WO2018183127A1

WO2018183127A1 - Mir-92 inhibitors for treatment of heart failure

Info

Publication number: WO2018183127A1
Application number: PCT/US2018/024167
Authority: WO
Inventors: Rabea Hinkel; Christian Kupatt; Stefanie Dimmeler
Original assignee: MiRagen Therapeutics, Inc.
Priority date: 2017-03-25
Filing date: 2018-03-23
Publication date: 2018-10-04
Also published as: EP3601565A1; EP3601565A4

Abstract

The present invention provides oligonucleotide inhibitors of miR-92 and methods of using said inhibitors for the treatment of heart failure.

Description

miR-92 INHIBITORS FOR TREATMENT OF HEART FAILURE

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. §1 19(e) of U.S. Provisional Patent Application No, 62/476,780, filed March 25, 2017, and U.S. Provisional Patent Application No. 62/477,385, filed March 27, 2017. The contents these applications are incorporated herein by reference in their entirety.

BACKGROUND

[0002] Heart failure can be classified based on heart function or which side of the heart is most affected. Left-sided heart failure describes a failure of the left-sided pumping action by which oxygen-rich blood from the lungs is moved through the left atrium into the left ventricle and then out into the rest of the body. Systolic failure and diastolic failure are two types of left-sided heart failure. The term right-sided heart failure is used for a failure of the right-sided pumping action which pumps blood that returns to the heart through the veins through the right atrium into the right ventricle and then back out into the lungs to have it replenished with oxygen. The sy mptoms of heart failure are further distinguished whether they have developed quickly (acute heart failure) or gradually over time (chronic heart failure). Congestive heart failure (CHF) describes the general condition in which the heart cannot pump enough blood to meet the needs of the body.

[0003] Heart failure with preserved ejection fraction (HFpEF) is a form of heart failure where the amount of blood pumped from the heart's left ventricle with each beat (ejection fraction) remains greater than 50%. Approximately half of people with heart failure have HFpEF, while the remainder display a reduction in ejection fraction, or heart failure with reduced ejection fraction (HFrEF). (Owan et al, (2006); The New England Journal of Medicine. 355 (3): 251-59).

[0004] MicroRNAs (miRNAs) are a class of small, endogenous and non-coding RNAs able to regulate posttranscriptional expression of target genes. MicroRNAs have been implicated in a number of bi ological processes including regulation and maintenance of cardiac function, vascular inflammation and development of vascular pathologies. In particular, micro-RNA 92 (miR-92) has been implicated as a therapeutic target in the treatment of cardi ovascular pathologies. Accordingly, modulating the function and/or activity of miR-92 may present as a therapeutic target in the development of effective treatments for particular types of heart failure and associated symptoms. [0005] There is a need for microRNA-based treatments of chronic heart failure where the chronic heart failure presents with either preserved or reduced ejection fraction volumes. The compositions and methods described herein address this need,

SUMMARY

[0006] The present invention provides oligonucleotide inhibitors of miR-92 and methods of using said inhibitors for the treatment of heart failure,

[0007] In one aspect provided herein is a method of treating heart failure in a subject comprising administering to the subject an oligonucleotide comprising a sequence that is at least partially complementary to a miR-92 inhibitor (e.g. SEQ ID NOs: 7 to 164 disclosed in Table 2), wherein the administration of the oligonucleotide reduces function or activity of miR-92, and wherein the administration of the oligonucleotide results in an improvement of left ventricular function. In some embodiments, the oligonucleotide comprises a sequence identical to SEQ ID NOs: 7-9. In some embodiments, the oligonucleotide is identical to SEQ ID NOs: 7-9.

[0008] In an aspect, there is provided an oligonucleotide comprising a sequence that is at least partially complementary to a miR-92 inhibitor (e.g. SEQ ID NOs: 7 to 164 disclosed in Table 2), for use in a method of treating heart failure.

[0009] In some embodiments, administration of the oligonucleotide results in an improvement of ejection fraction. In some embodiments, administration of the oligonucleotide results in a decrease of left ventricular end-diastoiic pressure (LVEDP). In some embodiments, the heart failure is characterized as heart failure with reduced ejection fraction (HFrEF). In some embodiments, the s bject has decreased left ventricular function having an ejection fraction that is less than 40%, less than 35%, less than 30%, less than 25%, or less than 20%.

[0010] In another aspect provided herein, is a method of treating heart failure in a subject comprising administering to the subject an oligonucleotide comprising a sequence that is at least partially complementary to miR-92, wherein the administration of the oligonucleotide reduces function or activity of miR-92, and wherein the subject has left ventricular function having an ejection fraction that is at least 40%. In some embodiments, the ejection fraction is at least 50%. In some embodiments, the heart failure is characterized as heart failure with preserved ejection fraction (HFpEF). [0011] In any of the embodiments provided herein, the heart failure is chronic heart failure. In any of the embodiments provided herein, the subject has ischemic cardiomyopathy. In any of the embodiments provided herein, the subject has chronic myocardial ischemia. In any of the embodiments provided herein, administration of the oligonucleotide results in enhanced capillar - density in the ischemic area. In any of the embodiments provided herein, administration of the oligonucleotide results in enhanced pericyte coverage in the ischemic area. In any of the embodiments provided herein, administration of the oligonucleotide results in reduced infarct size, in any of the embodiments provided herein, administration of the oligonucleotide results in improved coronary reserve. In any of the embodiments provided herein, administration of the oligonucleotide results in improved functional cardiac reserve. In any of the embodiments provided herein, administration of the oligonucleotide results in reduced myocardial fibrosis. In any of the embodiments provided herein, administration of the oligonucleotide results in reduced myocyte hypertrophy. In any of the embodiments provided herein, administration of the oligonucleotide results in enhanced neovascularization. In any of the embodiments provided herein, administration of the oligonucleotide results in prolonged cardioprotection. In any of the embodiments provided herein, administration of the oligonucleotide results in reduced endothelial ceil death. In any of the embodiments provided herein, administration of the oligonucleotide results in reduced inflammation. In any of the embodiments provided herein, administration of the oligonucleotide results in improved collateral growth. In any of the embodiments provided herein, administration of the oligonucleotide results in improved global myocardial function. In any of the embodiments provided herein, the improved global myocardial function results in a lower LVEDP. In any of the embodiments provided herein, the improved global myocardial function results in a higher ejection fraction. In any of the embodiments provided herein, the improved global myocardial function results in increased contraction velocity. In any of the embodiments provided herein, the improved global myocardial function results in improved diastolic function. In any of the embodiments provided herein, the improved global myocardial function results in improved regional myocardial function. In any of the embodiments provided herein, the subject is undergoing pressure-induced cardiac remodeling (pathologic hypertrophy). In any of the embodiments provided herein, administration of the oligonucleotide results in decreased heart weight to body weight ratio. In any of the embodiments provided herein, administration of the oligonucleotide results in decreased fibrosis. In any of the embodiments provided herein, the subject has non-ischemic cardiomyopathy. In any of the embodiments provided herein, the subject is a diabetic subject. In any of the embodiments provided herein, the subject is a non-diabetic subject.

[0012] In any of the embodiments provided herein, the miR-92 inhibitor is selected from the oligonucleotides of Table 2, in any of the embodiments provided herein, the oligonucleotide comprises at least one locked nucleic acid (LNA) containing a 2' to 4' methylene bridge. In any of the embodiments provided herein, the oligonucleotide comprising a sequence that is at least partially complementary to miR-92 comprises a sequence of at least 16 nucleotides, wherein the sequence comprises no more than three contiguous LNAs, wherein from the 5' end to the 3' end, positions 1 , 6, 10, 11, 13 and 16 of the sequence are LNAs; and optionally further comprises LNAs at positions 3, 9, and 14 or optionally further comprises LNAs at positions 3, 8, and 14; or optionally further comprises LNAs at positions 5, 8, and 15. In any of the embodiments provided herein, from the 5' end to the 3' end, the sequence further comprises a deoxyribonucleic acid (DNA) nucleotide at the second nucleotide position. In any of the embodiments provided herein, the DNA nucleotide at the second nucleotide position contains a chemically modified nitrogenous base. In any of the embodiments provided herein, the chemically modified nitrogenous base is 5- methylcytosine. In any of the embodiments provided herein, the oligonucleotide comprises at least one nucleotide that is 2'-deoxy, 2' O-alkyl or 2' halo modified. In any of the embodiments provided herein, the oligonucleotide has a 5' cap structure, 3' cap structure, or 5' and 3' cap structure. In any of the embodiments provided herein, the oligonucleotide comprises one or more phosphorothioate linkages. In any of the embodiments provided herein, the oligonucleotide is fully phosphorothioate-linked. In any of the embodiments provided herein, the oligonucleotide further comprises a pendent lipophilic group.

[0013] In any of the embodiments provided herein, the subject is a human.

[0014] In any of the embodiments provided herein, administration of the oligonucleotide is performed by intravenous administration, subcutaneous administration, intracardiac administration, or mtracoronary administration (either retrograde or anterograde).

[0015] In any of the embodiments provided herein, the dose by weight of the subject of the oligonucleotide is about 0.01 mg/kg, 0.015 mg/kg, 0.02 mg/kg, 0.03 mg/kg, 0.04 mg/kg, 0.05 mg/kg, 0.075 mg/kg, 0. 1 mg/kg, 0.15 mg/kg, 0.2 mg/kg, 0,3 mg/kg, 0.4mg/kg, 0.5 mg/kg, 1 mg/kg, or 1 .5 mg/kg. In any of the embodiments provided herein, the dose of the oligonucleotide is about 0.5 mg, 0.75 nig, 1 mg, 1.5 mg, 2 mg, 2.5 mg, 3.75 mg, 5 mg, 7,5 mg, 10 mg, 15 nig, 20 nig, 25 mg, 50 mg, or 75 mg.

[0016] In any of the embodiments provided herein, administration is performed intracoronarily one, two, or three times per year; or intravenously about once every week, every month, every quarter, every half-year, or ever year.

[0017] In any of the embodiments provided herein, the administration is repeated until an improvement of ejection fraction is observed.

[0018] In any of the embodiments provided herein, the subject suffers from hypertension, cardiac hypertrophy, myocardial infarction, coronary artery disease, cardiomyopathy, high blood pressure, aortic stenosis, or myocarditis.

BRIEF DESCRIPTIO OF THE DRAWINGS

[0019] FIG. 1 illustrates that miR-92a inhibition improves myocardial function.

[0020] FIG. 2 illustrates that miR-92a inhibition improves myocardial function. The treatment was applied retrograde to blood flow ("retro"), anterograde to blood flow at the same dosage ("ante"), or anterograde to blood flow at a higher dosage ("high ante").

[0021] FIG. 3 illustrates that miR-92a inhibition improves vascularization.

[0022] FIG. 4 illustrates prolonged cardioprotection after miR-92a inhibition.

[0023] FIG. 5 illustrates the utilized model of chronic myocardial ischemia in a pig.

[0024] FIG. 6 illustrates that miR-92a inhibition improves global myocardial function in chronic myocardial ischemia.

[0025] FIG. 7 illustrates that miR-92a inhibition improves regional myocardial function in chronic myocardial ischemia.

[0026] FIG. 8 illustrates that miR~92a inhibition increases angiogenesis in chronic myocardial ischemia,

[0027] FIG. 9 illustrates that miR-92a inhibition increases collateral growth in chronic myocardial ischemia.

[0028] FIG. 10 illustrates that miR-92a inhibition reduces myocardial fibrosis in chronic myocardial ischemia.

[0029] FIG. 11 illustrates that miR-92a inhibition reduces myocyte hypertrophy in chronic myocardial ischemia. [0030] FIG. 12 illustrates the utilized in vivo model of ischemia and reperfusion in diabetic pigs.

[0031] FIG. 13 illustrates the effect of miR-92a inhibition on infarct size in diabetic pigs following ischemia and reperfusion.

[0032] FIG. 14 illustrates the effect of miR-92a inhibition on global myocardial function in diabetic pigs following ischemia and reperfusion.

DETAILED DESCRIPTION

[0033] The present invention provides oligonucleotide inhibitors that inhibit the activity or function of miR-92 for use in the treatment of chronic ischemia, and treatment of heart failure. The heart failure can be characterized as either heart failure with reduced ejection fraction (HFrEF) or heart failure with preserved ejection fraction (HFpEF).

[0034] The term "about" as used herein is meant to encompass variations of +/- 10% and more preferably +/- 5%, as such variations are appropriate for practicing the present invention.

MicroRNA- (miR-92) Target

[0035] miR-92 is located in the miR- 17-92 cluster, which consists of miR-17-5p, miR-! 7-3p, miR-18a, miR-! 9a, miR-20a, miR-! 9b, and miR-92- 1 (Venturini et al, Blood 109 10:4399-4405

(2007)). The pre-miRNA sequence for miR-92 is processed into a mature sequence (3p) and a star

(i.e. minor or 5p) sequence. The star sequence is processed from the other arm of the stem loop structure. The mature and star miRNA sequences for human, mouse, and rat miR-92 are provided in Table 1.

Table 1: miR-92 Sequences

Human mature miR-92 (i.e. hsa-miR-92a-3p) (S EQ ID NO: 1) j

5 ' - UAUUGC ACUUGUCCCGGCCUGU-3 '

Human miR-92a-l* (i.e. hsa-miR-92a-l-5p) (SE Q ID NO: 2) j

5 ' - AGGUUGGGAUCGGUUGC AAUGCU-3 '

Human miR-92a-2* (i.e. hsa-miR-92a-2-5p) (SE Q ID NO: 3) j

5 ' -GGGUGGGGAUUUGUUGCAUUAC-3 '

Mouse mature miR-92 (i.e. mmu-miR-92a-3p) ( SEQ ID NO: 4) j

5 ' -UAUUGCACUUGUCCCGGCCUG-3 ' Mouse miR-92a-l * (i.e. mmu-miR-92a-l-5p) (SEQ ID NO: 5)

5 - AGGUUGGGAUUUGUCGCAAUGCU-3 "

Mouse nnR-92a-2* (i.e. mmu-miR-92a-2-5p) (SEQ ID NO: 6)

5 ' - AGGUGGGGAUUGGUGGC AUUAC-3 '

Rat mature miR-92 (i.e. mo-miR-92a-3p) (SEQ ID NO: 4)

5 ' -UAUUGC ACUUGUCCCGGCCUG-3 '

Rat miR-92a-l* (i.e. rno-miR-92a-l-5p) (SEQ ID NO: 5)

5 ' -AGGUUGGGAUUUGUCGCAAUGCU-3 '

Rat miR-92a-2* (i.e. rno-miR-92a-2-5p) (SEQ ID NO: 6)

5 ' -AGGUGGGGAUUAGUGCC AUUAC-3 '

[0036] The above sequences can be either ribonucleic acid sequences or deoxyribonucleic acid sequences or a combination of the two (i.e. a nucleic acid comprising both ribonucleotides and deoxyribonucleotides). It is understood that a nucleic acid comprising any one of the sequences described herein will ha ve a thymidine base in place of the uridine base for DNA sequences and a uridine base in place of a thymidine base for RNA sequences.

[0037] The term "miR-92" as used herein includes pri-miR-92, pre-miR-92, miR-92, miR-92a, miR-92b, miR-92a-3p, and hsa-miR-92a-3p.

Oligonucleotide Inhibitors of miR-92

[0038] Provided herein are oligonucleotides comprising a sequence at least partially complementary to miR-92. These oligonucleotides reduce the function or activity of miR-92.

[0039] In the context of the present invention, the term "oligonucleotide inhibitor", "antimiR", "antagonist", "antisense oligonucleotide or ASO", "oligomer", "anti-microRNA oligonucleotide or AMO", or "mixmer" is used broadly and encompasses an oligomer comprising ribonucleotides, deoxyribonucleotides, modified ribonucleotides, modified deoxyribonucleotides or a combination thereof, that inhibits the activity or function of the target miR-92 by fully or partially hybridizing to the miR-92, thereby repressing the function or activity of the target miR-92.

[0040] The activity of the oligonucleotide in modulating the function and/or activity of miR- 92 may be determined in vitro, ex vivo and/or in vivo. For example, when inhibition of miR-92 activity is determined in vitro, the activity may be determined using a dual luciferase assay. The dual luciferase assay can be any dual luciferase assay known in the art. The dual luciferase assay can be a commercially available dual luciferase assay . The dual luciferase assay, as exemplified by the commercially available product PsiCHECK™ (Promega), can involve placement of the miR recognition site in the 3' UTR of a gene for a detectable protein (e.g., renilla luciferase). The construct can be co-expressed with miR-92, such that inhibitor activity can be determined by change in signal. A second gene encoding a detectable protein (e.g., firefly luciferase) can be included on the same plasrmd, and the ratio of signals determined as an indication of the a timiR- 92 activity of a candidate oligonucleotide. In some embodiments, the oligonucleotide significantly inhibits such activity, as determined in the dual luciferase activity, at a concentration of about 50 nM or less, or in other embodiments, 40 nM or less, 20 nM or less, or 10 tiM or less. For example, the oligonucleotide may have an IC50 for inhibition of miR-92 activity of about 50 nM or less, 40 nM or less, 30 nM or less, or 20 nM or less, as determined in the dual luciferase assay.

[0041] Alternatively, or in addition, the in vivo efficacy of the oligonucleotide inhibitor of miR-92 may also be determined in a suitable animal model. The animal model can be a rodent model (e.g., mouse or rat model). The oligonucleotide may exhibit at least 50% miR-92 target derepression at a dose of 50 mg/kg or less, 25 mg/kg or less, 10 mg/kg or less or 5 mg/kg or less. In such embodiments, the oligonucleotide may be dosed, delivered or administered to the non-human animal intravenously or subcutaneously or delivered locally such as local injection, and the oligonucleotide may be formulated in saline. The oligonucleotide inhibitor of miR-92 as provided herein can have increased in vivo efficacy in a particular tissue as compared to other oligonucleotide inhibitors of miR-92.

[0042] In these or other embodiments, the oligonucleotides of the present invention can be stable after administration, being detectable in the circulation and/or target organ for at least three weeks, at least four weeks, at least five weeks, or at least six weeks, or more, following administration. Thus, the oligonucleotide inhibitors of miR-92 provided herein may provide for less frequent administration, lower doses, and/or longer duration of therapeutic effect as compared to other oligonucleotide inhibitors of miR-92.

[0043] The nucleotide sequence of the oligonucleotide can be substantially complementary to a nucleotide sequence of an RNA, such as a mRNA or miRNA. The nucleotide sequence of the oligonucleotide can be fully complementary to a nucleotide sequence of an RNA, such as a mRNA or miRNA. In some embodiments, the miRNA is miR-92 or miR-92a. The oligonucleotide comprises at least one LNA, such as at least two, at least three, at least five, at least seven or at least nine LNAs. In some embodiments, the oligonucleotide comprises a mix of LNA and non- locked nucleotides. For example, the oligonucleotide may contain at least five or at least seven or at least nine locked nucleotides, and at least one non-locked nucleotide.

[0044] Generally, the length of the oligonucleotide and number and position of locked nucleotides can be such that the oligonucleotide reduces miR-92 function and/or activity, in some embodiments, the length of the oligonucleotide and number and position of locked nucleotides is such that the oligonucleotide reduces miR-92 function and/or activity at an oligonucleotide concentration of about 50 nM or less in the in vitro luciferase assay, or at a dose of about 50 mg/kg or less, or about 25 mg/kg or less in a suitable mouse or rat model, each as described. In some embodiments, the length of the oligonucleotide and number and position of locked nucleotides is such that the oligonucleotide reduces miR-92 activity as determined by target de-repression, at a dose of about 50 mg/kg or less, or about 25 mg/kg or less in a suitable mouse or rat model, such as described herein.

[0045] The oligonucleotide of the present invention can comprise a sequence of nucleotides in which the sequence comprises at least five LNAs, a LNA at the 5' end of the sequence, a LNA at the 3' end of the sequence, or any combination thereof. In one embodiment, the oligonucleotide comprises a sequence of nucleotides in which the sequence comprises at least five LNAs, a LNA at the 5' end of the sequence, a LNA at the 3^! end of the sequence, or any combination thereof, wherein three or fewer of the nucleotides are contiguous LNAs. For example, the oligonucleotide comprises no more than three contiguous LN As. For example, the oligonucleotide may comprise a sequence with at least five LNAs, a LNA at the 5^! end, a LNA at the 3' end, and no more than three contiguous LNAs. The oligonucleotide may comprise a sequence with at least five LN As, a LNA at the 5' end, a LNA at the 3' end, and no more than three contiguous LNAs, wherein the sequence is at least 16 nucleotides in length. The sequence can be substantially or completely complementary to a RNA, such as mRNA, or miRNA, wherein a substantially complementary sequence may have from 1 to 4 mismatches (e.g., 1 or 2 mismatches) with respect to its target sequence. In one embodiment, the target sequence is a miRNA, such that the oligonucleotide is a miRNA inhibitor, or antimiR. In one embodiment, the target sequence is a miR-92 sequence as provided herein.

[0046] In yet another embodiment, the oligonucleotide of the present invention can comprise a sequence complementary to the seed region of miR-92, wherein the sequence comprises at least five LNAs. The "seed region of a miRNA" is the portion spanning bases 2 to 9 at the 5' end of the miRNA. The oligonucleotide comprising a sequence complementary to the seed region of a miR- 92, wherein the sequence comprises at least five LNAs, may comprise a LNA at the 5' end or a LNA at the 3' end, or both a LNA at the 5' end and 3' end. In one embodiment, the oligonucleotide comprising at least 5 LNAs, a LNA at the 5' end and/or a LNA at the 3' end, also has three or fewer consecutive LNAs, In some embodiments, the sequence is at least 16 nucleotides in length. The sequence complementary to the seed region of a miRNA can be substantially complementary or compl etely complementary ,

[0047] The oligonucleotides of the present invention may comprise one or more locked nucleic acid (LNAs) residues, or "locked nucleotides," The oligonucleotide of the present invention can contain one or more locked nucleic acid (LNAs) residues, or "locked nucleotides," The oligonucleotides of the present invention may comprise one or more nucleotides containing other sugar or base modifications. The terms "locked nucleotide," "locked nucleic acid unit," "locked nucleic acid residue," "LNA" or "LNA unit" may be used interchangeably throughout the disclosure and refer to a bicyclic nucleoside analogue. For instance, suitable oligonucleotide inhibitors can be comprised of one or more "conformationally constrained" or bicyclic sugar nucleoside modifications (BSN) that confer enhanced thermal stability to complexes formed between the oligonucleotide containing BSN and their complementary target strand. LNAs are described, for example, in U.S. Patent Nos. 6,268,490, 6,316,198, 6,403,566, 6,770,748, 6,998,484, 6,670,461, and 7,034,133, all of which are hereby incorporated by reference in their entireties. LNAs are modified nucleotides or ribonucleotides that contain an extra bridge between the 2' and 4' carbons of the ribose sugar moiety resulting in a "locked" conformation, and/or bicyclic structure. In one embodiment, the oligonucleotide contains one or more LNAs having the structure shown by structure A below. Alternatively or in addition, the oligonucleotide may contain one or more LNAs having the structure shown by structure B below. Alternatively or in addition, the oligonucleotide contains one or more LNAs having the structure shown by structure C below.

A B

c

[0048] When referring to substituting a DNA or RNA nucleotide by its corresponding locked nucleotide in the context of the present invention, the term "corresponding locked nucleotide" is intended to mean that the DNA/RNA nucleotide has been replaced by a locked nucleotide containing the same naturally-occurring nitrogenous base as the DNA/RNA nucleotide that it has replaced or the same nitrogenous base that is chemically modified. For example, the corresponding locked nucleotide of a DNA nucleotide containing the nitrogenous base C may contain the same nitrogenous base C or the same nitrogenous base C that is chemically modified, such as 5-methylcytosine.

[0049] The term "non-locked nucleotide" refers to a nucleotide different from a locked- nucleotide, i.e. the term "non-locked nucleotide" includes a DNA nucleotide, an RNA nucleotide as well as a modified nucleotide where a base and/or sugar is modified except that the modification is not a locked modification.

[0050] Other suitable locked nucleotides that can be incorporated in the oligonucleotide inhibitors of miR-92 of the present invention include those described in U.S. Patent Nos. 6,403,566 and 6,833,361 , both of which are hereby incorporated by reference in their entireties.

[0051] In exemplary embodiments, the locked nucleotides have a 2' to 4' methylene bridge, as shown in structure A, for example. In other embodiments, the bridge comprises a methylene or ethylene group, which may be substituted, and which may or may not have an ether linkage at the 2' position.

[0052] The oligonucleotide inhibitors of miR-92 of the present invention may include modified nucleotides that have a base modification or substitution. The natural or unmodified bases in RNA are the purine bases adenine (A) and guanine (G), and the pyrimidine bases cytosine (C) and uracil (U) (DNA has thymine (T)). Modified bases, also referred to as heterocyclic base moieties, include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5- hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2 -propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thioeytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine and other alkynyl derivatives of pyrimidme bases, 6-azo uracil, cytosine and thymine, 5- uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8- substituted adenines and guanines, 5-halo (including 5-bromo, 5-trifluoromethyl and other 5- substituted uracils and cytosines), 7-methylguanme and 7-methyladenine, 2-F-adenine, 2-aminoadenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. In certain embodiments, oligonucleotide inhibitors targeting miR-92 comprise one or more BSN modifications (i.e., LNAs) in combination with a base modification (e.g. 5 -methyl cytidine).

[0053] The oligonucleotide inhibitors of miR-92 of the present invention may include nucleotides with modified sugar moieties. Representative modified sugars include carbocyclic or acyclic sugars, sugars having substituent groups at one or more of their 2', 3' or 4' positions and sugars having substituents in place of one or more hydrogen atoms of the sugar. In certain embodiments, the sugar is modified by having a substituent group at the 2' position. In additional embodiments, the sugar is modified by having a substituent group at the 3' position. In other embodiments, the sugar is modified by having a substituent group at the 4' position. It is also contemplated that a sugar may have a modification at more than one of those positions, or that an oligonucleotide inhibitor may have one or more nucleotides with a sugar modification at one position and also one or more nucleotides with a sugar modification at a different position.

[0054] The oligonucleotide may comprise, consist essentially of, or consist of, an antisense sequence to miR-92. In one embodiment, the oligonucleotide comprises an antisense sequence directed to miR-92. For example, the oligonucleotide can comprise a sequence that is at least partially complementary to a mature miR-92 sequence, e.g. at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementary to a mature miR-92 sequence. In one embodiment, the oligonucleotide inhibitor as provided herein comprises a sequence that is at least 75% complementary to a mature miR-92 sequence. In one embodiment, the oligonucleotide inhibitor as provided herein comprises a sequence that is at least 85% complementary to a mature miR-92 sequence. In one embodiment, the oligonucleotide inhibitor as provided herein comprises a sequence that is at least 95% complementary to a mature miR-92 sequence. In one embodiment, the oligonucleotide inhibitor as provided herein comprises a sequence that is at least 75% complementary to a miR-92 inhibitor selected from those listed in Table 2 (i.e. SEQ ID NOs: 7 to 164). In one embodiment, the oligonucleotide inhibitor as provided herein comprises a sequence that is at least 85% complementary to a miR-92 inhibitor selected from those listed in Table 2 (i.e. SEQ ID NOs: 7 to 164). In one embodiment, the oligonucleotide inhibitor as provided herein comprises a sequence that is at least 95% complementary to a miR-92 inhibitor selected from those listed in Table 2 (i.e. SEQ ID NOs: 7 to 164). In one embodiment, the oligonucleotide inhibitor as provided herein comprises a sequence that is 100% or fully complementary to a mature miR-92 sequence. It is understood that the sequence of the oligonucleotide inhibitor is considered to be complementary to miR-92 even if the oligonucleotide inhibitor sequence includes a modified nucleotide instead of a naturally-occurring nucleotide. For example, if a mature sequence of miR- 92 comprises a guanosine nucleotide at a specific position, the oligonucleotide inhibitor may comprise a modified cytidme nucleotide, such as a locked cytidine nucleotide or 2'-fluoro- cytidine, at the corresponding position.

[0055] In certain embodiments, the oiigonucleotide comprises a nucleotide sequence that is completely complementary to a nucleotide sequence of miR-92. In particular embodiments, the oligonucleotide comprises, consists essentially of, or consists of the nucleotide sequence complementary to miR-92. In this context, "consists essentially of includes the optional addition of nucleotides (e.g., one or two) on either or both of the 5' and 3' ends, so long as the additional nucleotide(s) do not substantially affect (as defined by an increase in IC50 of no more than 20%) the oligonucleotide's inhibition of the target miRNA activity in the dual luciferase assay or animal (e.g., mouse) model.

[0056] The oligonucleotide can generally have a nucleotide sequence designed to target mature miR-92. The oligonucleotide may, in these or other embodiments, also or alternatively be designed to target the pre- or pri-miRNA forms of miR-92. In certain embodiments, the oligonucleotide may be designed to have a sequence containing from 1 to 5 (e.g., 1 , 2, 3, or 4) mismatches relative to the fully complementary (mature) miR-92 sequence. In certain embodiments, such antisense sequences may be incorporated into shRNAs or other RNA structures containing stem and loop portions, for example. [0057] The oligonucleotide can be from 8 to 20 nucleotides in length, from 15 to 50 nucleotides in length, from 18 to 50 nucleotides in length, from 10 to 18 nucleotides in length, or from 11 to 16 nucleotides in length. The oligonucleotide in some embodiments is about 8, about 9, about 10, about 1 1 , about 12, about 13, about 14, about 15, about 16, about 17, or about 18 nucleotides in length, in one embodiment, the present invention provides an oligonucleotide inhibitor of miR- 92 that has a length of 1 1 to 16 nucleotides. In various embodiments, the oligonucleotide inhibitor targeting miR-92 is 1 1 , 12, 13, 14, 15, or 16 nucleotides in length, in one embodiment, the oligonucleotide inhibitor of miR-92 has a length of 12 nucleotides, in some embodiments, the oligonucleotide inhibitor of miR-92 is at least 16 nucleotides in length.

[0058] Generally, the number and position of LNA can be such that the oligonucleotide reduces miR-92 activity or function. In one embodiment, the number and position of LNAs is such that the oligonucleotide has an increased efficacy relative to a control. In some embodiments, efficacy is a capacity for producing a beneficial or desired result (e.g., clinical result). The beneficial or desired result can be a reduction, amelioration, or removal of a symptom or symptoms of a disease or condition. The beneficial or desired result can be a inhibition, reduction, amelioration, or removal of the activity or function of miR-92. The increased efficacy can be increased in vivo, in vitro, or ex vivo. The control can be an oligonucleotide containing the same sequence as the oligonucleotide comprising LNAs as provided herein but no chemical modifications. The control can be an oligonucleotide containing the same sequence as the oligonucleotide comprising LNAs as provided herein but a different chemical modification motif or pattern. The control can be an oligonucleotide containing the same sequence as the oligonucleotide comprising LNAs as provided herein but a different number and/or position of LNAs. The control can be an oligonucleotide containing the same sequence as well as number and/or position of LNAs, but a different additional modification such as the presence of one or more 5-methylcytosines.

[0059] The oligonucleotide inhibitors of miR-92 as provided herein generally contain at least about 2, at least about 3, at least about 4, at least about 5, at least about 7, or at least about 9 LNAs, but in vari ous embodiments is not fully comprised of LN As. Generally, the number and position of LNAs is such that the oligonucleotide reduces mRNA or miRNA function or activity. In certain embodiments, the oligonucleotide does not contain a stretch of nucleotides with more than four, or more than three, contiguous LNAs. For example, the oligonucleotide comprises no more than three contiguous LNAs. In these or other embodiments, the oligonucleotide can comprise a region or sequence that is substantially or completely complementary to a miRNA seed region, in which the region or sequence comprises at least two, at least three, at least four, or at least five locked nucleotides.

[0060] In certain embodiments, the oligonucleotide inhibitor of miR-92 contains at least 1 , at least 2, at least 3, at least 4, or at least 5 DNA nucleotides. In one embodiment, the oligonucleotide inhibitor comprises at least one I.NA, wherein each non-locked nucleotide in the oligonucleotide inhibitor is a DNA nucleotide. In one embodiment, the oligonucleotide inhibitor comprises at least two LNAs, wherein each non-locked nucleotide in the oligonucleotide inhibitor is a DNA nucleotide. In one embodiment, at least the second nucleotide from the 5' end of the oligonucleotide inhibitor is a DNA nucleotide. In one embodiment, at least 1 , at least 2, at least 3, at least 4, or at least 5 DNA nucleotides in an oligonucleotide as provided herein contains a nitrogenous base that is chemically modified. In one embodiment, the second nucleotide from the 5' end of an oligonucleotide inhibitor as provided herein contains a nitrogenous base that is chemically modified. The chemically modified nitrogenous base can be 5-methylcytosine. In one embodiment, the second nucleotide from the 5' end is a 5-methylcytosine. In one embodiment, an oligonucleotide inhibitor as provided herein comprises a 5-methylcytosine at each LNA that is a cytosine.

[0061] In one embodiment, an oligonucleotide inhibitor of miR-92 as provided herein comprises a sequence of 12 to 16 nucleotides, wherein the sequence is at least partially or fully complementary to a mature sequence of miR-92, in which from the 5' end to the 3' end of the oligonucleotide, at least the first and last nucleotide positions are LNAs. In certain embodiments, the oligonucleotide inhibitor of miR-92 has a length of 12 nucleotides. In certain embodiments, the oligonucleotide inhibitor of miR-92 has a length of 13 nucleotides. In certain embodiments, the oligonucleotide inhibitor of miR-92 has a length of 14 nucleotides. In certain embodiments, the oligonucleotide inhibitor of miR-92 has a length of 15 nucleotides. In certain embodiments, the oligonucleotide inhibitor of miR-92 has a length of 16 nucleotides. The oligonucleotide can have a full or partial (i.e., one or more) phosphorothioate backbone. The oligonucleotide can further comprise any additional modification as provided herein including but not limited to one or more chemically modified nitrogenous bases, a 5' and/or 3' cap structure, a pendent lipophilic group and/or 2' deoxy, 2' O-alkyl or 2' halo modification(s). In certain embodiments, the oligonucleotide inhibitor of miR-92 comprising a sequence of from 12 to 16 nucleotides comprises at least one nucleotide with a chemically modified nitrogenous base. The chemically modified nitrogenous base can be a methylated base. In certain embodiments, the chemically modified nitrogenous base is 5-methyIcytosine. In one embodiment, each LNA that is a cytosme is a 5- methylcytosine. In certain embodiments, an oligonucleotide inhibitor as provided herein comprising at least one nucleotide with a chemically modified nitrogenous base (e.g., 5- methylcytosine) shows increased efficacy as compared to the same oligonucleotide inhibitor lacking the chemically modified nitrogenous base. The increased efficacy can be an increased reduction or inhibition of miR-92 function and/or activity. The increased efficacy can be in vivo, ex vivo and/or in vitro.

[0062] In one embodiment, the oligonucleotide can comprise a sequence of 13 to 16 nucleotides, in which from the 5' end to the 3' end of the oligonucleotide, positions 1, 6, 10, 11 and 13 are LNAs, and the remaining positions are non-locked nucleotides, wherein the oligonucleotide is at least partially complementary to a miRNA or a seed region of a miRNA, in which the miRNA may in some embodiments, be miR-92. The oligonucleotide can be fully complementar to the miRN A, in which the miRN A may in some embodiments, be miR-92. In some embodiments, at least one non-locked nucleotide comprises a nitrogenous base that is chemically modified. In certain embodiments, the oligonucleotide inhibitor comprises a nucleotide containing a chemically modified nitrogenous base at a second nucleotide position from the 5' end to the 3' end of the oligonucleotide. In certain embodiments, the second nucleotide position is a cytosme and the chemically modified nitrogenous base is a 5-methylcytosine. In one embodiment, the presence of the chemically modified nitrogenous base(s) (e.g., 5-methylcytosine) in the oligonucleotide inhibitor has increased in vivo or in vitro efficacy as compared to an oligonucleotide with the same number and/or position of LNAs but no chemically modified nitrogenous base (e.g., 5- methylcytosine). The increased efficacy can be an increased reduction of miR-92 function and/or activity.

[0063] In another embodiment, the oligonucleotide can comprise at least 16 nucleotides, in which from the 5' end to the 3' end of the oligonucleotide, positions 1 , 3, 6, 8, 10, 11, 13, 14, and 16 are LNAs, and the remaining positions are non-locked nucleotides, the oligonucleotide is at least partially complementary to a miRNA or a seed region of a miRNA, in which the miRNA may in some embodiments, be miR-92. The oligonucleotide can be fully complementary to the miRNA, in which the miRNA may in some embodiments, be miR-92. In some embodiments, the second nucleotide from the 5' end comprises a nitrogenous base that is chemically modified (e.g. 5- methylcytosine). In one embodiment, the presence of the chemically modified nitrogenous base(s) (e.g., 5-methylcytosine) in the oligonucleotide inhibitor has increased in vivo or in vitro efficacy as compared to an oligonucleotide with the same number and/or position of LNAs but no chemically modified nitrogenous base (e.g., 5-methylcytosine). The increased efficacy can be an increased reduction of miR-92 function and/or activity.

[0064] In another embodiment, the oligonucleotide can comprise at least 16 nucleotides, in which from the 5' end to the 3' end of the oligonucleotide, positions 1 , 5, 6, 8, 10, 1 1 , 13, 1 5, and 16 are LNAs, and the remaining positions are non-locked nucleotides, the oligonucleotide is at least partially complementary to a miRNA or a seed region of a miRNA, in which the miRNA may in some embodiments, be miR-92. The oligonucleotide can be fully complementary to the miRNA, m which the miRNA may in some embodiments, be miR-92. In some embodiments, the second nucleotide from the 5' end comprises a nitrogenous base that is chemically modified (e.g. 5- methylcytosine). In one embodiment, the presence of the chemically modified nitrogenous base(s) (e.g., 5-methylcytosine) in the oligonucleotide inhibitor has increased in vivo or in vitro efficacy as compared to an oligonucleotide with the same number and/or position of LNAs but no chemically modified nitrogenous base (e.g., 5-methylcytosine). The increased efficacy can be an increased reduction of miR-92 function and/or activity.

[0065] In another embodiment, the oligonucleotide can comprise at least 16 nucleotides, in which from the 5' end to the 3^! end of the oligonucleotide, positions 1, 3, 6, 9, 10, 11, 13, 14, and 16 are LNAs, and the remaining positions are non-locked nucleotides, the oligonucleotide is at least partially complementary to a m iRNA or a seed region of a m iRNA, in which the miRN A may in some embodiments, be miR-92. The oligonucleotide can be fully complementary to the miRNA, in which the miRNA may in some embodiments, be miR-92. In some embodiments, the second nucleotide from the 5' end comprises a nitrogenous base that is chemically modified (e.g. 5- methylcytosine). In one embodiment, the presence of the chemically modified nitrogenous base(s) (e.g., 5-methylcytosine) in the oligonucleotide inhibitor has increased in vivo or in vitro efficacy as compared to an oligonucleotide with the same number and/or position of LNAs but no chemically modified nitrogenous base (e.g., 5-methylcytosine). The increased efficacy can be an increased reduction of miR-92 function and/or activity. [0066] In some embodiments, an oligonucleotide inhibitor of miR-92 shows at least about 0.5%, at least about 1 %, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 99% greater inhibition of the function and/or activity of the target miR-92 as compared to other inhibitors of the target miR-92. The improvement or increase can be in vitro, ex vivo and/or in vivo.

[0067] In some embodiments, an oligonucleotide inhibitors of miR-92 comprising a 5- methylcytosine produces at least about 0.5%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 10%, at least about 1 5%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 99% of an increase or improvement in the reduction of function and/or activity of the target miR-92 as compared to an oligonucleotide with the same nucleotide sequence as well as LNA/DNA pattern but lacking a 5-methylcyotsine. The improvement or increase can be in vitro, ex vivo and/or in vivo. In some cases, all LNA cytosines in an oligonucleotide inhibitor as provided herein is a 5-methylcytosine LNA.

[0068] In some embodiments for non-locked nucleotides, the nucleotide may contain a 2' modification with respect to a 2' hydroxyl. For example, the 2' modification may be 2' deoxy. Incorporation of 2' -modified nucleotides in antisense oligonucleotides may increase resistance of the oligonucleotides to nucleases. Incorporation of 2'-modified nucleotides in antisense oligonucleotides may increase their thermal stability with complementary RNA. Incorporation of 2'-modified nucleotides in antisense oligonucleotides may increase both resistance of the oligonucleotides to nucleases and their thermal stability with complementary RNA. Various modifications at the 2' positions may be independently selected from those that provide increased nuclease sensitivity, without compromising molecular interactions with the RNA target or cellular machinery. Such modifications may be selected on the basis of their increased potency in vitro, ex vivo or in vivo. Exemplary methods for determining increased potency (e.g., IC50) for miR-92 inhibition are described herein, including, but not limited to, the dual luciferase assay and in vivo miR-92 abundance or target de-repression. [0069] In some embodiments the 2' modification may be independently selected from O-alkyl (which may be substituted), halo, and deoxy (H). Substantially all, or all, nucleotide 2' positions of the non-locked nucleotides may be modified in certain embodiments, e.g. , as independently selected from O-alkyl (e.g., O-methyl), halo (e.g., fluoro), deoxy (H), and ammo. For example, the 2' modifications may each be independently selected from O-methyl (OMe) and fluoro (F). In exemplary embodiments, purine nucleotides each have a 2' OMe and pyrimidine nucleotides each have a 2'-F. In certain embodiments, from one to about five 2' positions, or from about one to about three 2' positions are left unmodified (e.g., as 2' hydroxyls).

[0070] 2' modifications in accordance with the invention can also include small hydrocarbon substituents. The hydrocarbon substituents include alkyl, alkenyl, alkynyl, and alkoxyalkvl, where the alkyl (including the alkyl portion of alkoxy), alkenyl and alkynyl may be substituted or unsubstituted. The alkyl, alkenyl, and alkynyl may be CI to CIO alkyl, alkenyl or alkynyl, such as CI, C2, or C3. The hydrocarbon substituents may mclude one or two or three non-carbon atoms, which may be independently selected from nitrogen (N), oxygen (O), and/or sulfur (S). The 2' modifications may further include the alkyl, alkenyl, and alkynyl as O-alkyl, O-alkenyl, and O- alkynyl.

[0071] Exemplary 2' modifications in accordance with the invention can include 2'-0-alkyl (CI -3 alkyl, such as 2' OMe or 2'OEt), 2'-0-methoxyethyl (2^!-0-MOE), 2'-0-aminopropyl (2'-0- AP), 2^!-0-dimethylaminoethyl (2'-Q-DMAOE), 2^!-0-dimethyiaminopropyi (2'-0-DMAP), 2^!-0- dimethyiaminoethyloxyethyl (2^!-0-DMAEOE), or 2^!-0-N-methylacetamido (2'-0-NMA) substitutions.

[0072] In certain embodiments, the oligonucleotide contains at least one 2' -halo modification (e.g., in place of a 2' hydroxyl), such as 2' -fluoro, 2'-chloro, 2'-bromo, and 2'-iodo. In some embodiments, the 2' halo modification is fluoro. The oligonucleotide may contain from 1 to about 5 2' -halo modifications (e.g., fluoro), or from 1 to about 3 2'-halo modifications (e.g., fluoro). In some embodiments, the oligonucleotide contains all 2'-fluoro nucleotides at non-locked positions, or 2'-fluoro on all non-locked pyrimidine nucleotides. In certain embodiments, the 2' -fluoro groups are independently di-, tri-, or un-methylated.

[0073] The oligonucleotide may have one or more 2' -deoxy modifications (e.g., H for 2' hydroxy!), and in some embodiments, contains from 2 to about 10 2'-deoxy modifications at non- locked positions, or contains 2' deoxy at all non-locked positions. [0074] In exemplary embodiments, the oligonucleotide contains 2' positions modified as 2'OMe in non-locked positions. Alternatively, non-locked purine nucleotides can be modified at the 2' position as 2'OMe, with non-locked pyrimidine nucleotides modified at the 2' position as 2'-fluoro.

[0075] in certain embodiments, the oligonucleotide further comprises at least one terminal modification or "cap." The cap may be a 5' and/or a 3 '-cap structure. The terms "cap" or "end- cap" include chemical modifications at either terminus of the oligonucleotide (with respect to terminal ribonucleotides), and includes modifications at the linkage between the last two nucleotides on the 5' end and the last two nucleotides on the 3' end. The cap structure as described herein may increase resistance of the oligonucleotide to exonucleases without compromising molecular interactions with the target miR-92 or cellular machinery. Such modifications may be selected on the basis of their increased potency in vitro or in vivo. The cap can be present at the 5'-terminus (5'-cap) or at the 3 '-terminus (3'-cap) or can be present on both ends. In certain embodiments, the 5'- and/or 3 '-cap is independently selected from phosphorothioate monophosphate, abasic residue (moiety), phosphorothioate linkage, 4'-thio nucleotide, carbocyclic nucleotide, phosphorodithioate linkage, inverted nucleotide or inverted abasic moiety (2'-3' or 3'- 3'), phosphorodithioate monophosphate, and methylphosphonate moiety. The phosphorothioate or phosphorodithioate Imkage(s), when part of a cap structure, are generally positioned between the two terminal nucleotides on the 5' end and the two terminal nucleotides on the 3' end.

[0076] In certain embodiments, the oligonucleotide has at least one terminal phosphorothioate monophosphate. The phosphorothioate monophosphate may support a higher potency by inhibiting the action of exonucleases. The phosphorothioate monophosphate may be at the 5' and/or 3' end of the oligonucleotide. A phosphorothioate monophosphate is defined by the following structures, where B is base, and R is a 2' modification as described above:

[0077] Where the cap structure can support the chemistry of a locked nucleotide, the cap structure may incorporate a LNA as described herein.

[0078] Phosphorothioate linkages may be present in some embodiments, such as between the last two nucleotides on the 5' and the 3' end (e.g., as part of a cap structure), or as alternating with phosphodi ester bonds, in these or other embodiments, the oligonucleotide may contain at least one terminal abasic residue at either or both the 5' and 3' ends. An abasic moiety does not contain a commonly recognized purine or pyrimidine nucleotide base, such as adenosine, guanine, cytosme, uracil or thymine. Thus, such abasic moieties lack a nucleotide base or have other non- nucleotide base chemical groups at the Γ position. For example, the abasic nucleotide may be a reverse abasic nucleotide, e.g., where a reverse abasic phosphoramidite is coupled via a 5' amidite (instead of 3' amidite) resulting in a 5 '-5' phosphate bond. The structure of a reverse abasic nucleoside for the 5' and the 3' end of a polynucleotide is shown below.

[0079] The oligonucleotide may contain one or more phosphorothioate linkages, Phosphorothioate linkages can be used to render oligonucleotides more resistant to nuclease cleavage. For example, the polynucleotide may be partially phosphorothioate-linked, for example, phosphorothioate linkages may alternate with phosphodiester linkages. In certain embodiments, however, the oligonucleotide is fully phosphorothioate-linked. In other embodiments, the oligonucleotide has from one to five or one to three phosphate linkages.

[0080] In some embodiments, the nucleotide has one or more carboxamido-raodified bases as described in PCT/XJS 11/59588, which is hereby incorporated by reference, including with respect to all exemplar pyrimidine carboxamido modifications disclosed therein with heterocyclic substituents.

[0081] The synthesis of oligonucleotides, including modified polynucleotides, by solid phase synthesis is well known and is reviewed in Caruthers et al, Nucleic Acids Symp. Ser. 7:215-23 (1980).

[0082] In some embodiments, the oligonucleotide comprises a sequence selected from Table 2, in which "+" or "1" indicates the nucleotide is a LNA; "d" indicates the nucleotide is a DNA; "s" indicates a phosphorothioate linkage between the two nucleotides; and "mdC" indicates the nucleotide is a 5-methyl cytosine DNA:

[0083] In one embodiment, the oligonucleotide comprises a sequence selected from Table 2, and comprises at least one non-locked nucleotide that is 2' O-alkyi or 2' halo modified. In some embodiments, the oligonucleotide comprises at least one LNA that has a 2' to 4' methylene bridge. In some embodiments, the oligonucleotide has a 5' cap structure, 3' cap structure, or 5' and 3 ' cap structure. In yet other embodiments, the oligonucleotide comprises a pendent lipophilic group. [0084] The oligonucleotide may be incorporated within a variety of macromolecular assemblies or compositions. Such complexes for delivery may include a variety of liposomes, nanoparticles, and micelles, formulated for delivery to a subject. The complexes may include one or more fusogenic or lipophilic molecules to initiate cellular membrane penetration. Such molecules are described, for example, in US Patent No. 7,404,969 and US Patent No. 7,202,227, which are hereby incorporated by reference in their entireties. Alternatively, the oligonucleotide may further comprise a pendant lipophilic group to aid cellular delivery, such as those described in WO 2010/129672, which is hereby incorporated by reference.

Methods o f Use

[0085] Provided herein are methods for delivering any one of the oligonucleotide inhibitors of miR-92 disclosed herein to heart cells (e.g. , as part of a composition or formulation described herein) for reducing or inhibiting activity or function of miR-92 in the heart cells, e.g. for the treatment of heart failure, in one embodiment, the heart failure is chronic heart failure. In one embodiment, the heart failure is characterized as either heart failure with reduced ejection fraction (HFrEF). In one embodiment, the heart failure is characterized as heart failure with preserved ejection fraction (HFpEF).

[0086] Provided herein are methods of treating heart failure in a subject. As used herein, the term "subject" refers to any vertebrate including, without limitation, humans and other primates (e.g., chimpanzees and other apes and monkey species), farm animals (e.g. , cattle, sheep, pigs, goats and horses), domestic mammals (e.g. , dogs and cats), laboratory animals (e.g. , rodents such as mice, rats, and guinea pigs), and birds (e.g., domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like). In some embodiments, the subject is a mammal. In other embodiments, the subject is a human. The subject may have a condition associated with, mediated by, or resulting from, expression of miR-92.

[0087] In one embodiment, the subject has ischemic cardiomyopathy. In one embodiment, the subject has diabetic cardiomyopathy. In one embodiment, the subject is undergoing pressure- induced cardiac remodeling (pathologic hypertrophy). In one embodiment, the subject has nonischemic cardiomyopathy. In one embodiment, the subject is a diabetic subject. In one embodiment, the subject is a non-diabetic subject. In one embodiment, the subject is undergoing heart failure that is characterized as heart failure with preserved ejection fraction (HFpEF), e.g. greater than 50% ejection fraction volume. In one embodiment, the subject is undergoing heart failure that is characterized as heart failure with reduced ejection fraction (HFrEF), e.g. less than 50% ejection fraction volume.

[0088] Provided herein are methods of treating heart failure in a subject comprising administering to the subject any one of the oligonucleotides provided herein (comprising a sequence that is at least partially complementary to miR-92), wherein the administration of the oligonucleotide reduces function or activity of miR-92, and wherein the administration of the oligonucleotide results the treatment of the heart failure. In one embodiment, the oligonucleotide is selected from Table 2. In some embodiments, the oligonucleotide comprises a sequence at least partially identical to SEQ ID NOs: 7-9. In some embodiments, the oligonucleotide comprises a sequence identical to SEQ ID NOs: 7-9. In some embodiments, the oligonucleotide is identical to SEQ ID NOs: 7-9.

[0089] Provided herein are methods of treating heart failure in a subject comprising administering to the subject any one of the oligonucleotides provided herein (comprising a sequence that is at least partially complementary to miR-92), wherein the administration of the oligonucleotide reduces function or activity of miR-92, and wherein the administration of the oligonucleotide results in an improvement of left ventricular function. In one embodiment the administration of the oligonucleotide results in a decrease of left ventricu lar end-diastolic pressure (LVEDP). In one embodiment the heart failure is characterized as heart failure with reduced ejection fraction (HFrEF). In one embodiment the subject has decreased left ventricular function having an ejection fraction that is less than 40%, less than 35%, less than 30%, less than 25%, or less than 20%. In one embodiment the administration of the oligonucleotide results in an improvement of ejection fraction. In one embodiments, the improvement of ejection fraction results in the subject having an ejection fraction greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, or greater than 75%. In one embodiment, the oligonucleotide is selected from Table 2.

[009Θ] Provided herein are methods of treating heart failure in a subject comprising administering to the subject any one of the oligonucleotides described herein (comprising a sequence that is at least partially complementary to miR-92), wherein the administration of the oligonucleotide reduces function or activity of miR-92, and wherein the subject has left ventricular function having an ejection fraction that is at least 40%. In some embodiments, the subject has left ventricular function having an ejection fraction that is at least 45%, or at least 50%. In some embodiments, the heart failure is characterized as heart failure with preserved ejection fraction (HFpEF). In one embodiment, the oligonucleotide is selected from Table 2.

[0091] Provided herein are methods of treating chronic heart failure in a subject comprising administering to the subject any one of the oligonucleotides provided herein (comprising a sequence that is at least partially complementary to miR-92), wherein the administration of the oligonucleotide reduces function or activity of miR-92, and wherein the administration of the oligonucleotide results the treatment of the heart failure. In one embodiment, the chronic heart failure is a result of myocardial ischemia. In one embodiment, the chronic heart failure is a result of a non-ischemic event. In one embodiment, the subject has congestive heart failure. In one embodiment, the subject has left-sided heart failure. In one embodiment, the subject has right- sided heart failure. In one embodiment the heart failure is characterized as heart failure with preserved ejection fraction (HFpEF). In one embodiment the heart failure is characterized as heart failure with reduced ejection fraction (HFrEF). In one embodiment, the subject has ischemic cardiomyopathy. In one embodiment, the subject is undergoing pressure-induced cardiac remodeling (pathologic hypertrophy). In one embodiment, the subject has non-ischemic cardiomyopathy. In one embodiment, the subject is a diabetic subject. In one embodiment, the subject is a non-diabetic subject. In one embodiment the subject has HFrEF and the administration of the oligonucleotide results in a decrease of left ventricular end-diastolic pressure (LVEDP). In one embodiment the subject has decreased left ventricular function having an ejection fraction that is less than 40%, less than 35%, less than 30%, less than 25%, or less than 20%. In one embodiment the administration of the oligonucleotide results in an improvement of ejection fraction. In one embodiment, the improvement of ejection fraction results in the subject having an ejection fraction greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, or greater than 75%. In one embodiment, the subject has chronic myocardial ischemia. In one embodiment, administration of the oligonucleotide results in enhanced capillar)' density in the ischemic area. In one embodiment, administration of the oligonucleotide results in enhanced pericyte coverage in the ischemic area. In one embodiment, administration of the oligonucleotide results in reduced infarct size. In one embodiment, administration of the oligonucleoti de results in improved coronary reserve. In one embodiment, administration of the oligonucleotide results in improved functional cardiac reserve, in one embodiment, administration of the oligonucleotide results in reduced myocardial fibrosis. In one embodiment, administration of the oligonucleotide results in reduced myocyte hypertrophy. In one embodiment, administration of the oligonucleotide results in enhanced neovascularization. In one embodiment, administration of the oligonucleotide results in prolonged cardioprotection. In one embodiment, administration of the oligonucleotide results in reduced endothelial cell death. In one embodiment, administration of the oligonucleotide results in reduced inflammation. In one embodiment, administration of the oligonucleotide results in improved collateral growth. In one embodiment, admmistration of the oligonucleotide results in improved global myocardial function. In one embodiment, administration of the oligonucleotide results in improved regional myocardial function. In one embodiment, administration of the oligonucleotide results in a lower LVEDP. In one embodiment, administration of the oligonucleotide results in a higher ejection fraction. In one embodiment, administration of the oligonucleotide results in increased contraction velocity. In one embodiment, administration of the oligonucleotide results in improved diastolic function. In one embodiment, administration of the oligonucleotide results in decreased heart weight to body weight ratio. In one embodiment, administration of the oligonucleotide results in decreased fibrosis. In one embodiment, the oligonucleotide is selected from Table 2.

Pharmaceutical Use

[0092] The present invention provides pharmaceutical compositions comprising an oligonucleotide disclosed herein for use in the treatment of heart failure. Where clinical applications are contemplated, pharmaceutical compositions can be prepared in a form appropriate for the intended application. Generally, this can entail preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals.

[0093] In one embodiment, the pharmaceutical composition comprises an effective dose of a miR-92 oligonucleotide inhibitor and a pharmaceutically acceptable carrier. For instance, the pharmaceutical composition comprises an effective dose or amount of an oligonucleotide of the present invention or a pharmaceuticaliy-acceptabie salt thereof, and a pharmaceutically-acceptable carrier or diluent. The oligonucleotide can be selected any one of those oligonucleotides presented in Table 2.

[0094] In some embodiments, an "effective dose" is an amount sufficient to affect a beneficial or desired clinical result, e.g. treatment of heart failure. An "effective dose" can be an amount sufficient or required to substantially reduce, eliminate or ameliorate a symptom or symptoms of a disease and/or condition. This can be relative to an untreated subject. An "effective dose" can be an amount sufficient or required to slow, stabilize, prevent, or reduce the severity of the heart failure or symptoms of heart failure in a subject. This can be relative to an untreated subject. An effective dose of an oligonucleotide disclosed herein may be from about 0.001 mg/kg to about 100 mg/kg, about 0.01 mg/kg to about 10 mg/kg, about 0.1 mg/kg to about 10 mg/kg, about 1 mg/kg to about 10 mg/kg, about 2,5 mg/kg to about 50 mg/kg, or about 5 mg/kg to about 25 mg/kg. The precise determination of what would be considered an effective dose may be based on factors individual to each subject, including their size, age, and nature of the oligonucleotide {e.g. melting temperature, LNA content, etc.). Therefore, dosages can be readily ascertained by those of ordinary skill in the art from this disclosure and the knowledge in the art. In some embodiments, the methods comprise administering an effective dose of the pharmaceutical composition 1, 2, 3, 4, 5, or 6 times a day. In some embodiments, administration is 1, 2, 3, 4, 5, 6, or 7 times a week. In other embodiments, administration is biweekly or monthly.

[0095] Colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes, may be used as delivery vehicles for the oligonucleotide inhibitors of miR-92 function. Commercially available fat emulsions that are suitable for delivering the nucleic acids of the invention to cardiac and skeletal muscle tissues include Intralipid™, Liposyn™, Liposyn™ II, Liposyn™ III, Nutrilipid, and other similar lipid emulsions. A preferred colloidal system for use as a delivery vehicle in vivo is a liposome (i.e., an artificial membrane vesicle). The preparation and use of such systems is well known in the art. Exemplary formulations are also disclosed in U.S. Pat. Nos, 5,981 ,505; 6,217,900 6,383,512; 5,783,565; 7,202,227; 6,379,965; 6,127,170; 5,837,533; 6,747,014; and WO03/093449, all of which are hereby incorporated by reference in their entireties.

[0096] In certain embodiments, liposomes used for delivery are amphoteric liposomes such SMARTICLES® (Marina Biotech, Inc.) which are described in detail in U.S. Pre-grant Publication No, 20110076322. The surface charge on the SMARTICLES® is fully reversible which make them particularly suitable for the delivery of nucleic acids. SMARTICLES® can be delivered via injection, remain stable, and aggregate free and cross cell membranes to deliver the nucleic acids. [0097] Any of the miR~92 oligonucleotide inhibitors described herein can be delivered to a target cell (e.g., a heart cell, fibrocyte, fibroblast, keratinocyte or endothelial cell) by delivering to the cell an expression vector encoding the miR~92 oligonucleotide inhibitor. A "vector" is a composition of matter which can be used to deliver a nucleic acid of interest to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes an autonomously replicating plasmid or a virus. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like. In one particular embodiment, the viral vector is a lentiviral vector or an adenoviral vector. An expression construct can be replicated in a living cell, or it can be made synthetically. For purposes of this application, the terms "expression construct," "expression vector," and "vector," are used interchangeably to demonstrate the application of the invention in a general, illustrative sense, and are not intended to limit the invention.

[0098] In one embodiment, an expression vector for expressing a miR-92 oligonucleotide inhibitor described herein comprises a promoter operably linked to a polynucleotide sequence encoding the oligonucleotide inhibitor. The phrase "operably linked" or "under transcriptional control" as used herein means that the promoter is in the correct location and orientation in relation to a polynucleotide to control the initiation of transcription by RNA polymerase and expression of the polynucleotide.

[0099] As used herein, a "promoter" refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene. Suitable promoters include, but are not limited to RNA pol I, pol II, pol III, and viral promoters (e.g. human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, and the Rous sarcoma virus long terminal repeat). In one embodiment, the promoter is a fibrop!ast specific promoter such as the FSP1 promoter, etc. In another embodiment, the promoter is an endothelial specific promoter such as the ICAM-2 promoter, etc.

[001ΘΘ] In certain embodiments, the promoter operably linked to a polynucleotide encoding an oligonucleotide inhibitor described herein (e.g. , oligonucleotide inhibitors of miR-92a) can be an inducible promoter. Inducible promoters are known in the art and include, but are not limited to, tetracycline promoter, metal lothionein IIA promoter, heat shock promoter, steroid/thyroid hormone/retinoic acid response elements, the adenovirus late promoter, and the inducible mouse mammary tumor virus LTR.

[00101] Methods of delivering expression constructs and nucleic acids to cells are known in the art and can include, for example, calcium phosphate co-precipitation, electroporation, microinjection, DEAE-dextran, lipofection, transfection employing polyamine transfection reagents, cell sonication, gene bombardment using high velocity microprojectiles, and receptor- mediated transfection.

[00102] One will generally desire to employ appropriate salts and buffers to render delivery vehicles stable and allow for uptake by target cells. Aqueous compositions of the present invention can comprise an effective amount of the delivery vehicle comprising the inhibitor polynucleotides (e.g. liposomes or other complexes or expression vectors) dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. The phrases "pharmaceutically acceptable" or "pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human. As used herein, "pharmaceutically acceptable earner" includes solvents, buffers, solutions, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like acceptable for use in formulating pharmaceuticals, such as pharmaceuticals suitable for administration to humans. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients of the present invention, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions, provided they do not inactivate the oligonucleotides of the compositions.

[00103] The oligonucleotide inhibitors of miR-92 of the present invention include classic pharmaceutical preparations. Administration of these compositions according to the present invention may be via any common route so long as the target tissue is available via that route. This includes oral, nasal, or buccal. Alternatively, administration may be by intradermal, subcutaneous, intramuscular, intraperitoneal, intraarterial, or intravenous injection. In some embodiments, the pharmaceutical composition is directed injected into lung or cardiac tissue. In any of the embodiments provided herein, administration of the oligonucleotide is performed by intravenous administration, subcutaneous administration, intracardiac administration, or intracoronary administration. In some embodiments, the intracoronary administration is retrograde. In some embodiments, the intracoronary administration is anterograde.

[00104] Pharmaceutical compositions comprising a miR-92 inhibitor may also be administered by catheter systems or systems that isolate coronary/pulmonary circulation for delivering therapeutic agents to the heart and lungs. Various catheter systems for delivering therapeutic agents to the heart and coronary vasculature are known in the art. Some non-limiting examples of catheter-based delivery methods or coronary isolation methods suitable for use in the present invention are disclosed in U. S. Patent No, 6,416,510; U.S. Patent No. 6,716,1 96; U.S. Patent No. 6,953,466, WO 2005/082440, WO 2006/089340, U.S. Patent Publication No, 2007/0203445, U.S. Patent Publication No. 2006/0148742, and U.S. Patent Publication No. 2007/0060907, which are all herein incorporated by reference i their entireties. Such compositions would normally be administered as pharmaceutically acceptable compositions as described herein.

[00105] The oligonucleotide inhibitors of miR-92 may also be administered parenterally or intraperitoneally. By way of illustration, solutions of the oligonucleotide inhibitors of miR-92 as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinaiy conditions of storage and use, these preparations generally contain a preservative to prevent the growth of microorganisms.

[00106] The pharmaceutical forms suitable for injectable use, catheter delivery, or inhalational delivery include, for example, sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (e.g. aerosols, nebulizer solutions). Generally, these preparations are sterile and fluid to the extent that easy injectability or aerosolization/nebulization exists. Preparations should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Appropriate solvents or dispersion media may contain, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof!, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial an antifungal agents, for example, parabens, chJorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin,

[00107] Sterile injectable solutions may be prepared by incorporating the active compounds in an appropriate amount into a solvent along with any other ingredients (for example as enumerated above) as desired, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the desired other ingredients, e.g., as enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation include vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient(s) plus any additional desired ingredient from a previously sterile-filtered solution thereof. In some embodiments, sterile powders can be administered directly to the subject (i.e. without reconstitution in a diluent), for example, through an insufflator or inhalation device.

[00108] The compositions of the present invention generally may be formulated in a neutral or salt form. Pharmaceuticaliy-acceptabie salts include, for example, acid addition salts (formed with the free amino groups of the protein) derived from inorganic acids (e.g., hydrochloric or phosphoric acids), or from organic acids (e.g., acetic, oxalic, tartaric, mandeiic, and the like). Salts formed with the free carboxyl groups of the protein can also be derived from inorganic bases (e.g., sodium, potassium, ammonium, calcium, or ferric hydroxides) or from organic bases (e.g., isopropylamine, trimethyiamme, histidme, procaine and the like).

[0100] Upon formulation, solutions are preferably administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations may easily be administered in a variety of dosage forms such as injectable solutions, drug release capsules, unit dose inhalers, and the like. For parenteral administration in an aqueous solution, for example, the solution generally is suitably buffered and the liquid diluent first rendered isotonic for example with sufficient saline or glucose. Such aqueous solutions may be used, for example, for intravenous, intramuscular, subcutaneous, intraarterial, and intraperitoneal administration. Preferably, sterile aqueous media are employed as is known to those of skill in the art, particularly in light of the present disclosure. By way of illustration, a single dose may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 1 5th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicit , and general safety and purity standards as required by FDA Office of Biologies standards.

[0101] The composition or formulation may employ a plurality of therapeutic oligonucleotides, including at least one described herein. For example, the composition or formulation may employ at least 2, 3, 4, or 5 miR-92 inhibitors described herein. In another embodiment, an oligonucleotide of the present invention may be used in combination with other therapeutic modalities. Combinations may also be achieved by contacting a cell with more than one distinct composition or formulation, at the same time. Alternatively, combinations may be administered sequentially.

[0102] In one embodiment of the present invention, an oligonucleotide inhibitor of miR-92 is used m combination with other therapeutic modalities. Examples of combination therapies include any of the foregoing. Combinations may be achieved with a single composition or pharmacological formulation that includes both agents, or with two distinct compositions or formulations, at the same time, wherein one composition includes the oligonucleotide inhibitor of miR-92 and one more other agents. Alternatively, the therapy using an oligonucleotide inhibitor of miR-92 may precede or follow administration of the other agent(s) by intervals ranging from minutes to weeks. In embodiments where the other agent and oligonucleotide inhibitor of miR-92 are applied separately to the cell, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agent and the oligonucleotide inhibitor of miR-92 would still be able to exert an advantageously combined effect on the cell. In such instances, it is contemplated that one would typically contact the cell with both modalities within about 12-24 hours of each other, within about 6-12 hours of each other, or with a delay time of only about 12 hours. In some situations, it may be desirable to extend the time period for treatment significantly, however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations.

[0103] In one embodiment, more than one administration of the oligonucleotide inhibitor of miR-92 or the other agent(s) will be desired. In this regard, various combinations may be employed. By way of illustration, where the oligonucleotide inhibitor of miR-92 is "A" and the other agent is "B," the following permutations based on 3 and 4 total administrations are provided as examples: A/B/A, B/A , B/B/A, A/A/B, B/A/A, A/B B, B/B B/A, B/B/AB, A/A/B/B, A B/A/B, A/B/B/A, B/B/A/A, B/ A/B/A, B/ A/A/B, B/B/B/A, A/A/AB, B/A/A/A, AB/A/A, A/ A/B/A, A/B/B/B, B/A/B/B, B/B/A/B. Other combinations are likewise contemplated,

[0104] The methods of the present invention can also include methods for altering the treatment regimen of a therapeutic. Altering the treatment regimen can include but is not limited to changing and/or modifying the type of therapeutic intervention, the dosage at which the therapeutic intervention is administered, the frequency of administration of the therapeutic intervention, the route of administration of the therapeutic intervention, as well as any other parameters that would be well known by a physician to change and/or modify.

[0105] In some embodiments, the treatment efficacy can be used to determine whether to continue a therapeutic intervention. In some embodiments the treatment efficacy can be used to determine whether to discontinue a therapeutic intervention. In some embodiments the treatment efficacy can be used to determine whether to modify a therapeutic intervention. In some embodiments the treatment efficacy can be used to determine whether to increase or decrease the dosage of a therapeutic intervention. In some embodiments the treatment efficacy can be used to determine whether to change the dosing frequency of a therapeutic intervention. In some embodiments, the treatment efficacy can be used to determine whether to change the number or the frequency of administration of the therapeutic intervention. In some embodiments, the treatment efficacy can be used to determine whether to change the number of doses per day, per week, times per day. In some embodiments the treatment efficacy can be used to determine whether to change the dosage amount.

[0106] This invention is further illustrated by the following additional examples that should not be construed as limiting. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made to the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. [0107] The methods provided herein apply to all subsequent examples.

Pig Ischemia-Reperfusion Model

[0108] All pig experiments were conducted at the Walter-Brendel Center for Experimental Medicine at the University of Munich. A percutaneous transluminal coronary angioplasty (PTCA) balloon catheter was advanced via guiding catheter and the balloon was placed in the left anterior descending artery (LAD) distal to the first diagonal branch and inflated with 6atm (0.41 MPa) for 60 minutes. Correct localization of the coronary occlusion and patency of the first diagonal branch were ensured by injection of contrast agent via guiding catheter. In all groups, the PTCA balloon was deflated after 60 minutes of ischemia; the onset of reperfusion was documented angiographically. After either 24h of reperfusion, hemodynamic measurements were performed and the pigs were sacrificed for further analysis of the infarct size, inflammation and apoptosis. Oligonucleotide Application

[0109] miR-92 inhibition was achieved with the use of an oligonucleotide with a length of 16 nucleotides, with a sequence that is complementar to miR-92, comprising locked nucleic acids (LNAs) at positions 1, 6, 10, 11, 13 and 16 (SEQ ID NO: 7; referred to as LNA-92a in FIGS. 1- 14).

[0110] In some experiments, the oligonucleotide was delivered by regional anterograde ("LNA-92a ante"). Local anterograde application was performed via the "over the wire" PTCA balloon catheter (5 mg/kg heart weight). Here the vessel was reopened after 60 min of ischemia, the "over the wire" PTCA balloon was slightly inflated again and the infusion of the LNA-92a (SEQ ID NO: 7) anterograde was performed for 5 min.

Hemodynamic Measurements

[0111] Left ventricular end diastolic pressure (LVEDP) and ejection fraction (EF) measurements were performed before ischemia and after 24 hours of reperfusion. Additionally, regional myocardial function was obtained at 24 hours of reperfusion via ultrasound crystals (Sonometrics, USA). The ultrasound crystals were implanted in the ischemic and non-ischemic tissue after sternotomy and opening of the pericardium. Subendocardial segment shortening (SES) was assessed in the ischemic and non-ischemic region at rest and under increased heart rated (150 bpm). Infarct size

[0112] Infarct size was assessed by methylene blue exclusion, tetrazolium red viability staining. Before explanation of the heart, the LAD was ligated at the side of infarct induction and methylene blue was injected into the left ventricle. After excision of the heart, tetrazolium red was injected into the LAD distal of the occlusion site, thereby staining the viable myocytes in the ischemic area. Then the heart was cut into 5 slices and digital pictures were taken for planimetric analysis of the infarct size and the area at risk (AAR).

Example 2 - miR-92 Inhibition in Myocardial Ischemia is Cardioprotective

[0113] The effect of regional miR-92 inhibition on myocardial ischemia in a porcine model was quantified.

Results

[0114] miR-92 inhibition with the use of LNA-92a (SEQ ID NO: 7) resulted in reduced expression of miR92a in the heart; and resulted in reduced infarct size.

[0115] Hibernating myocardium in the pigs was induced by a reduction stent, inducing occlusion of a coronary artery. Regional LNA-92a (SEQ ID NO: 7) treatment was introduced by retroinfusion into the coronary vein accompanying the occluded arteiy. Global myocardial function and perfusion were obtained at day 28 and 56. At day 56 regional myocardial function and collateral growth were analyzed. In addition histological analysis of microcirculation was performed.

[0116] Inhibition of miR-92a enhanced capillary density and pericyte coverage in the ischemic area. Improved collateral growth (3±1 control vs. 6±1 in LNA-92a) and distal perfusion of the occluded coronary artery (3±1 control vs. 6±1 in LNA-92a) were observed. Global myocardial function was improved after LNA-92a (SEQ ID NO: 7) treatment (LVEDP control: 6±1 vs. LNA- 92a 15±lmmHg). Regional myocardial function, obtained in the ischemic area under increased heart rate, was significantly improved after miR-92a inhibition (SES at 150bpm: 19±9 % LNA- 92a vs. 70±1 % control; improved myocardial function is depicted in FIGS. 1 -3).

[0117] Subendocardial segment shortening (SES) measurements (FIG. 2, right panel) demonstrate that LNA~92a (SEQ ID NO: 7) treatment improves the cardiac reserve during the electric stimulation at 150bpm. Thus, surprisingly, antimiR-92 therapy counteracts the decrease in cardiac reserve associated with heart failure. [0118] Left ventricular end diastolic pressure (LVEDP) measurements (FIG, 2, left panel) demonstrate that a decrease in delta LVEDP after LNA-92a (SEQ ID NO: 7) treatment. The pressure at the end of the filling is correlated to the dysfunction of the left ventricular. Excessive delta LEVDP may induce a lung congestion and an acute pulmonary oedema. Thus, surprisingly, antimiR-92 therapy counteracts an increase in delta LVEDP associated with heart failure.

[0119] Regional reduction of miR~92a levels via LNA-92a (SEQ ID NO: 7) is cardioprotective in a pig model of chronic myocardial ischemia. (FIG. 4). A single application of LNA-92a (SEQ ID NO: 7) was able to reduce the ischemia-induced microcirculatory rarefaction and impaired myocardial function.

Example 3 - miR-92 Inhibition in Cardiac Pathophysiology

[0120] The effects of miR-92 inhibition with regional application of a miR-92 inhibitor in three large animal models of cardiac pathophysiology myocardial ischemia were quantified. The three large animal models of cardiac pathophysiology were: (1) acute myocardial ischemia (myocardial infarction), (2) chronic myocardial ischemia (hibernating myocardium), and (3) pressure-induced cardiac remodeling (pathologic hypertrophy).

Acute Myocardial Ischemia (Myocardial Infarction)

[0121] In a model of acute myocardial infarction, recover}' of left ventricular function was assessed 24h and 7d after application of LNA-92a (SEQ ID NO: 7) into the coronary vessels (anterogradely or retrogradelv). Regional application improved functional recovery (e.g. ejection fraction) and limited infarct size (normalized to area at risk). This effect was not observed after intravenous injection.

Chronic Myocardial Ischemia (Hibernating Myocardium)

[0122] Hibernating myocardium was induced by a reduction stent, inducing eventual occlusion of a coronary artery. Regional LNA-92a (SEQ ID NO: 7) treatment was induced by retroinfusion into the coronary vein accompanying the occluded artery. The model is depicted in FIG. 5.

[0123] Global and regional myocardial function of the ischemic area improved (FIGS. 6-7). This concurred with enhanced collateral formation and angiogenesis, indicating improved coronary perfusion (FIGS. 8-9). Myocardial fibrosis and myocyte hypertrophy were also reduced (FIGS. 10-11).

Pressure-Induced Cardiac Remodeling (Pathologic Hypertrophy) [0124] As opposed to ischemic cardiomyopathy, pathologic hypertrophy is caused by dysregulated cardiomyocyte growth, leading to dysfunction, fibrosis and capillary rarefaction. In a porcine model of percutaneously induced transverse aortic constriction (pTAC), LNA~92a (SEQ ID NO: 7) application improved regional myocardial function, decreased heart weight to body weight ratio and decreased fibrosis. libition m ischemia am lepertusion habetic Animals

[0125] Diabetic cardiomyopathy is a clinically relevant form of heart disease with distinct pathophysiology compared to heart failure in non-diabetic patients. In order to determine whether results in non-diabetic subjects could be expected to diabetic subjects, additional experiments were performed. The diabetic pig has a microvascular cardiac dysfunction that resembles diabetic cardiomyopathy in humans. Using this apporach angiogenesis induced by miR-92 inhibition in diabetic and non-diabetic subjects was compared.

[0126] The effects of miR-92 inhibition in an in vivo model of ischemia and reperfusion in diabetic (db) pigs was examined. miR-92 inhibition was achieved by anterograde application of the oligonucleotide. The diabetic pigs were INS^C94y transgenic pigs (Renner et al, Diabetes 2013). FIG. 12 depicts the in vivo model utilized in this example. FIG 13 shows the effect of LNA-92a (SEQ ID NO: 7) administration on infarct size in the diabetic pigs. FIGS. 14 shows the effects of LNA-92a (SEQ ID NO: 7) administration on global myocardial function in the diabetic pigs.

[0127] These experiments in db pigs demonstrate that miR-92 inhibition, surprisingly, has the same effect on the cardiac function in diabetic patients and non-diabetic subjects. Therefore, it is possible to ameliorate sequela of diabetic cardiomyopathy (e.g., decrease infarct size) using miR- 92 therapy.

[0128] It is understood that the disclosed invention is not limited to the particular methodology, protocols and materials described as these can vary. It is also understood that the terminology used herein is for the purposes of describing particular embodiments only and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

The invention may be further defined by reference to the following illustrative clauses: An oligonucleotide comprising a sequence that is at least partially complementary to a miR~92 inhibitor, for use in a method of treating heart failure, wherein the

oligonucleotide reduces function or activity of miR-92.

The oligonucleotide for use according to clause 1, wherein the oligonucleotide comprises a sequence that is at least partially complementary to a miR-92 inhibitor selected from the group consisting of SEQ ID NOs: 7 to 164.

The oligonucleotide for use according to clause 1 or 2, wherein the oligonucleotide comprises:

(a) a sequence that is at least 75%, 85%, or 95% complementary to a miR-92 inhibitor; and/or

(b) a sequence containing 1, 2, 3, 4, or 5 mismatches relative to a miR-92 inhibitor,

The oligonucleotide for use according to clause 1, wherein the oligonucleotide comprises a sequence identical to SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.

The oligonucleotide for use according to any preceding clause, wherein the method of treating results in:

(a) an improvement of left ventricular function; and/or

(b) an improvement of ejection fraction; and/or

(c) a decrease of left ventricular end-diastolic pressure (LVEDP).

The oligonucleotide for use according to any preceding clause, wherein:

(a) the heart failure is characterized as heart failure with reduced ejection fraction (HFrEF); or

(b) the subject has decreased left ventricular function having an ejection fraction that is less than 40%, less than 35%, less than 30%, less than 25%, or less than 20%>; or

(c) the subject has left ventricular function having an ejection fraction that is at least 40%; preferably wherein the ejection fraction is at least 50%; and optionally wherein the heart failure is characterized as heart failure with preserved ejection fraction (HFpEF).

The oligonucleotide for use according to any preceding clause, wherein: (a) the heart failure is chronic heart failure; and/or

(b) the subject has ischemic cardiomyopathy or non-ischemic cardiomyopathy; and/or

(c) the subject has chronic myocardial ischemia; optionally wherein

administration of the oligonucleotide results in

(i) enhanced capillary density in the ischemic area; or

(ii) enhanced pericyte coverage in the ischemic area; or

(iii) reduced infarct size.

The oligonucleotide for use according to any preceding clause, wherein administration of the oligonucleotide results in improved coronary reserve, improved functional cardiac reserve, reduced myocardial fibrosis, reduced myocyte hypertrophy, enhanced neovascularization, prolonged cardioprotection, reduced endothelial cell death, reduced inflammation, and/or improved collateral growth.

The oligonucleotide for use according to any preceding clause, wherein administration of the oligonucleotide results in improved global myocardial function, optionally wherein the improved global myocardial function results in a lower LVEDP, a higher ejection fraction, increased contraction velocity, improved diastolic function, or improved regional myocardial function.

The oligonucleotide for use according to any preceding clause, wherein the method of treatment further comprises pressure- i duced cardiac remodeling (pathologic hypertrophy), optionally wherein the use of the oligonucleotide results in decreased heart-weight-to-body-weight ratio or decreased fibrosis.

The oligonucleotide for use according to any preceding clause, wherein the

oligonucleotide is for use in treating:

(a) a diabetic subject or a non-diabetic subject; and/or

(b) a human; and/or

(c) a subject who suffers from hypertension; and/or

(d) a subject who suffers from cardiac hypertrophy; and/or (e) a subject suffers from myocardial infarction, coronary artery disease, cardiomyopathy, high blood pressure, aortic stenosis, or myocarditis.

The oligonucleotide for use according to any preceding clause, wherein the miR-92 inhibitor is SEQ ID NO: 7,

The oligonucleotide for use according to any preceding clause, wherein the

oligonucleotide:

(a) comprises at least one locked nucleic acid (I.NA) containing a 2' to 4' methylene bridge; and/or

(b) comprises a sequence that is at least partially complementary to miR-92 comprises a sequence of at least 16 nucleotides, wherein the sequence comprises no more than three contiguous LNAs, wherein from the 5' end to the 3' end, positions 1, 6, 10, 11, 13 and 16 of the sequence are LNAs; optionally wherein from the 5' end to the 3' end, the sequence further comprises LNAs at positions 3, 9, and 14; or at positions 3, 8, and 14; or at positions 5, 8, and 1 5; and/or

(c) comprises at least one nucleotide that is 2'-deoxy, 2' O-alkyl or 2' halo modified; and/or

(d) has a 5' cap structure, 3' cap structure, or 5' and 3' cap structure; and/or

(e) comprises one or more phosphorothioate linkages; optionally wherein the oligonucleotide is fully phosphorothioate-linked; and/or

(f) further comprising a pendent lipophilic group. The oligonucleotide for use according to any preceding clause, wherein the

oligonucleotide is administered to a subject, optionally wherein the administration is repeated until an improvement of ejection fraction is observed, and optionally

(a) wherein administration of the oligonucleotide is performed by intravenous administration, optionally wherein the administration is performed about once every week, every month, every quarter, every half-year, or ever year; or

(b) wherein administration of the oligonucleotide is performed by subcutaneous administration or intracardiac administration; or (c) wherein administration of the oligonucleotide is performed by intracoronary administration; optionally

(i) wherein the intracoronary administration is retrograde and/or anterograde; and/or

(ii) wherein the administration is performed one, two, or three times per year.

15. The oligonucleotide for use according to any preceding clause, wherein the dose by

weight of the subject of the oligonucleotide is about 0.01 mg/kg, 0.015 mg/kg, 0.02 mg/kg, 0.03 mg/kg, 0,04 mg/kg, 0.05 mg/kg, 0.075 mg/kg, 0. 1 mg/kg, 0.15 mg/kg, 0,2 mg/kg, 0.3 mg/kg, 0.4mg/kg, 0.5 mg/kg, 1 mg/kg, or 1 ,5 mg/kg; or wherein the dose of the oligonucleotide is about 0.5 mg, 0.75 mg, 1 mg, 1.5 mg, 2 mg, 2,5 mg, 3,75 mg, 5 mg, 7,5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 50 mg, or 75 mg.

16. The oligonucleotide for use according to clause 1, wherein the oligonucleotide

comprises a sequence identical to SEQ ID NO: 7, and wherein the oligonucleotide is for use in treating heart failure which is characterized as heart failure with reduced ejection fraction (HFrEF).

17. The oligonucleotide for use according to clause 1, wherein the oligonucleotide

comprises a sequence identical to SEQ ID NO: 7, and wherein the oligonucleotide is for use in treating heart failure which is characterized as heart failure with preserved ejection fraction (HFpEF).

18. The oligonucleotide for use according to clause 1 , wherein the oligonucleotide comprises a sequence identical to SEQ ID NO: 7, and wherein the oligonucleotide is for use in treating a subject who has diabetic cardiomyopathy.

[0130] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

1. A method of treating heart failure in a subject comprising administering to the subject an oligonucleotide comprising a sequence that is at least partially complementary to a miR- 92 inhibitor selected from the group consisting of SEQ ID NOs: 7 to 164, wherein the administration of the oligonucleotide reduces function or activity of miR-92, and wherein the administration of the oligonucleotide results in an improvement of left ventricular function.

2. The method of claim 1, wherein the administration of the oligonucleotide results in an improvement of ejection fraction,

3. The method of any one of claims 1.-2, wherein the administration of the oligonucleotide results in a decrease of left ventricular end-diastolic pressure (LVEDP).

4. The method of any one of claims 1 -3, wherein the heart failure is characterized as heart failure with reduced ejection fraction (HFrEF).

5. The method of any one of claims 1-4, wherein the subject has decreased left ventricular function having an ejection fraction that is less than 40%, less than 35%, less than 30%, less than 25%, or less than 20%.

6. A method of treating heart failure in a subject comprising administering to the subject an oligonucleotide comprising a sequence that is at least partially complementary to miR-92, wherein the administration of the oligonucleotide reduces function or activity of miR-92, and wherein the subject has left ventricular function having an ejection fraction that is at least 40%.

7. The method of claim 6, wherein the ejection fraction is at least 50%.

8. The method of claim 7, wherein the heart failure is characterized as heart failure with preserved ejection fraction (HFpEF).

9. The method of any one of claims 1 to 8, wherein the heart failure is chronic heart failure.

10. The method of any one of claims 1 to 9, wherein the subject has ischemic

cardiomyopathy.

11. The method of any one of claims 1 to 10, wherein the subject has chronic myocardial ischemia.

12. The method of claim 1 1 , wherein administration of the oligonucleotide results in enhanced capillary density in the ischemic area.

13. The method of claim 11, wherein administration of the oligonucleotide results in

enhanced pericyte coverage in the ischemic area.

14. The method of claim 1 1 , wherein administration of the oligonucleotide results in reduced infarct size.

1.5. The method of any one of claims 1 to 14, wherein administration of the oligonucleotide results in improved coronary reserve.

16. The method of any one of claims 1 to 1 5, wherein administration of the oligonucleotide results in improved functional cardiac reserve.

1.7. The method of any one of claims 1 to 16, wherein administration of the oligonucleotide results in reduced myocardial fibrosis.

18. The method of any one of claims 1 to 17, wherein administration of the oligonucleotide results in reduced myocyte hypertrophy.

1.9. The method of any one of claims 1 to 18, wherein administration of the oligonucleotide results in enhanced neovascularization.

20. The method of any one of claims 1 to 19, wherein administration of the oligonucleotide results in prolonged cardioprotection.

21. The method of any one of claims 1 to 20, wherein administration of the oligonucleotide results in reduced endothelial cell death.

22. The method of any one of claims 1 to 21, wherein administration of the oligonucleotide results in reduced inflammation.

23. The method of any one of claims 1 to 22, wherein administration of the oligonucleotide results in improved collateral growth.

24. The method of any one of claims 1 to 23, wherein administration of the oligonucleotide results in improved global myocardial function.

25. The method of claim 24, wherein the improved global myocardial function results in a lower LVEDP.

26. The method of claim 24, wherein the improved global myocardial function results in a higher ejection fraction,

27. The method of claim 24, wherein the improved global myocardial function results in increased contraction velocity.

28. The method of claim 24, wherein the improved global myocardial function results in improved diastolic function.

29. The method of claim 24, wherein the improved global myocardial function results in improved regional myocardial function.

30. The method of any one of claims 1 to 29, wherem the subject is undergoing pressure- induced cardiac remodeling (pathologic hypertrophy).

31. The method of claim 30, wherein administration of the oligonucleotide results in

decreased heart weight to body weight ratio.

32. The method of claim 30, wherein administration of the oligonucleotide results in

decreased fibrosis.

33. The method of any one of claims 1 to 9, wherein the subject has non-ischemic

cardiomyopathy.

34. The method of any one of claims 1 to 33, wherem the subject is a diabetic subject.

35. The method of any one of claims 1 to 33, wherem the subject is a non-diabetic subject.

36. The method of any one of claims 1 to 35, wherein the miR-92 inhibitor is SEQ ID NO: 7.

37. The method of any one of claims 1 - 36, wherem the oligonucleotide comprises at least one locked nucleic acid f LNA) containing a to 4' methylene bridge.

38. The method of any one of claims 1 to 37, wherein the oligonucleotide comprising a

sequence that is at least partially complementary to miR-92 comprises a sequence of at least 16 nucleotides, wherein the sequence comprises no more than three contiguous LNAs, wherein from the 5' end to the 3' end, positions 1, 6, 10, 1 1, 13 and 16 of the sequence are LNAs.

39. The method of claim 38, wherem from the 5' end to the 3' end, the sequence further comprises LN As at positions 3, 9, and 14.

40. The method of claim 38, wherein from the 5' end to the 3' end, the sequence further comprises LNAs at positions 3, 8, and 14.

41. The method of claim 38, wherein from the 5' end to the 3' end, the sequence further comprises LNAs at positions 5, 8, and 15.

42. The method of any one of claims 1-41, wherein the oligonucleotide comprises at least one nucleotide that is 2'-deoxy, O-alkyl or 2' halo modified.

43. The method of any one of claims 1-42, wherein the oligonucleotide has a 5' cap structure, 3' cap structure, or 5' and 3' cap structure.

44. The method of any one of claims 1-43, wherein the oligonucleotide comprises one or more phosphorothioate linkages.

45. The oligonucleotide of claim 44, wherein the oligonucleotide is fully phosphorothioate- linked.

46. The method of any one of claims 1-45, further comprising a pendent lipophilic group.

47. The method of any one of claims 1-46, wherein the subject is a human.

48. The method of any one of claims 1-47, wherein administration of the oligonucleotide is performed by intravenous administration.

49. The method of any one of claims 1-47, wherein administration of the oligonucleotide is performed by subcutaneous administration.

50. The method of any one of claims 1-47, wherein administration of the oligonucleotide is performed by intracardiac administration.

51. The method of any one of claims 1-47, wherein administration of the oligonucleotide is performed by intracoronary administration.

52. The method of claim 51, wherein the intracoronary administration is retrograde.

53. The method of claim 51 , wherein the intracoronary administration is anterograde.

54. The method of any of claims 1 -53, wherein the dose by weight of the subject of the

oligonucleotide is about 0.01 mg/kg, 0.015 mg/kg, 0.02 mg/kg, 0.03 mg/kg, 0.04 mg/kg, 0,05 mg/kg, 0.075 mg/kg, 0. 1 mg/kg, 0.15 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4mg/kg, 0.5 mg/kg, 1 mg/kg, or 1.5 mg/kg.

55. The method of any of claims 1 -53, wherein the dose of the oligonucleotide is about 0.5 mg, 0.75 mg, 1 mg, 1 .5 mg, 2 mg, 2.5 mg, 3.75 mg, 5 rag, 7,5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 50 mg, or 75 mg.

56. The method of any of claims 51-55, wherein the administration is performed one, two, or three times per year.

57. The method of claim 48, wherein the administration is performed about once every week, every month, every quarter, every half-year, or ever year.

58. The method of any of claims 1-57, wherein the administration is repeated until an

improvement of ejection fraction is observed.

59. The method of any of claims 1-58, wherein the subject suffers from hypertension.

60. The method of any of claims 1 -59, wherein the subject suffers from cardiac hypertrophy

61. The method of any of claims 1-60, wherein the subject suffers from myocardial

infarction, coronary artery disease, cardiomyopathy, high blood pressure, aortic stenosis, or myocarditis.

62. A method of treating heart failure in a subject comprising administering to the subject an oligonucleotide comprising a sequence identical to SEQ ID NOs: 7 to 9, wherein the administration of the oligonucleotide reduces function or activity of miR-92, and wherein the administration of the oligonucleotide results in an improvement of left ventricular function.

63. The method of claim 62 wherein the oligonucleotide comprises a sequence identical to SEQ ID NOs: 7.

64. The method of claim 62 wherein the oligonucleotide comprises a sequence identical to SEQ ID NOs: 8.

65. The method of claim 62 wherein the oligonucleotide comprises a sequence identical to SEQ ID NOs: 9.

66. The method of any of claim 1-62 wherein the oligonucleotide is SEQ ID NOs: 7, is SEQ ID NOs: 8, or is SEQ ID NOs: 9.