embodiments the oligonucleotide is LNA-224 comprising a nucleic acid sequence which has at least 80% sequence identity to SEQ ID NO: 4. In some embodiments the oligonucleotide is LNA-224 comprising a nucleic acid sequence which has at least 85% sequence identity to SEQ ID NO: 4. In some embodiments the oligonucleotide is LNA-224 comprising a nucleic acid sequence which has at least 90% sequence identity to SEQ ID NO: 4. In some embodiments the oligonucleotide is LNA-224 comprising a nucleic acid sequence which has at least 95% sequence identity to SEQ ID NO: 4. In some embodiments the oligonucleotide is LNA-224 comprising a nucleic acid sequence which has at least 96% sequence identity to SEQ ID NO: 4. In some embodiments the oligonucleotide is LNA-224 comprising a nucleic acid sequence which has at least 97% sequence identity to SEQ ID NO: 4. In some embodiments the oligonucleotide is LNA-224 comprising a nucleic acid sequence which has at least 98% sequence identity to SEQ ID NO: 4. In some embodiments the oligonucleotide is LNA-224 comprising a nucleic acid sequence which has at least 99% sequence identity to SEQ ID NO: 4. In some embodiments the oligonucleotide is LNA-224 comprising a nucleic acid sequence which has at least 99.5% sequence identity to SEQ ID NO: 4. In some embodiments the oligonucleotide is LNA-224 comprising a nucleic acid sequence which has at least 99.9% sequence identity to SEQ ID NO: 4. In some embodiments the oligonucleotide is LNA-224 comprising a nucleic acid sequence of SEQ ID NO: 4.

[0092] In some embodiments, the methods comprise administering an oligonucleotide, wherein the oligonucleotide comprises a nucleic acid modification. In some embodiments, the nucleic acid modification enhances stability. In some embodiments, the nucleic acid modification comprises a 2'-0-methoxyethyl sugar, a 2'-fluoro sugar modification, a 2'-0- methyl sugar, a bicyclic sugar moiety, a cholesterol, a phosphorothioate, or combination thereof. In some embodiments, the nucleic acid modification comprises a 2'-0-methoxyethyl sugar. In some embodiments, the nucleic acid modification comprises a 2'-fluoro sugar modification. In some embodiments, the nucleic acid modification comprises a 2'-0-methyl sugar. In some embodiments, the nucleic acid modification comprises a bicyclic sugar moiety. In some embodiments, the nucleic acid modification comprises a cholesterol modification. In some embodiments, the nucleic acid modification comprises a phosphorothioate modification.

[0093] The disclosure relates to a variety of methods for inhibiting miR-224, treating, preventing, or reversing pulmonary hypertension, and for delivering miRNA inhibitors. The methods described herein also may be useful in evaluating individuals of particular subject populations, for example, in some embodiments, the subject has, is suspected of having, or is at risk of having an indication (e.g., disease or disorder). In some embodiments, the disorder is pulmonary hypertension.

Administration, Viral Deliver , Compositions , and Kits

Administration

[0094] To practice any of the methods disclosed herein, an effective amount of the composition described above can be administered to a subject (e.g., a human) in need of the treatment via a suitable route (as discussed herein below).

[0095] The subject to be treated by the methods described herein can be any subject in need of the agents disclosed herein (e.g., miR-224 inhibitors), for example, mammals, or more preferably a human. A subject who needs the treatment may be a subject who has, is at risk of having, or is suspected of having pulmonary hypertension or an indication (e.g., disease or disorder) related to miR-224. A subject having pulmonary hypertension or an indication ( e.g ., disease or disorder) related to miR-224, can be identified by routine medical examination (e.g., laboratory tests, organ functional tests, CT scans, ultrasounds, etc.). A subject suspected of having any of such indication (e.g., disease or disorder) might show one or more symptoms of the indication (e.g., disease or disorder). A subject at risk for the disorder can be a subject having one or more of the risk factors for that disorder.

[0096] Any of the agents disclosed herein (e.g., miR-224 inhibitors) may be administered by any administration route known in the art. For example, in some embodiments, one of ordinary skill in the art of medicine, can administer the agents via conventional routes, e.g., administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term “parenteral” as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrastemal, intrathecal, intralesional, and intracranial injection or infusion techniques. In addition, it can be administered to the subject via injectable depot routes of administration such as using 1-, 3-, or 6-month depot injectable or biodegradable materials and methods. In some embodiments, the administration route is enteral or gastrointestinal (e.g., oral) and the formulation is formulated for enteral or gastrointestinal administration (e.g., oral). In some embodiments, the administration route is parenteral and the formulation is formulated for parenteral administration. In some embodiments, the administration route is via injection and the formulation is formulated for injection. In some embodiments, the administration route is sublingual and the formulation is formulated for sublingual administration. In some embodiments, the administration route is buccal and the formulation is formulated for buccal administration. In some embodiments, the administration route is nasal and the formulation is formulated for nasal administration. In some embodiments, the administration route is transdermal and the formulation is formulated for transdermal administration. In some embodiments, the administration route is subcutaneous and the formulation is formulated for subcutaneous administration. In some embodiments, the administration route is perivascular and the formulation is formulated for perivascular administration. In some embodiments, the administration route is topical and the formulation is formulated for topical administration. In some embodiments, the administration route is rectal (e.g., intrarectal) and the composition is formulated for rectal administration. In some embodiments, the administration route is intravenously (i.e., by venous or arterial puncture), and the formulation is formulated for intravenous (i.e., by venous or arterial puncture) administration. [0097] A dosing regimen may comprise administering an initial dose of about 3 mg/kg to about 7 mg/kg of body weight. The doses may be administered subcutaneously, over a 5 week period. The doses may be administered weekly. In some embodiments, the dose is about 5 mg/kg, administered weekly, over the course of 5 weeks. However, other dosage regimens may be useful, depending on the pattern of pharmacokinetic decay that the practitioner ( e.g ., skilled artisan) wishes to achieve. For example, dosing from one to seven times a week is contemplated. In some embodiments, dosing ranging from about 3 pg/mg to about 10 mg/kg (such as about 3 pg/mg, about 10 pg/mg, about 30 pg/mg, about 100 pg/mg, about 300 pg/mg, about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 5 mg/kg, about 7 mg/kg, and about 10 mg/kg) may be used. In some embodiments, dosing frequency is once every week, every 2 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, or every 10 weeks; or once every month, every 2 months, or every 3 months, or longer. The progress of this therapy is easily monitored by conventional techniques and assays. The dosing regimen (including the agents used) can vary over time. [0098] Targeted delivery of therapeutic compositions containing an oligonucleotide (e.g., miRNA inhibitor), or expression vector can also be used. Receptor-mediated DNA delivery techniques are described in, for example, Findeis et al, Trends Biotechnol. (1993) 11:202; Chiou et al, Gene Therapeutics: Methods And Applications Of Direct Gene Transfer (J. A. Wolff, ed.) (1994); Wu etal, J. Biol. Chem. (1988) 263:621; Wu etal, J. Biol. Chem.

(1994) 269:542; Zenke et al, Proc. Natl. Acad. Sci. USA (1990) 87:3655; Wu et al, J. Biol. Chem. (1991) 266:338.

[0099] Therapeutic compositions containing an oligonucleotide (e.g., those encoding any of the agents described herein (e.g., miRNA inhibitors)) are administered in a range of about 100 ng to about 200 mg of nucleic acids for local administration in a gene therapy protocol.

In some embodiments, concentration ranges of about 500 ng to about 50 mg, about 1 pg to about 2 mg, about 5 pg to about 500 pg, and about 20 pg to about 100 pg of nucleic acids or more can also be used during a gene therapy protocol.

[0100] In some embodiments, any of the agents described herein can be administered to a subject in single or divided doses. In some embodiments, any of the agents described herein is administered to a subject in a single dose. In some embodiments, any of the agents described is administered to a subject in divided doses (e.g., multiple or sequential doses). The skilled artisan (e.g., physician) in any event may determine the actual dosage which will be most suitable for any subject, which will vary with the age, weight, and the particular indications ( e.g ., disease or disorder) to be treated or prevented.

[0101] Aspects of the disclosure relate to a method of modulating expression of target mRNA, for example, by administering miRNA inhibitors (e.g., miR-224 inhibitors) to modulate the effect of the miRNA on its target (e.g., mRNA). To perform such a method, an effective amount of the miRNA inhibitor (e.g., miR-224 inhibitor) as described herein can be administered as described herein. In some embodiments, the method described herein comprises administering an effective amount of a miRNA inhibitor (e.g., miR-224 inhibitor) or a composition comprising one or more miRNA inhibitor (e.g., miR-224 inhibitor) to a subject in need of treatment. The subject may be a human subject who has, or is at risk of, any indication (e.g., disease or disorder) related at least one of the target miRNA. Such a subject may be on additional treatments (e.g., have undergone, or currently being treated, by at least one other treatment). In some embodiments, the miRNA inhibitors (e.g., miR-224 inhibitor) as described herein can be administered at a specific period before, during, or after a diagnosis or suspicion of a diagnosis has occurred in the subject. In some embodiments, miRNA inhibitors (e.g., miR-224 inhibitor) as described herein is administered prior to manifestation of one or more symptoms of the target indication (e.g., disease or disorder). In other embodiments, the miRNA inhibitors (e.g., miR-224 inhibitor) as described herein are administered to the subject during or after manifestation of one or more symptoms of an indication (e.g., disease or disorder), or during or after occurrence of the an indication (e.g., disease or disorder), such as within 12 or 24 hours of an manifestation of one or more symptoms of an indication (e.g., disease or disorder). In some embodiments, the miRNA inhibitors (e.g., miR-224 inhibitor) as described herein are administered to the subject within 7 days (e.g., within 7, 6, 5, 4, 3, 2, or 1 days) after the subject is infected with a pathogen such as a bacterium or a virus, or manifests a symptom of the infection. In some embodiments, the miRNA inhibitors (e.g., miR-224 inhibitor) as described herein can be administered at a specific period before, during, or after another treatment. In some embodiments, the miRNA inhibitors (e.g., miR-224 inhibitor) as described herein are administered prior to another treatment. In other embodiments, the miRNA inhibitors (e.g., miR-224 inhibitor) as described herein are administered to the subject during or after another treatment, such as within 12 or 24 hours of the another treatment. In some embodiments, the miRNA inhibitors (e.g., miR-224 inhibitor) as described herein are administered to the subject within 6 months (e.g., within 3 months, within 2 months, within 1 month, or with 2 weeks) after the subject is treated. Diagnosis can be made using any method known in the art. In some embodiments, the subject has one or more symptoms of the indication ( e.g ., disease or disorder). In some embodiments, the subject is not, or has not, manifested any symptoms.

[0102] The agents (e.g., miRNA inhibitors) described herein can be delivered using gene delivery vehicles. The gene delivery vehicle can be of viral or non-viral origin (see generally, Jolly, Cancer Gene Therapy (1994) 1:51; Kimura, Human Gene Therapy (1994) 5:845; Connelly, Human Gene Therapy (1995) 1:185; and Kaplitt, Nature Genetics (1994) 6:148). Expression of such coding sequences can be induced using endogenous mammalian or heterologous promoters and/or enhancers. Expression of the coding sequence can be either constitutive or regulated.

[0103] The particular dosage regimen, e.g., dose, timing and repetition, used in the method described herein will depend on the particular subject and that subject's medical history. [0104] In some embodiments, more than one miRNA inhibitor (e.g., miR-224 inhibitor) and another suitable therapeutic agent, may be administered to a subject in need thereof. The miRNA inhibitor (e.g., miR-224 inhibitor) can also be used in conjunction with other agents that serve to enhance and/or complement the effectiveness of the agents.

[0105] In some embodiments, the subject is a patient and is under the care of (e.g., treatment or supervision) of a medical professional (e.g., doctor). In some embodiments, the subject is a patient. In some embodiments, the doctor is a medical doctor.

Viral Delivery

[0106] In an aspect, the disclosure relates to methods comprising administering (e.g., delivering) one or more nucleic acids (e.g., oligonucleotides, miRNA inhibitors), such as or one or more miRNA inhibitors (e.g., miR-224 inhibitors) as described herein to a subject in need thereof. Various means of delivering the agents of the disclosure the subjects, for example, by means of a vector encoding one or more components of agents (e.g., miRNA inhibitors) described herein. In some embodiments, the viral vectors are delivered to a host cell. In some embodiments, the disclosure further relates to cells produced by such methods, and organisms (such as animals, plants, or fungi) comprising or produced from such host cells. In some embodiments, an agent as described herein delivered to a host cell. Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids into mammalian cells or target tissues. Such methods can be used to administer nucleic acids encoding components of the agents disclosed herein (e.g., miRNA inhibitors) to cells in culture, or in a host organism. Non-viral vector delivery systems include DNA plasmids, RNA (e.g. a transcript of a vector described herein), naked nucleic acid, and nucleic acid complexed with a delivery vehicle, such as a liposome. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. For a review of gene therapy procedures, see Anderson, Science 256:808-813 (1992); Nabel & Feigner, TIB TECH 11:211-217 (1993); Mitani & Caskey,

TIB TECH 11:162-166 (1993); Dillon, TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992); Van Brunt, Biotechnology 6(10): 1149- 1154 (1988); Vigne, Restorative Neurology and Neuroscience 8:35-36 (1995); Kremer & Perricaudet, British Medical Bulletin 51(1):31-44 (1995); Haddada el al, in Current Topics in Microbiology and Immunology Doerfler and Bihm (eds) (1995); and Yu et al, Gene Therapy 1:13-26 (1994).

[0107] Methods of non- viral delivery of nucleic acids include lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipidmucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Lipofection is described in e.g., United States Patent: 5,049,386; 4,946,787; and 4,897,355, and lipofection reagents are sold commercially (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides (e.g., oligonucleotides) include those of Feigner: WO 91/17424 and WO 91/16024. Delivery can be to cells (e.g. in vitro or ex vivo administration) or target tissues (e.g. in vivo administration).

[0108] The preparation of lipidmucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to one of skill in the art (e.g., Crystal, Science 270:404-410 (1995); Blaese et al, Cancer Gene Ther. 2:291-297 (1995); Behr et al, Bioconjugate Chem. 5:382-389 (1994); Remy et al, Bioconjugate Chem. 5:647-654 (1994); Gao et al, Gene Therapy 2:710-722 (1995); Ahmad et al, Cancer Res. 52:4817-4820 (1992); United States Patent Numbers: 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

[0109] The use of RNA or DNA viral based systems for the delivery of nucleic acids take advantage of highly evolved processes for targeting a virus to specific cells in the body and trafficking the viral payload to the nucleus. Viral vectors can be administered directly to subjects (in vivo ) or they can be used to treat cells in vitro, and the modified cells may optionally be administered to subjects (ex vivo). Conventional viral based systems could include retroviral, lentivirus, adenoviral, adeno-associated, and herpes simplex virus vectors for gene transfer. Integration in the host genome is possible with the retrovirus, lentivirus, and adeno-associated virus (AAV) gene transfer methods, often resulting in long term expression of the inserted transgene. Additionally, high transduction efficiencies have been observed in many different cell types and target tissues.

[0110] The tropism of a viruses can be altered by incorporating foreign envelope proteins, expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors that are able to transduce or infect non-dividing cells and typically produce high viral titers. Selection of a retroviral gene transfer system would therefore depend on the target tissue. Retroviral vectors are comprised of cis-acting long terminal repeats (LTRs) with packaging capacity for up to 6-10 kb of foreign sequence. The minimum cis-acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the therapeutic gene into the target cell to provide permanent transgene expression. Widely used retroviral vectors include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immuno deficiency virus (SIV), human immuno deficiency vims (HIV), and combinations thereof (see, e.g., Buchscher et al, J. Virol. 66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992); Sommnerfelt et al., Virol. 176:58-59 (1990); Wilson etal, J. Virol. 63:2374-2378 (1989); Miller etal, J. Virol. 65:2220-2224 (1991); PCT/US94/05700). In applications where transient expression is preferred, adenoviral based systems may be used. Adenoviral based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. With such vectors, high titer and levels of expression have been obtained. This vector can be produced in large quantities in a relatively simple system. Adeno-associated vims (AAV) vectors may also be used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures (see, e.g., West et al, Virology 160:38-47 (1987); United States Patent Number 4,797,368; WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest.

94:1351 (1994). Constmction of recombinant AAV vectors are described in a number of publications, including: United States Patent Number 5,173,414; Tratschin et al, Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin, etal, Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:6466-6470 (1984); and Samulski et al, J. Virol. 63:03822-3828 (1989).

[0111] Packaging cells are typically used to form vims particles that are capable of infecting a host cell. Such cells include 293 cells, which package adenovims, and y2 cells or PA317 cells, which package retrovims. Viral vectors used in gene therapy are usually generated by producing a cell line that packages a nucleic acid vector into a viral particle. The vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host, other viral sequences being replaced by an expression cassette for the oligonucleotide(s) to be expressed. The missing viral functions are typically supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically only possess inverted terminal repeat (ITR) sequences from the AAV genome which are required for packaging and integration into the host genome. Viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences. The cell line may also be infected with adenovirus as a helper. The helper vims promotes replication of the AAV vector and expression of AAV genes from the helper plasmid. The helper plasmid is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by various means, for example heat treatment to which adenovirus is more sensitive than AAV. Additional methods for the delivery of nucleic acids to cells are known to those skilled in the art (see, e.g., US20030087817, incorporated herein by reference).

Compositions

[0112] In some embodiments, the agents disclosed herein (e.g., miR-224 inhibitors) may further comprise a pharmaceutically acceptable composition. In some embodiments, the agents disclosed herein (e.g., miR-224 inhibitors) can be formulated for administration to a subject as a pharmaceutically acceptable composition, which, as used herein, comprises the agents disclosed herein (e.g., miR-224 inhibitors) and another pharmaceutically acceptable carrier, diluent, or excipient). A carrier, diluent, or excipient that is “pharmaceutically acceptable” includes one that is sterile and pyrogen free. Suitable pharmaceutical carriers, diluents, and excipients are well known in the art. The carrier(s) must be “acceptable” in the sense of being compatible with the inhibitor and not deleterious to the recipients thereof. [0113] Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. Aqueous solutions may be suitably buffered (preferably to a pH of from about 3 to about 9). The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well-known to those skilled in the art.

[0114] Injectable compositions may contain various carriers such as vegetable oils, dimethylactamide, dimethyformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, and the like). For intravenous injection, water soluble antibodies can be administered by the drip method, whereby a pharmaceutical formulation containing the agents and a physiologically acceptable excipients is infused. Physiologically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, Ringer’s solution or other suitable excipients. Intramuscular preparations, e.g., a sterile formulation of a suitable soluble salt form of the agents, can be dissolved and administered in a pharmaceutical excipient such as Water-for- Injection, 0.9% saline, or 5% glucose solution.

[0115] Any of the agents disclosed herein (e.g., miR-224 inhibitors) may be administered by any administration route known in the art, such as parenteral administration, oral administration, buccal administration, sublingual administration (e.g., tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavoring or coloring agents, for immediate-, delayed- or controlled-release applications) topical administration, or inhalation, in the form of a pharmaceutical formulation (e.g., comprising a composition) comprising the active ingredient, optionally in the form of a non-toxic organic, or inorganic, acid, or base, addition salt, in a pharmaceutically acceptable dosage form. Suitable tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably com, potato or tapioca starch), sodium starch glycolate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and acacia. Solid compositions of a similar type may also be employed as fillers in gelatin capsules. Preferred excipients in this regard include lactose, starch, a cellulose, milk sugar or high molecular weight polyethylene glycols. For aqueous suspensions and/or elixirs, the compounds (e.g., miR-224 inhibitors) of the disclosure may be combined with various sweetening or flavoring agents, coloring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.

[0116] The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules or vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier immediately prior to use.

Kits

[0117] In an aspect, the disclosure relates to kits for administering one or more agents (e.g., miR-224 inhibitors) to a subject for the treatment of disorder related to miR-224 (e.g., pulmonary hypertension. In some embodiments, the indication ( e.g ., disease or disorder) is pulmonary hypertension. The representative kits include one or more dosage units comprising an effective amount of one or more agents described herein for administration to a subject, at a given frequency, and/or in a given manner (e.g., route of administration).

[0118] In some embodiments, the kits comprise one or more nucleic acid constructs encoding the various components of the miRNA inhibitors (e.g., miR-224 inhibitor) described herein.

In some embodiments, the nucleotide sequence comprises a heterologous promoter that drives expression of the miRNA inhibitors (e.g., miR-224 inhibitor).

[0119] In some embodiments, the kits provide cells comprising any of the agents (e.g., constructs, miRNA inhibitors) disclosed herein. In some embodiments, a host cell is transiently or non-transiently transfected with one or more vectors described herein. In some embodiments, a cell is transfected as it naturally occurs in a subject. In some embodiments, a host cell that is transfected is taken from a subject. In some embodiments, the host cell is derived from cells not taken from a subject, such as a cell line. A wide variety of cell lines for tissue culture are known in the art. Examples of cell lines include, but are not limited to, C8161, CCRF-CEM, MOLT, mIMCD-3, NHDF, HeLa-S3, Huhl, Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panel, PC-3, TF1, CTLL-2, C1R, Rat6, CV1, RPTE, A10, T24, J82, A375, ARH-77, Calul, SW480, SW620, SKOV3, SK-UT, CaCo2, P388D1, SEM-K2, WEHI-231, HB56, TIB55, Jurkat, J45.01, LRMB, Bcl-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E, MRC5, MEF, Hep G2, HeLa B, HeLa T4, COS, COS-1, COS- 6, COS-M6A, BS-C-1 monkey kidney epithelial, BALB/3T3 mouse embryo fibroblast, 3T3 Swiss, 3T3-L1, 132-d5 human fetal fibroblasts; 10.1 mouse fibroblasts, 293-T, 3T3, 721, 9L, A2780, A2780ADR, A2780cis, A 172, A20, A253, A431, A-549, ALC, B16, B35, BCP-1 cells, BEAS-2B, bEnd.3, BHK-21, BR 293. BxPC3. C3H-10T1/2, C6/36, Cal-27, CHO, CHO-7, CHO-IR, CHO-K1, CHO-K2, CHO-T, CHO Dhfr -/-, COR-L23, COR-L23/CPR, COR-L23/5010, COR-L23/R23, COS-7, COV-434, CML Tl, CMT, CT26, D17, DH82, DU145, DuCaP, EL4, EM2, EM3, EMT6/AR1, EMT6/AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, HEK-293, HeLa, Hepalclc7, HL-60, HMEC, HT-29, Jurkat, JY cells, K562 cells, Ku812, KCL22, KG1, KYOl, LNCap, Ma-Mel 1-48, MC-38, MCF-7, MCF-IOA, MDA-MB-231, MDA-MB-468, MDA-MB-435, MDCK II, MDCK 11, MOR/0.2R, MONO- MAC 6, MTD-1A, MyEnd, NCI-H69/CPR, NCI-H69/LX10, NCI-H69/LX20, NCI- H69/LX4, NIH-3T3, NALM-1, NW-145, OPCN/OPCT cell lines, Peer, PNT-1A/PNT 2, RenCa, RIN-5F, RMA/RMAS, Saos-2 cells, Sf-9, SkBr3, T2, T-47D, T84, THP1 cell line, U373, U87, U937, VCaP, Vero cells, WM39, WT-49, X63, YAC-1, YAR, and transgenic varieties thereof. Cell lines are available from a variety of sources known to those with skill in the art ( e.g ., the American Type Culture Collection (ATCC) (Manassus, Va.)). In some embodiments, a host cell transfected with one or more vectors described herein is used to establish a new cell line comprising one or more vector-derived sequences. In some embodiments, a cell transiently transfected with the components of the miRNA inhibitors (e.g., vectors encoding the miRNA inhibitor) of the disclosure (such as by transient transfection of one or more vectors, or transfection with RNA). In some embodiments, host cells transiently or non-transiently transfected with one or more vectors described herein, or cell lines derived from such cells are used in assessing one or more test compounds.

[0120] Instructions for performing the claimed methods and administering the agent may also be included in the kits described herein.

[0121] The kits may be organized to indicate a single formulation containing an agent described herein or combination of formulations, each containing an agent described herein. The composition may be sub-divided to contain appropriate quantities of an agent described herein. The unit dosage can be packaged compositions such as packeted (i.e., contained in a packet) powders, vials, ampoules, prefilled syringes, tablets, caplets, capsules, or sachets containing liquids.

[0122] The agents described herein may be a single dose or for continuous or periodic discontinuous administration. For continuous administration, a kit may include an agent described herein in each dosage unit. When varying concentrations of an agent described herein, the components of the composition containing the agent described herein, or relative ratios of the agent described herein or other agents within a composition over time is desired, a kit may contain a sequence of dosage units.

[0123] The kit may contain packaging or a container with an agent described herein formulated for the desired delivery route. The kit may also contain dosing instructions, an insert regarding the agent described herein, instructions for monitoring circulating levels of the agent, or combinations thereof. Materials for using the agent may further be included and include, without limitation, reagents, well plates, containers, markers, or labels, and the like. Such kits may be packaged in a manner suitable for treatment of a desired indication (e.g., disease or disorder).

[0124] Other suitable components to include in such kits will be readily apparent to one of skill in the art, taking into consideration the desired indication and the delivery route. The kits also may include, or be packaged with, instruments for assisting with the injection/administration of the agent to the subject. Such instruments include, without limitation, an inhalant, syringe, pipette, forceps, measuring spoon, eye dropper, or any such medically approved delivery means. Other instrumentation may include a device that permits reading or monitoring reactions in vitro.

[0125] The agent may be provided in dried, lyophilized, or liquid forms. When reagents or components are provided as a dried form, reconstitution generally is by the addition of a solvent. The solvent may be provided in another packaging means and may be selected by one skilled in the art.

[0126] A number of packages or kits are known to those skilled in the art for dispensing pharmaceutical agents. In certain embodiments, the package is a labeled blister package, dial dispenser package, or bottle.

EXAMPLES

[0127] Pulmonary hypertension (PH) is a disease characterized by progressive remodeling of the distal pulmonary arteries, resulting in the loss of vascular cross-sectional area and elevated pulmonary vascular resistance. Without intervention, PH is usually progressive, leading to right heart failure and death. The present invention provides a new therapeutic strategy to prevent and inhibit PH based on microRNA-224 inhibition. It was found that miR- 224 levels are significantly increased in patients with clinical PAH, and in different PAH animal models. In vitro , miR-224 overexpression induces an increase in human pulmonary artery smooth muscle cell (hPASMC) proliferation, whereas miR-224 inhibition decreases hPASMC proliferation. The overexpression of miR-224 in vivo exacerbates the PAH phenotype of mice exposed to Sugen/Hypoxia. The results indicate that miR-224 inhibition, using chemically modified oligonucleotides or AAVl-Tough decoy miRNA, reverses pulmonary vascular remodeling and Sugen/Hypoxia-induced PAH.

Example 1: miR-224 expression in the lung [0128] First, miR-224 expression profile across different organs was determined. The expression of miR-224 was quantitated by real-time qPCR in a spectrum of ten normal rat tissues that included specimens derived from kidney, lung, right ventricle, left ventricle, liver, brain, spleen, skeletal muscle, pancreas, and stomach. Interestingly, it was found that miR- 224 is mainly expressed in lung (FIG. 1A). [0129] A quantitative PCR analysis of miR-224 expression in human pulmonary artery smooth muscle cells (hPASMC), endothelial cells and lung fibroblasts revealed that miR-224 is predominantly expressed in hPASMCs (FIG. IB).

[0130] These preliminary results indicate that miR-224 is enriched in the lung and that its levels are significantly increased in patients with clinical PAH, and in different animal models of PH.

Example 2: miR-224 upregulation in PAH

[0131] Next, it was determined whether miR-224 is dysregulated in PAH. The expression levels of miR-224 in non-PAH subjects and patients with clinical PAH was investigated. miR-224 levels were determined in a cohort of five patients with idiopathic PAH (iPAH), five patients with heritable PAH (hPAH) and five controls (non-PAH). The expression of miR-224 increased in lungs of patients with clinical PAH (FIG. 2A), confirming the microarray data published by Zhao and colleagues.²⁶ The expression of miR-224 in lungs obtained from wild-type (WT) mice treated with the VEGF inhibitor Sugen and exposed to six weeks of chronic hypoxia (10% O2) was assessed. To evaluate whether miR-224 expression is altered during response to injury in these tissues, the expression of miR-224 in lungs from the hypoxic mice was compared to those obtained from normoxic (i.e., normal levels of oxygen in environment, tissue, or blood) controls. An analysis revealed a modest but significant upregulation of miR-224 in response to Sugen/Hypoxia (FIG. 2B). miR-224 expression profile was explored in another in vivo PAH animal model: Monocrotaline (MCT)-induced PAH in rats. The expression level of miR-224 in lungs extracted from rats six weeks after MCT injection was compared to lungs from control rats. In line with the human data, quantitative PCR analysis revealed a significant increase in miR-224 level in diseased rat lungs (FIG. 2C). In order to have a robust picture of miR-224 expression in PAH disease and keeping in mind the future translational aspect of the project, the next step was to quantify miR-224 expression in PH diseased pigs. A thoracotomy was performed on small (10 kg) domestic swine and the inferior pulmonary vein was banded (post-capillary PH model). As the animals grow, the pulmonary vein gets more stenotic and the pressure in the pulmonary vasculature increases.³¹ Four months after surgery, all pigs developed PH and right heart failure. In line with human and rodent data, it was found that miR-224 was upregulated in PH diseased pigs compared to sham operated animals (FIG. 2D). These preliminary results indicate that miR-224 levels are significantly increased in patients with clinical PAH, and in different animal models of PH. Example 3: miR-224 inhibition attenuates pulmonary artery smooth muscle cell proliferation

[0132] The effects of miR-224 overexpression and knockdown on smooth muscle cells proliferation was determined. Therefore, human Pulmonary Artery Smooth Muscle Cells (hPASMCs) were first transfected with miR-224 mimic (a synthetic miR-224 molecule, miR- 224, 50 nM) or microRNA control (a mimic with scrambled sequence, miR-Ctrl, 50 nM). Forty-eight hours later, cells were harvested, RNA was extracted and the levels of miR-224 were assessed by real-time quantitative PCR. miR-224 levels were found to be significantly increased after miR-224 transfection, showing the efficiency of miR-224 mimic (FIG. 3A). Serum (FBS: Fetal Bovine Serum) stimulation increased proliferation of hPASMCs by 3- fold, and overexpression of miR-224 using the synthetic mimic induced an additional significant increase in hPASMC proliferation, as assessed by a BrdU proliferation assay (FIG. 3B).

[0133] It was then assessed whether miR-224 inhibition could attenuate the proliferation of hPASMCs. In order to inhibit specifically miR-224, an inhibitor molecule designed to specifically inactivate endogenous miRNA-224 was used. hPASMCs was first transfected with Anti-miR-224 (50 nM) or a control molecule (Anti-miR-Ctrl, 50 nM) and performed a real-time quantitative PCR. Anti-miR-224 transfection significantly decreased miR-224 levels showing the efficiency of anti-miR-224 (FIG. 3C). Next, a proliferation assay was completed where it was found that inhibition of miR-224 confers an anti-proliferative phenotype (FIG. 3D). These results suggest that miR-224 overexpression increases hPASMC proliferation whereas miR-224 inhibition attenuates serum-induced hPASMC proliferation.

Example 4: miR-224 overexpression exacerbates Sugen/Hxpoxia-induced PEI [0134] It was assessed whether modulating miR-224 expression has any effect on pathological vascular remodeling. As an in vivo model, the Sugen/Hypoxia model in mice was used first, imitating moderate PH disease in humans. In this model, mice are subjected to chronic hypoxia combined with the VEGF receptor blocker Sugen (SU5416, SU) (FIG. 4A). The advantage of this model over the “classic” hypoxia model in mouse is that the mice develop more severe PAH. This murine model of PAH, established by Ciuclan and colleagues,³² displays many of the hallmarks of the human disease. Combination of SU5416 and exposure to three weeks of chronic hypoxia has been proven to cause PH with angioobliterative lesions in the pulmonary arterioles that are similar to the “plexiform” lesions found in human idiopathic PAH.^{32 33} It was first investigated whether increasing miR-224 levels in diseased hypoxic mice would exacerbate the PAH phenotype. In order to overexpress miR-224 in the lung, the previous approach was adopted for adeno-associated vims (AAV)-mediated expression of a microRNA.³⁴

[0135] Adeno-associated virus serotype 1 (AAV1) was used, because it shows good tropism for pulmonary vascular cells.^{35 38} Upon generation and intratracheal injection of AAVl-miR- 224 or an AAV1 control (2xlOⁿ genome copies per mouse) into PAH diseased mice, a 2-fold increase was observed in pulmonary miR-224 (compared to control, FIG. 4B), whereas right ventricular miR-224 remained unchanged (FIG. 4B). In AAV 1 -Ctrl-treated mice, hypoxia resulted in an increase in right ventricular (RV) weight, Fulton index, and RV systolic pressure (FIG. 4C-4D). Under Sugen/Hypoxia, AAVl-miR-224-treated mice showed enhanced myocardial hypertrophy and RV systolic pressure (FIG. 4C-4D). These results indicate that miR-224 exacerbates Sugen/Hypoxia-induced PH.

Example 5: miR-224 inhibition reverses Su en/Hvpoxia- induced PAH [0136] It was assessed whether in vivo Sugen/Hypoxia-induced upregulation of miR-224 contributed to the PAH phenotype and whether miR-224 inhibition could reverse this. In order to inhibit miR-224 in vivo, a chemically modified antisense oligonucleotide specific for miR-224 (LNA-224) was used. In a first approach, the efficiency of LNA-224 was validated in vitro by transfecting HEK293 cells with LNA-224 or LNA-Ctrl, and performing a real time PCR. Cells transfected with LNA-224 displayed a 90% decrease in miR-224 level, suggesting a high efficiency of LNA-224 in inhibiting miR-224 (FIG. 5A). In a second approach, the efficacy of different delivery methods was tested. Two different delivery routes were tested for the evaluation of LNA-224 delivery efficacy: the commonly used intraperitoneal injection (IP), as well as Intratracheal injection (ITT) using an intratracheal aerolizer. In addition, two different LNA-224 concentrations were tested: 10 mg/kg (this dose is commonly used for microRNAs inhibition in the lung) and 5 mg/kg. Three weeks after LNA delivery, mice were sacrificed, RNA was extracted from the lungs, and miR-224 expression was measured by reverse transcription polymerase PCR (RT-PCR). Mice injected intraperitoneally displayed 50%-70% decrease in pulmonary miR-224 levels, whereas mice injected intratracheally displayed 95% decrease in pulmonary miR-224 level (FIG. 5B). No significant difference was observed between the two different doses when LNA-224 was injected intratracheally (FIG. 5B). Thus, intratracheal injection seems to be the most efficient delivery method and 5 mg/kg appears to be the optimal concentration to use.

[0137] It was then determined whether miR-224 inhibition, using LNA-224 (5 mg/kg, FIG. 6A), is able to reverse Sugen/Hypoxia-induced PAH. Mice were maintained in hypoxia for three weeks with a weekly injection of SU5416 (20 mg/kg) and were then randomized to receive LNA-224 or LNA-Ctrl (5 mg/kg) for three weeks (FIG. 6B). Hemodynamics and morphometric measurements were performed three weeks after LNAs injection. A single injection of the chemically modified antisense oligonucleotide LNA-224 lead to a 95% reduction of pulmonary miR-224 in the Sugen/Hypoxia model (FIG. 6C). LNA-Ctrl-treated mice displayed all the hallmarks of PAH (i.e., increased RV weight, Fulton index, and RV systolic pressures) (FIG. 6D), whereas LNA-224-treated mice displayed a marked decrease in these parameters (FIG. 6D). Diseased mice treated with LNA-Ctrl developed cardiomyocyte hypertrophy, whereas LNA-224 treatment significantly reversed PAH-induced cardiomyocyte hypertrophy, as determined by histological analysis of cardiac tissue (FIG.

6E). In addition, LNA-224-treated mice showed a significant reduction in the percentage of medial thickness in pulmonary arteries (FIG. 6F). Together, these data demonstrate that miR- 224 inhibition, via LNA- 224 delivery, reverses chronic hypoxia-induced PH in mice.

[0138] The inhibition of miR-224 using chemically modified antisense oligonucleotide is a potent method but has the disadvantage of transient inhibition. To manipulate miR-224 specifically in the lung in vivo, and to pave the way for a translational application of miR- 224 inhibition in PAH, AAV 1 was used, which targets almost exclusively pulmonary vascular cells within the lung.^{35 38} In addition, the Tough Decoy (TuD) technique was used. TuD inhibitors are emerging as a highly effective method for microRNA inhibition due to their resistance to endonucleolytic degradation, high miRNA binding affinity, and efficient delivery. Proved to be superior to chemically modified oligonucleotides and Sponge Decoys, the TuD is the most effective method of miRNA inhibition.³⁹ When delivered through a viral vector, TuD confers the longest duration of miRNA suppression.⁴⁰ Using this method, miR- 224 TuD (TuD-224) and combined it with the AAV1 system to achieve long-term activity (FIG. 7A). After a successful in vitro testing, we assessed the therapeutic efficacy of AAV1- TuD-miR-224 in the mouse model of Sugen/Hypoxia. Mice were maintained in hypoxia for three weeks with a weekly injection of SU5416 (20 mg/kg) and were then randomized to intratracheally receive AAVl-TuD-224 or AAVl-Ctrl (2x1011 genome copies per mouse) for three weeks (FIG. 7B). Consistent with our findings in LNA-224 -treated mice, AAV1- TuD-224 protected from cardiac hypertrophy at the tissue and cellular level (FIGs. 7C-7D). Morphometric analysis of distal pulmonary arteries demonstrated a significant decrease in medial thickness of AAVl-TuD-224-treated animals (FIG. 7E). These results indicate that AAVl-TuD-224 reverses Sugen/Hypoxia-induced PAH.

Example 6: miR-224 targets several key signaling pathways in PH [0139] To elucidate the functional role of miR-224, it is critical to identify its direct targets and its mechanism of action. Multiple experimental approaches are now available to identify miRNA targets, each having its own advantages and disadvantages. Overexpression of miRNA by use of synthetic miRNA mimics followed by high-throughput analysis of change in gene expression by RN A- Sequencing (RNA-seq) can give a direct assessment of target genes. To this purpose we transfected hPASMCs with miR-224 or control (miR-Ctrl), extracted the RNA and performed RNA sequencing (FIG. 8A). RNA-seq data from miR-224 overexpression revealed that 1,568 mRNAs are significantly downregulated (cutoff of two fold decrease and p-value less than 0.05). To narrow down the list of potential miRNA targets, we next combined the results of multiple target prediction programs (Targetscan, miRDB and Pictar); the most likely targets are shared amongst all sets of results. By combining the RNA-seq data with the results of the prediction programs, 21 mRNAs were identified as potential miR-224 targets (FIG. 8B). Interestingly, the first disease identified as related to the 21 identified mRNAs, using DAVID 6.8 algorithm that is used for diseases and pathways analysis, was pulmonary hypertension (FIG. 8C). To identify the pathways associated with miR-224, we next performed an enrichment analysis of the 21 identified genes using the Reactome pathway database. Using this approach, we found several pathways to be significantly enriched (FDR < 0.05) with these 21 genes and they mainly relate to the TGF-b and BMP signaling pathways (FIG. 8D). Of note, using different pathways analysis databases, we also found TGF-b and BMP signaling pathways as the most related to the 21 identified genes. Targets of miR-224 within the TGF-b and BMP signaling pathways, that have miR-224 seed sequence in their 3'-UTR, are the Bone Morphogenetic Proteins (BMP- 11 and BMP- 14), the BMP Receptors type lb and 2 (BMPRlb and BMPR2), three Smad family members (Smad4, Smad5, and Smad8), and the DNA-binding protein inhibitors Idl and Id3 (FIG. 8E). To verify if the RNA Seq candidates are true miR-224 targets, six targets were selected for validation by real-time PCR. Messenger RNA (mRNA) was isolated from hPASMCs that had been transfected with miR-224 or miR-ctrl and subjected it to real-time PCR. The results show that the mRNA levels of all tested targets are reduced in miR-224-transfected cells (FIG. 8F).

[0140] In addition, of the 21 genes having genetic evidence of mutations associated with PH, 8 genes were found to be predicted targets of miR-224. Importantly, the RNA seq data showed decreased mRNA levels of these predicted targets upon miR-224 overexpression in hPASMCs. Thus, decreased levels of these genes upon miR-224 overexpression may contribute to the detrimental consequences of increased miR-224 levels. Therefore, it was investigated, by real-time quantitative PCR (qPCR), whether miR-224 regulates the expression of these genes. It was found the mRNA levels of all tested genes to be reduced in miR-224-transfected hPASMCs (FIG. 9).

Methods for Examples 1 -6 Cell Culture

[0141] Human pulmonary artery smooth muscle cells (PASMCs) were purchased from Lonza, Inc. (Allendale, NJ). PASMCs were cultured in SmBM medium supplemented with 5% fetal bovine serum (FBS) and SmGM-2 SingleQuots (Lonza). Cells were grown in 5% CO2 at 37°C and passaged at confluence.

Cell Proliferation

[0142] Proliferation of PASMCs was measured by 5-bromo-2'-deoxyuridine (BrdU) incorporation for 48 hours using the Cell Proliferation ELISA, BrdU (colorimetric) assay (Roche, Indianapolis, IN), according to the manufacturer’s instructions.

Sugen Hypoxia mouse studies

[0143] All animal experiments were approved by the Icahn School of Medicine at Mount Sinai institutional animal use and care committee and were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Ten-week old WT mice subcutaneously received 20 mg/kg of SU5416 and were maintained in hypoxia for three weeks. SU5416 was injected once a week during the next two weeks. Mice were then randomized to intratracheally receive, for three weeks, LNA-antimiR-Ctrl, LNA-antimiR- 224, LNA-Ctrl, or LNA-224. Hemodynamic measurements were performed three weeks later.

Hemodynamic Studies

[0144] Mice were anesthetized with 1% isoflurane, intubated via a tracheotomy, and mechanically ventilated. The abdominal cavity and diaphragm were opened, a Scisense catheter was inserted directly into the right ventricle, and an ultrasonic flow probe (flow probe 2.5S176; Transonic Systems Inc., Ithaca, NY) was placed in the right ventricle (RV). The right ventricular end-systolic and diastolic pressures were measured directly. Hemodynamic data were recorded by using a Scisense P-V Control Unit (Scisense, Ontario, Canada).

Lung tissue histology

[0145] Lungs were inflated with OCT/PBS (50/50) at a pressure of 20cm H2O injected through the myocardium prior to tissue harvest. The lungs were then frozen, embedded in optimal cutting temperature compound (OCT), sectioned, and 8 pm sections were fixed with ice-cold acetone. Sections were stained using hematoxylin and eosin and examined by light microscopy. Pulmonary arterioles located distal to terminal bronchioles were identified. The external diameter and the cross-sectional medial wall thickness were measured in 30 muscular arteries per animal ranging in size from less than 50 pm and up to 50 pm in external diameter. Fibrosis and collagen deposition was examined in lung tissue frozen sections (8pm) that were fixed in 1% paraformaldehyde and stained with Masson’s trichrome stain. Sections were visualized and collagen deposition was quantified using ImageJ software.

Right ventricular histology

[0146] Hearts were dissected immediately after sacrifice and weighed. The weight ratio of the right ventricle (RV) to the left ventricle (LV) plus septum [RV/(LV+S)] was calculated as an index of right ventricular hypertrophy (RVH). The RV sections were then fixed with ice- cold acetone, embedded in OCT, and hematoxylin and eosin staining was performed on 8 pm-thick sections that were subsequently examined using light microscopy. Fibrosis and collagen deposition was examined in frozen sections (8pm) that were fixed in 1% paraformaldehyde and stained with Masson’s trichrome stain. Sections were visualized and collagen deposition was quantified using ImageJ software. Cardiomyocyte cross-sectional area was measured using fluorescence-tagged wheat germ agglutinin (Life Technologies) that binds to saccharides of cellular membranes. Images of RV cardiomyocyte cell membranes were captured digitally and analyzed by image analysis using ImageJ software.

Quantification of miR-224

[0147] Total RNA was prepared using TriFast (peqLab™) and 10 ng were reverse- transcribed, using the Universal cDNA Synthesis Kit II (Qiagen™). The cDNAs were quantified using the FastStart universal SYBR Green Master Mix (Roche™), and modified primers for miR- 224 (miRCURY LNA PCR primer sets) or for U6 snRNA (Qiagen™) were used for qPCR quantification in a StepOnePlus Real-Time-PCR System (Applied Biosystems™), with parameters recommended by Exiqon™.

Example 7: MiR-224 inhibition reverses Su en/Hvpoxia- induced PH in rats [0148] To evaluate the in vivo effect of miR-224 inhibition in a more severe model of PH, the Sugen/Hypoxia rat model was used. Rats received a single injection of SU5416 (20 mg/kg) and were maintained in hypoxia for 3 weeks. The rats were then randomized at day 21 to receive LNA-224 or LNA-Ctrl (5 mg/kg) for 3 weeks (FIG. 10A). Hemodynamics and morphometric measurements were performed 3 weeks after LNAs injection. A single injection of the chemically modified antisense oligonucleotide LNA-224 lead to a 99% reduction of pulmonary miR-224 in the Sugen/Hypoxia model (FIG. 10B). LNA-Ctrl-treated rats displayed all the hallmarks of PH ( i.e ., increased RVSP, Fulton index, and pulmonary artery pressures) (FIGs. 10C-10D), whereas LNA-224-treated rats displayed a marked decrease in these parameters (FIGs. 10C-10D). Treatment with LNA-224 also reduced pulmonary arterial media wall thickness (FIG. 10E). These results indicate that LNA-224 reverses Sugen/Hypoxia -induced PH development.

Example 8: Pharmacological inhibition ofmiR-224 improves survival and reverses MCT- induced PAH in rats.

[0149] It was then determined whether miR-224 inhibition would reverse PH induced by MCT administration. Rats were randomly assigned to LNA-224 or LNA-2242 weeks after MCT treatment (FIG. 11A). Hemodynamics and morphometric measurements were performed 4 weeks after MCT injection. A single injection of the chemically modified antisense oligonucleotide LNA-224 lead to a 98% reduction of pulmonary miR-224 level (FIG. 1 IB). Kaplan-Meier survival curves demonstrated that MCT rats treated with LNA- 224 had a significantly higher survival rate than those treated with LNA-Ctrl (FIG. 11C). MCT administration resulted in a marked increase in RV hypertrophy, RVSP, and pulmonary artery pressures (FIGs. 1 ID-1 IF). LNA-224-treated rats displayed lower RVSP, Fulton index and pulmonary arterial pressures (FIGs. 11D-11F). In addition, morphometric analysis of distal pulmonary arteries demonstrated a significant decrease in medial thickness of LNA- 224-treated animals (FIG. 11G). These results indicate that miR-224 inhibition improves survival and protects against the development of MCT-induced PH.

Methods for Examples 7-8 Sugen/Hypoxia rat studies

[0150] All animal experiments were approved by the Icahn School of Medicine at Mount Sinai institutional animal use and care committee and were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Sprague Dawley rats subcutaneously received 20 mg/kg of SU5416 and were maintained in hypoxia for 3 weeks. Rats were then randomized at day 21 to intratracheally receive for LNA-Ctrl or LNA-224 (5 mg/kg each). Hemodynamic and morphometric analyses were performed 3 weeks later. Monocrotaline Rat Studies

[0151] All animal experiments were approved by the Icahn School of Medicine at Mount Sinai institutional animal use and care committee and were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Adult male Sprague- Dawley rats (Charles River), weighing 150 to 200 g, received a single injection of MCT (60 mg/kg). Two weeks after MCT injection, rats were randomly assigned to receive LNA-Ctrl or LNA-224 (5 mg/kg each) for 2 weeks. Hemodynamics and morphometric measurements were performed 4 weeks after MCT injection.

Right Ventricle and Pulmonary artery Hemodynamic Studies

[0152] Animals were anaesthetized with 3-4% isoflurane, intubated via a tracheotomy, and mechanically ventilated. Next, the thoracic cavity was opened and a catheter (Transonic Systems Inc.) was inserted directly into the right ventricle or into the pulmonary artery. The pulmonary artery systolic pressure, pulmonary artery diastolic pressure, right ventricular end- systolic and diastolic pressures were measured directly. Hemodynamic data were recorded using an ADVantage P-V Control Unit (Transonic Systems).

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Exemplary Sequences

[0198] This Table ( i.e ., Table 1) exhibits some exemplary sequences as disclosed by the instant Specification, but is not limiting. This Specification includes a Sequence Listing submitted concurrently herewith as a text file in ASCII format. The Sequence Listing and all of the information contained therein are expressly incorporated herein and constitute part of the instant Specification as filed.

Table 1: Exemplary Sequences

* Unless otherwise specified, nucleic acid sequences are described 5' to 3' and amino acid sequences are described N-terminus to C-terminus ** ‘NT’ denotes a nucleic acid sequence; ‘AA’ denotes an amino acid sequence

Other Embodiments

[0199] Embodiment 1. A method of treating, preventing, or reversing pulmonary hypertension in subject in need thereof, comprising administering to the subject an effective amount of a miR-224 inhibitor.

[0200] Embodiment 2. The method of embodiment 1, wherein the miR-224 inhibitor comprises an oligonucleotide that has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence which is fully complementary to SEQ ID NO: 1.

[0201] Embodiment 3. The method of embodiment 2, wherein the oligonucleotide is at least 15 nucleotides, at least 15 +1 nucleotides, at least 15 + 2 nucleotides, at least 15 + 3 nucleotides, at least 15 + 4 nucleotides, at least 15 + 5 nucleotides, at least 15 + 6 nucleotides, at least 15 + 7 nucleotides.

[0202] Embodiment 4. The method of embodiment 2, wherein the oligonucleotide is an antagomir, antisense molecule, small hairpin RNA molecule, small interfering RNA molecule, microRNA sponge, tiny seed-targeting locked nucleic acid (LNA), or an RNA decoy.

[0203] Embodiment 5. The method of embodiment 4, wherein the RNA decoy is a tough decoy (TuD). [0204] Embodiment 6. The method of embodiment 2, wherein the oligonucleotide is TuD- 224 (SEQ ID NO: 3) or LNA-224 (SEQ ID NO: 4).

[0205] Embodiment 7. The method of embodiment 2, wherein the oligonucleotide comprises a nucleic acid modification to enhance stability.

[0206] Embodiment 8. The method of embodiment 7, wherein the nucleic acid modification is a 2'-0-methoxyethyl sugar, a 2'-fluoro sugar modification, a 2 '-O-methyl sugar, a bicyclic sugar moiety, a cholesterol, or a phosphorothioate.

[0207] Embodiment 9. The method of embodiment 1, wherein the subject in need thereof comprises a mutation in the Bone Morphogenetic Protein Receptor 2 (BMPR2) gene.

[0208] Embodiment 10. The method of embodiment 1, wherein the administering of the miR-224 inhibitor results in a decrease in human pulmonary artery smooth muscle cells (hPASMC) proliferation.

[0209] Embodiment 11. The method of embodiment 1, wherein the miR-224 inhibitor is one which reverses Sugen/Hypoxia-induced pulmonary hypertension in a mouse model.

[0210] Embodiment 12. A method of treating, preventing, or reversing pulmonary hypertension in a subject in need thereof, comprising administering to the subject an effective amount of an miRNA inhibitor comprising a nucleic acid sequence that has at least 90% sequence identity to a sequence which is fully complementary to an miR-224 sequence. [0211] Embodiment 13. A method of treating, preventing, or reversing pulmonary hypertension in a subject having a BMPR2 mutation, comprising: (a) obtaining a genetic test result on a subject sample to confirm the presence of a BMPR2 mutation; and (b) administering to the subject an effective amount of a miR-224 inhibitor.

[0212] Embodiment 14. The method of embodiment 13, wherein the genetic test result is obtained by a PCR-based method, a sequencing-based method, or a microarray-based method.

[0213] Embodiment 15. The method of embodiment 13, wherein the miR-224 inhibitor is an oligonucleotide that has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence which is fully complementary to SEQ ID NO: 1.

[0214] Embodiment 16. The method of embodiment 15, wherein the oligonucleotide is at least 15 nucleotides, at least 15 +1 nucleotides, at least 15 + 2 nucleotides, at least 15 + 3 nucleotides, or at least 15 + 4 nucleotides, at least 15 + 5 nucleotides, at least 15 + 6 nucleotides, at least 15 + 7 nucleotides.

[0215] Embodiment 17. The method of embodiment 15, wherein the oligonucleotide is an antagomir, antisense molecule, small hairpin RNA molecule, small interfering RNA molecule, microRNA sponge, tiny seed-targeting locked nucleic acid (LNA) oligonucleotide, or an RNA decoy.

[0216] Embodiment 18. The method of embodiment 17, wherein the RNA decoy is a tough RNA decoy (TuD).

[0217] Embodiment 19. The method of embodiment 15, wherein the oligonucleotide is TuD- 224 (SEQ ID NO: 3) or LNA-224 (SEQ ID NO: 4).

[0218] Embodiment 20. The method of embodiment 15, wherein the oligonucleotide comprises a nucleic acid modification to enhance stability.

[0219] Embodiment 21. The method of embodiment 20, wherein the nucleic acid modification is a 2'-0-methoxyethyl sugar, a 2'-fluoro sugar modification, a 2 '-O-methyl sugar, a bicyclic sugar moiety, a cholesterol, or a phosphorothioate.

[0220] Embodiment 22. A method for treating, preventing, or reversing pulmonary hypertension in a subject comprising: (a) identifying a subject suitable for treatment, wherein a suitable subject is one who has an increased level of miR-224 in a biological sample; (b) optionally obtaining a genetic test result on the biological sample to confirm the presence of a BMPR2 mutation; and (c) administering an effective amount of an miR-224 inhibitor to a subject having an increased level of miR-224, and optionally a BMPR2 mutation.

[0221] Embodiment 23. The method of embodiment 22, wherein the genetic test result is obtained by a PCR-based method, a sequencing-based method, or a microarray-based method.

[0222] Embodiment 24. The method of embodiment 22, wherein the miR-224 inhibitor is an oligonucleotide that has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence which is fully complementary to SEQ ID NO: 1.

[0223] Embodiment 25. The method of embodiment 24, wherein the oligonucleotide is at least 15 nucleotides, at least 15 +1 nucleotides, at least 15 + 2 nucleotides, at least 15 + 3 nucleotides, or at least 15 + 4 nucleotides, at least 15 + 5 nucleotides, at least 15 + 6 nucleotides, at least 15 + 7 nucleotides.

[0224] Embodiment 26. The method of embodiment 22, wherein the oligonucleotide is an antagomir, antisense molecule, small hairpin RNA molecule, small interfering RNA molecule, microRNA sponge, tiny seed-targeting locked nucleic acid (LNA) oligonucleotide, or RNA decoy.

[0225] Embodiment 27. The method of embodiment 26, wherein the RNA decoy is a tough decoy (TuD). [0226] Embodiment 28. The method of embodiment 22, wherein the oligonucleotide is TuD- 224 (SEQ ID NO: 3) or LNA-224 (SEQ ID NO: 4).

[0227] Embodiment 29. The method of embodiment 15, wherein the oligonucleotide comprises a nucleic acid modification to enhance stability.

[0228] Embodiment 30. The method of embodiment 29, wherein the nucleic acid modification is a 2'-0-methoxyethyl sugar, a 2'-fluoro sugar modification, a 2 '-O-methyl sugar, a bicyclic sugar moiety, a cholesterol, or a phosphorothioate.

[0229] Embodiment 31. A pharmaceutical composition comprising an miR-224 inhibitor and a pharmaceutically acceptable carrier.

[0230] Embodiment 32. A kit comprising the pharmaceutical composition of embodiment 31, and an oligonucleotide capable of being used to detect a BMPR2 mutation.

[0231] In addition to the embodiments expressly described herein, it is to be understood that all of the features disclosed in this disclosure may be combined in any combination ( e.g ., permutation, combination). Each element disclosed in the disclosure may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

[0232] From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, and can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the claims. Equivalents and Scope

[0233] In the claims articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The disclosure includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0234] Furthermore, the disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists ( e.g ., in Markush group format), each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the disclosure, or aspects of the disclosure, is/are referred to as comprising particular elements and/or features, certain embodiments of the disclosure or aspects of the disclosure consist, or consist essentially of, such elements and/or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein. It is also noted that the terms “comprising” and “containing” are intended to be open and permits the inclusion of additional elements or steps. Where ranges are given, endpoints are included in such ranges unless otherwise specified. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0235] This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the disclosure that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the disclosure can be excluded from any claim, for any reason, whether or not related to the existence of prior art.

[0236] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the disclosure, as defined in the following claims.

Claims

CLAIMS What is claimed is:

1. A method of treating, preventing, or reversing pulmonary hypertension in subject in need thereof, comprising administering to the subject an effective amount of a miR-224 inhibitor.

2. The method of claim 1, wherein the miR-224 inhibitor comprises an oligonucleotide that has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence which is fully complementary to SEQ ID NO: 1.

3. The method of claim 2, wherein the oligonucleotide is at least 15 nucleotides, at least 15 +1 nucleotides, at least 15 + 2 nucleotides, at least 15 + 3 nucleotides, at least 15 + 4 nucleotides, at least 15 + 5 nucleotides, at least 15 + 6 nucleotides, at least 15 + 7 nucleotides.

4. The method of claim 2, wherein the oligonucleotide is an antagomir, antisense molecule, small hairpin RNA molecule, small interfering RNA molecule, microRNA sponge, tiny seed-targeting locked nucleic acid (LNA), or an RNA decoy.

5. The method of claim 4, wherein the RNA decoy is a tough decoy (TuD).

6. The method of claim 2, wherein the oligonucleotide is TuD-224 (SEQ ID NO: 3) or LNA-224 (SEQ ID NO: 4).

7. The method of claim 2, wherein the oligonucleotide comprises a nucleic acid modification to enhance stability.

8. The method of claim 7, wherein the nucleic acid modification is a 2'-0-methoxyethyl sugar, a 2'-fluoro sugar modification, a 2 '-O-methyl sugar, a bicyclic sugar moiety, a cholesterol, or a phosphorothioate.

9. The method of claim 1, wherein the subject in need thereof comprises a mutation in the Bone Morphogenetic Protein Receptor 2 (BMPR2) gene.

10. The method of claim 1, wherein the administering of the miR-224 inhibitor results in a decrease in human pulmonary artery smooth muscle cells (hPASMC) proliferation.

11. The method of claim 1, wherein the miR-224 inhibitor is one which reverses Sugen/Hypoxia-induced pulmonary hypertension in a mouse model.

12. A method of treating, preventing, or reversing pulmonary hypertension in a subject in need thereof, comprising administering to the subject an effective amount of an miRNA inhibitor comprising a nucleic acid sequence that has at least 90% sequence identity to a sequence which is fully complementary to an miR-224 sequence.

13. A method of treating, preventing, or reversing pulmonary hypertension in a subject having a BMPR2 mutation, comprising:

(a) obtaining a genetic test result on a subject sample to confirm the presence of a BMPR2 mutation; and

(b) administering to the subject an effective amount of a miR-224 inhibitor.

14. The method of claim 13, wherein the genetic test result is obtained by a PCR-based method, a sequencing-based method, or a microarray-based method.

15. The method of claim 13, wherein the miR-224 inhibitor is an oligonucleotide that has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence which is fully complementary to SEQ ID NO: 1.

16. The method of claim 15, wherein the oligonucleotide is at least 15 nucleotides, at least 15 +1 nucleotides, at least 15 + 2 nucleotides, at least 15 + 3 nucleotides, or at least 15 + 4 nucleotides, at least 15 + 5 nucleotides, at least 15 + 6 nucleotides, at least 15 + 7 nucleotides.

17. The method of claim 15, wherein the oligonucleotide is an antagomir, antisense molecule, small hairpin RNA molecule, small interfering RNA molecule, microRNA sponge, tiny seed-targeting locked nucleic acid (LNA) oligonucleotide, or an RNA decoy.

18. The method of claim 17, wherein the RNA decoy is a tough RNA decoy (TuD).

19. The method of claim 15, wherein the oligonucleotide is TuD-224 (SEQ ID NO: 3) or LNA-224 (SEQ ID NO: 4).

20. The method of claim 15, wherein the oligonucleotide comprises a nucleic acid modification to enhance stability.

21. The method of claim 20, wherein the nucleic acid modification is a 2'-0- methoxyethyl sugar, a 2'-fluoro sugar modification, a 2 '-O-methyl sugar, a bicyclic sugar moiety, a cholesterol, or a phosphorothioate.

22. A method for treating, preventing, or reversing pulmonary hypertension in a subject comprising:

(a) identifying a subject suitable for treatment, wherein a suitable subject is one who has an increased level of miR-224 in a biological sample;

(b) optionally obtaining a genetic test result on the biological sample to confirm the presence of a BMPR2 mutation; and

(c) administering an effective amount of an miR-224 inhibitor to a subject having an increased level of miR-224, and optionally a BMPR2 mutation.

23. The method of claim 22, wherein the genetic test result is obtained by a PCR-based method, a sequencing-based method, or a microarray-based method.

24. The method of claim 22, wherein the miR-224 inhibitor is an oligonucleotide that has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence which is fully complementary to SEQ ID NO: 1.

25. The method of claim 24, wherein the oligonucleotide is at least 15 nucleotides, at least 15 +1 nucleotides, at least 15 + 2 nucleotides, at least 15 + 3 nucleotides, or at least 15 + 4 nucleotides, at least 15 + 5 nucleotides, at least 15 + 6 nucleotides, at least 15 + 7 nucleotides.

26. The method of claim 22, wherein the oligonucleotide is an antagomir, antisense molecule, small hairpin RNA molecule, small interfering RNA molecule, microRNA sponge, tiny seed-targeting locked nucleic acid (LNA) oligonucleotide, or RNA decoy.

27. The method of claim 26, wherein the RNA decoy is a tough decoy (TuD).

28. The method of claim 22, wherein the oligonucleotide is TuD-224 (SEQ ID NO: 3) or LNA-224 (SEQ ID NO: 4).

29. The method of claim 15, wherein the oligonucleotide comprises a nucleic acid modification to enhance stability.

30. The method of claim 29, wherein the nucleic acid modification is a 2'-0- methoxyethyl sugar, a 2'-fluoro sugar modification, a 2 '-O-methyl sugar, a bicyclic sugar moiety, a cholesterol, or a phosphorothioate.

31. A pharmaceutical composition comprising an miR-224 inhibitor and a pharmaceutically acceptable carrier.

32. A kit comprising the pharmaceutical composition of claim 31, and an oligonucleotide capable of being used to detect a BMPR2 mutation.