WO2009058818A2 - Compositions comprising a micro-rna and methods of their use in regulating cardiac remodeling - Google Patents

Abstract

The present invention relates to the identification of a microRNA, miR-21, that alters energy metabolism in cardiomyocytes and thus contributes to cardiac remodeling. Inhibition of this function is proposed as a treatment for cardiac hypertrophy, heart failure, and/or myocardial infarction.

Description

COMPOSITIONS COMPRISING A MICRO-RNA AND METHODS OF THEIR USE IN REGULATING CARDIAC REMODELING

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 60/983,490, filed October 29, 2007, which is herein incorporated by reference in its entirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with grant support under grant no. HL53351-06 from the National Institutes of Health. The government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates generally to the fields of developmental biology and molecular biology. More particularly, it concerns gene regulation and cellular physiology in cardiomyocytes and cardiac fibroblasts. Specifically, the invention relates to the identification of an miRNA that results in altered energy metabolism in cardiomyocytes and proliferation of fibroblasts which contributes to pathologic cardiac remodeling.

BACKGROUND OF THE INVENTION Heart disease and its manifestations, including coronary artery disease, myocardial infarction, congestive heart failure, and cardiac hypertrophy, clearly presents a major health risk in the United States today. The cost to diagnose, treat and support patients suffering from these diseases is well into the billions of dollars. Two particularly severe manifestations of heart disease are myocardial infarction and cardiac hypertrophy. With respect to myocardial infarction, typically an acute thrombocytic coronary occlusion occurs in a coronary artery as a result of atherosclerosis and causes myocardial cell death. Because cardiomyocytes, the heart muscle cells, are terminally differentiated and generally incapable of cell division, they are generally replaced by scar tissue when they die during the course of an acute myocardial infarction. Scar tissue is not contractile, fails to contribute to cardiac function, and often plays a detrimental role in heart function by expanding during cardiac contraction, or by increasing the size and effective radius of the ventricle, for example, becoming hypertrophic.

With respect to cardiac hypertrophy, one theory regards this as a disease that resembles aberrant development and, as such, raises the question of whether developmental signals in the heart can contribute to hypertrophic disease. Cardiac hypertrophy is an adaptive response of the heart to virtually all forms of cardiac disease, including those arising from hypertension, mechanical load, myocardial infarction, cardiac arrhythmias, endocrine disorders, and genetic mutations in cardiac contractile protein genes. While the hypertrophic response is initially a compensatory mechanism that augments cardiac output, sustained hypertrophy can lead to dilated cardiomyopathy (DCM), heart failure, and sudden death. In the United States, approximately half a million individuals are diagnosed with heart failure each year, with a mortality rate approaching 50%. The causes and effects of cardiac hypertrophy have been extensively documented, but the underlying molecular mechanisms have not been elucidated. Understanding these mechanisms is a major concern in the prevention and treatment of cardiac disease and will be crucial as a therapeutic modality in designing new drugs that specifically target cardiac hypertrophy and cardiac heart failure.

Treatment with pharmacological agents represents the primary mechanism for reducing or eliminating the manifestations of heart failure. Diuretics constitute the first line of treatment for mild-to-moderate heart failure. If diuretics are ineffective, vasodilatory agents, such as angiotensin converting (ACE) inhibitors (e.g., enalopril and lisinopril) or inotropic agent therapy (i.e., a drug that improves cardiac output by increasing the force of myocardial muscle contraction) may be used. Unfortunately, many of these standard therapies have numerous adverse effects and are contraindicated in some patients. Thus, the currently used pharmacological agents have severe shortcomings in particular patient populations. The availability of new, safe and effective agents would undoubtedly benefit patients who either cannot use the pharmacological modalities presently available, or who do not receive adequate relief from those modalities.

The adult heart is a dynamic organ capable of significant remodeling and hypertrophic growth as a means of adapting function to altered workloads or injury. Hemodynamic stress or neuroendocrine signaling associated with myocardial infarction, hypertension, aortic stenosis, and valvular dysfunction evoke a pathologic remodeling response through the activation of intracellular signaling pathways and transcriptional mediators in cardiac myocytes. Activation of these molecular pathways enhances cardiomyocyte size and protein synthesis, induces the assembly of sarcomeres, and causes reexpression of fetal cardiac genes. Although aspects of the hypertrophic response after acute and chronic stress may initially augment cardiac output, prolonged hypertrophy is a major predictor of heart failure and sudden death. There have been major advances in the identification of genes and signaling pathways involved in this disease process, but the overall complexity of hypertrophic remodeling suggests that additional regulatory mechanisms remain to be identified. MicroRNAs have recently been implicated in a number of biological processes including regulation of developmental timing, apoptosis, fat metabolism, and hematopoietic cell differentiation among others. MicroRNAs (miRNAs or miRs) are small, non-protein coding RNAs of about 18 to about 25 nucleotides in length that regulate gene expression in a sequence-specific manner. miRNAs are transcribed by RNA polymerase II as primary transcripts that are usually several thousand bases in length that are derived from from individual miRNA genes, from introns of protein coding genes, or from poly-cistronic transcripts that often encode multiple, closely related miRNAs. See review of Carrington et al. (2003). MiRs act as repressors of target mRNAs by promoting their degradation, when their sequences are perfectly complementary, or by inhibiting translation, when their sequences contain mismatches. miRNAs are transcribed by RNA polymerase II (pol II) or RNA polymerase III (pol III; see Qi et al. (2006) Cellular & Molecular Immunology Vol. 3:411-419) and arise from initial transcripts, termed primary miRNA transcripts (pri-miRNAs), that are generally several thousand bases long. Pri-miRNAs are processed in the nucleus by the RNase Drosha into about 70- to about 100-nucleotide hairpin-shaped precursors (pre-miRNAs). Following transport to the cytoplasm, the hairpin pre-miRNA is further processed by Dicer to produce a double-stranded miRNA (Lee et al., 1993). The mature miRNA strand is then incorporated into the RNA-induced silencing complex (RISC), where it associates with its target mRNAs by base-pair complementarity. In the relatively rare cases in which a miRNA base pairs perfectly with an mRNA target, it promotes mRNA degradation. More commonly, miRNAs form imperfect heteroduplexes with target mRNAs, affecting either mRNA stability or inhibiting mRNA translation.

The 5' portion of a miRNA spanning bases 2-8, termed the 'seed' region, is especially important for target recognition (Krenz and Robbins, 2004; Kiriazis and Kranias, 2000). The sequence of the seed, together with phylogenetic conservation of the target sequence, forms the basis for many current target prediction models. Although increasingly sophisticated computational approaches to predict miRNAs and their targets are becoming available, target prediction remains a major challenge and requires experimental validation. Ascribing the functions of miRNAs to the regulation of specific mRNA targets is further complicated by the ability of individual miRNAs to base pair with hundreds of potential high and low affinity mRNA targets and by the targeting of multiple miRNAs to individual mRNAs.

The high sequence conservation of many miRNAs across metazoan species suggests strong evolutionary pressure and participation in essential biologic processes (Reinhart et al., 2000; Stark et ah, 2005). Indeed, miRNAs have been shown to play fundamental roles in diverse biological and pathological processes, including cell proliferation, differentiation, apoptosis, and carcinogenesis in species ranging from Caenorhabditis elegans and Drosophila melanogaster to humans. However, there remains limited information on the role that miRNAs play in cardiogenesis and molecular events that can contribute to heart disease.

SUMMARY OF THE INVENTION

The present invention provides a method of treating pathologic cardiac hypertrophy, heart failure, or myocardial infarction in a subject in need thereof. In one embodiment, the method comprises (a) identifying a subject having cardiac hypertrophy, heart failure or myocardial infarction; and (b) inhibiting expression or activity of miR-21 in heart cells of the subject. In another embodiment, the method further comprises administering to the subject a second therapy. The second therapy may be, for example, a beta blocker, an ionotrope, a diuretic, ACE inhibitor, All antagonist, BNP, a Ca++-blocker, and ERA, or an HDAC inhibitor.

In some embodiments of the invention, inhibiting the expression or activity of miR- 21 comprises administering an antagomir of miR-21. In one embodiment, the present invention provides a miR-21 antagomir. In another embodiment, miR-21 expression or activity is inhibited by administering an antisense oligonucleotide that targets the mature miR-21 sequence. In yet another embodiment, miR-21 expression or activity is inhibited by administering an inhibitory RNA molecule, wherein the inhibitory RNA molecule comprises a double stranded region that is at least partially identical and complementary to the mature miR-21 sequence. The inhibitory RNA molecule may be a ribozyme, siRNA or shRNA molecule.

The present invention also provides a method of preventing pathologic hypertrophy or heart failure in a subject in need thereof comprising identifying a subject at risk of developing pathologic cardiac hypertrophy or heart failure; and inhibiting expression or activity of miR-21 in heart cells (e.g myocytes, fibroblasts, endothelial cells) of said subject. In one embodiment, inhibiting comprises delivering to the heart cells an inhibitor of miR-21. In another embodiment, the subject at risk may exhibit one or more risk factors selected from the group consisting of long standing uncontrolled hypertension, uncorrected valvular disease, chronic angina, recent myocardial infarction, congenital predisposition to heart disease, and pathological hypertrophy.

Antagomirs, antisense oligonucleotides, inhibitory RNA molecules, or other modulators of miR-21 expression or activity may be administered by any method known to those in the art suitable for delivery to the targeted organ, tissue, or cell type. For example, in certain embodiments of the invention, the modulator of miR-21 may be administered by parenteral administration, such as intravenous injection, intraarterial injection, intrapericardial injection, or subcutaneous injection, or by direct injection into the tissue (e.g., cardiac tissue). In some embodiments, the modulator of miR-21 may be administered by oral, transdermal, intraperitoneal, subcutaneous, sustained release, controlled release, delayed release, suppository, or sublingual routes of administration. In other embodiments, the modulator of miR-21 may be administered by a catheter system.

The present invention also encompasses a transgenic, non-human mammal, the cells of which fail to express a functional miR-21. In another embodiment, the invention provides a transgenic, non-human mammal, the cells of which comprise a miR-21 coding region under the control of a heterologous promoter active in the cells of said non-human mammal. In some embodiments, the mammal is a mouse. The present invention provides a method for identifying a modulator of miR-21. In one embodiment, the method comprises contacting a cell with a candidate compound; assessing miR-21 activity or expression; and comparing the activity or expression in step (b) with the activity or expression in the absence of the candidate compound, wherein a difference between the measured activities or expression indicates that the candidate compound is a modulator of miR-21. The cell may be contacted with the candidate compound in vitro or in vivo. The candidate compound may be a protein, a peptide, a polypeptide, a polynucleotide, an oligonucleotide, or small molecule. The modulator of miR- miR-21 may be an agonist or inhibitor of miR-21. The modulator of miR-21 may be an agonist or inhibitor of an upstream regulator of miR-21.

The present invention also provides a pharmaceutical composition comprising an inhibitor of miR-21. In one embodiment, the composition is formulated for injection. In another embodiment, the pharmaceutical composition is combined with a kit for administration, such as parenteral or catheter administration. In another embodiment, the present invention provides a method of treating cancer in a subject in need thereof comprising administering to the subject an inhibitor of miR-21. In some embodiments, the inhibitor may be delivered in conjunction with a second cancer therapy, such as chemotherapy, radiotherapy, immunotherapy, hormonal therapy or gene therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

Figure 1. MiRNA expression during cardiac hypertrophy. A. H&E stained sections of representative hearts from mice following sham and thoracic aortic banding (TAB) for 21 days and from calcineurin transgenic (CnA Tg) mice. Scale bar equals 2 mm. Venn diagrams illustrating the numbers of microRNAs that changed in expression in each type of heart are shown in the bottom panel. B. Northern blots for particular microRNAs that were upregulated (top panel) or downregulated (bottom panel) during hypertrophy. C. Bar graph indicating the fold change in expression of miRNAs of interest during both TAB- (blue) and CnA- (green) induced hypertrophy compared to baseline. Histology sections of each type of hypertrophic heart are shown on the left of the bar graph. D. Changes in expression of miR-21 during TAB and calcineurin-induced hypertrophy. Expression of U6 RNA was used as a loading control.

Figure 2. miRNA 21 is conserved among species and shows a ubiquitous expression pattern. A. Intergenic miR-21 is located immediately after the 3 'UTR of the TMEM49 gene and is expressed as a separate 3.4 kb transcript. Both the mature sequence (SEQ ID NO: 1) and star sequence (SEQ ID NO: 2) are conserved among species (SEQ ID NOs: 3-8). B. Northern blot analysis for miR-21 in different tissues shows that miR-21 is broady expressed. U6 expression was used as a loading control.

Figure 3. MiR-21 expression in response to cardiac stress. A. The first five panels show up-regulation of miR-21 in mouse hearts following thoracic aortic banding (TAB), expressing activated calcineurin (CnA), following myocardial infarction (MI), and chronic delivery of either angiotensin II (Angll) or isoproterenol (ISO). Excersize-induced hypertrophy did not induce miR-21 expression. Northern blot analysis of miR-21 in primary cardiomyocytes in vitro treated with serum free medium (SF) or phenylephrine (PE). Baseline expression of miR-21 in fibroblasts, (far right panel). B. MiR-21 expression in cardiac tissue from hypertensive rats at different ages (2 mos, 15 mos, and 24 mos). C. Cardiac hypertrophy in the same hypertensive rats as in B. Cardiac hypetroiphy was measured by heart weight (HW) and left ventricular (LV) weight to body weight (BW) ratios.

Figure 4. Stress responsiveness of miR-21 is regulated by MEF2. Electrophoretic mobility shift analysis indicates the presence of a functional MEF2 binding site upstream of the miR-21 transcript. MEF2 is a stress responsive transcription factor the activity of which increases during cardiac stress.

Figure 5. MiR-21 expression is transcribed independently of TMEM49 expression. A. Northern blot analysis showing miR-21 to be ubiquitously expressed in various tissues. RT-PCR analysis of TMEM49 on the same tissues show a different expression pattern than that for miR-21. B. Northern blot analysis for miR-21 expression and RT-PCR analysis for TMEM49 expression in cardiac tissue in animals who underwent thoracic aortic banding (TAB) or myocardial infarction (MI). The stress inducibility of miR- 21 is not mirrored by a comparable increase in expression of TMEM49. Expression of GAPDH was used as a control.

Figure 6 Conditional gene deletion miR-21. A. Targeting strategy to generate a conditional gene deletion of miR-21. B. Southern blot analysis showing a successful ES cell targeting event.

Figure 7. Genomic deletion of miR-21 results in viable and fertile animals. A. Realtime PCR analysis for miR-21 expression in wild-type (WT), heterozygous (Het), and homozygous (KO) animals. B. Northern blot analysis of lung tissue from wild-type animals or animals heterozygous or homozygous for deletion of miR-21. C. RT-PCR for TMEM49 in wild-type, heterozygous, and homozygous animals. Genetic deletion of miR-21 does not affect the expression of TMEM49.

Figure 8. Genes regulated in cardiac tissue upon global deletion of miR-21. Microarray analysis of cardiac tissue reveals genes that are significantly upregulated and downregulated in response to miR-21 deletion. Fold-expression is compared to expression in wild-type animals.

Figure 9. MiR-21 regulates cardiac levels of PTEN. Western blot analysis of cardiac tissue isolated from wild- type (WT) and miR-21 knockout (KO) animals. Blots were probed with a mouse antibody to PTEN or Sproutyl (SRPYl). GAPDH expression was used as a loading control. Figure 10. MiR-21 influences the cardiac stress response. A. Northern blot analysis of cardiac tissue from wild-type (WT) or miR-21 knockout (KO) animals in sham conditions and after thoracic aortic banding procedures (TAB). B. The ratio between heart weight and body weight, as a measure for hypertrophy, is shown for WT and KO animals under sham (unstressed) conditions and TAB (stress) conditions. C. Realtime PCR analysis for the stress responsive gene, βMHC, in cardiac tissue of WT animals and KO animals before and after stress. D. Realtime mRNA analysis indicates an increase of thioredoxin- interacting protein (Txnip) in miR-21 knockout animals as compared to wild-type animals.

Figure 11. MiR-21 is regulated by stress responsive factor. A. The stress responsive factor (SRF) binding site located upstream of the pri-miRNA-21 is conserved among species. B. Electrophoretic mobility shift analysis indicates a functional SRF binding site upstream of miR-21. Mutation of this site abolishes SRF binding. Figure 12. SRF can activate transcription of miR-21. A. Luciferase expression in cells transfected with a construct in which the luciferase reporter gene was under the control of the miR-21 regulatory sequence. Cells were exposed to increasing concentrations of myocardin, an activator of SRF. B. Schematic model of the regulation of the cardiac stress response by miR-21. In response to stress, the heart secretes pro-fibrotic cytokines/hormones, which stimulate the activity of SRF. Activation of SRF leads to the induction of miR-21, which in turn reduces the level of PTEN. PTEN normally blocks the proliferation and migration of fibroblasts. Thus, inhibition of PTEN expression by increased levels of miR-21 results in fibroblast proliferation and the onset of cardiac fibrosis. Figure 13. In vivo modulation of miR-21. A. Sequence of a synthetic oligonucleotide targeted to the mature miR-21 sequence. The mismatch control sequence contains four base mismatches compared to the anti miR-21 sequence. B. Northern blot analysis of miR-21 expression in different tissues following intravenous injection (IV) of 80 mg/kg anti miR-21, mismatch miR-21 (scrambled miR-21), or saline. Figure 14. C ardio myocyte specific overexpression of miR-21 induces cardiac remodeling. A. Northern blot analysis on hearts from wild-type (WT) and miR-21 transgenic (Tg) mice shows efficient cardiac overexpression is achieved in the transgenic mice using the myocyte specific αMHC promoter. B. H&E stained sections of the heart show that over- expression of miR-21 induces cardiac growth and fibrosis. The lower panels show a higher magnification of the H&E sections, which indicate severe myocyte disorganization in the miR-21 Tg animals compared to WT. C. Heart weight (HW) to body weight (BW) ratios and realtime PCR analysis for atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) in cardiac tissue from WT and miR-21 Tg animals.

Figure 15. Microarray analysis of miR-21 transgenic hearts compared to wild type. Microarray analysis was performed on mRNA isolated from wild type and miR-21 transgenic hearts at 6 weeks of age. The most downregulated genes are shown. MiR-21 regulated genes are biased toward genes involved in cardiac metabolism, including the PP ARa gene, which encodes a transcription factor that regulates fatty acid metabolism.

Figure 16. MiR-21 targets PP ARa, a master regulator of cardiac metabolism. A. Putative miR-21 binding site located within the 3' UTR of the PP ARa gene. B. Realtime PCR analysis for PP ARa and PGC lα in cardiac tissue from miR-21 transgenic (Tg) and wild-type (WT) animals.

Figure 17. MiR-21 overexpression in myocytes induces mitochondrial abnormalities. H & E stained sections of hearts from miR-21 trangenic animals and wild- type animals. The transgenic animals have gross mitochondrial abnormalities compared to wild-type littermates.

Figure 18. Model for the control of cardiac energy metabolism by miR-21. In the normal heart, miR-21 represses expression of PP ARa, causing a reduction in metabolic enzymes. In response to stress, miR-21 expression increases, and PP ARa expression decreases with a shift from oxidative to glycolytic metabolism.

Figure 19. Additional targets of miR-21. Although miR-21 regulates cardiac metabolism through PP ARa, the effect of overexpression of miR-21 on cardiac remodeling may be dependent on the downregulation of Sproutyl and -2.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, in part, on the discovery that miR-21 is upregulated during pathological cardiac remodeling and is associated with heart failure. Cardiac overexpression of miR-21 contributes to energy utilization changes by down- regulating expression of PP ARa and is sufficient to induce hypertrophy and eventually heart failure. Additionally miR-21 overexpression may influence fibroblast proliferation by regulating expression of PTEN or Sprouty. In view of these findings, the invention describes strategies to modulate the expression of miR-21 as a means to prevent pathological cardiac remodeling in humans.

Thus, the present invention provides a method of treating pathologic cardiac hypertrophy, heart failure, or myocardial infarction in a subject in need thereof by inhibiting miR-21 expression or activity. In one embodiment, the method comprises identifying a subject having cardiac hypertrophy, heart failure, or myocardial infarction; and inhibiting expression or activity of miR-21 in heart cells of said subject. The method may optionally include identifying a subject having cardiac overexpression of miR-21. MiRNA-21 (miR-21) is an intergenic miRNA expressed as a primary transcript of 3.4 kb and efficiently processed into the mature miR-21 sequence (Xuezhong et al., RNA 2004). MiR-21 is located immediately after the 3'UTR of the TMEM49 (vacuole membrane protein) gene on human chromosome 17 and is expressed as a separate transcript (see Fig. 2A). The mature miR-21 sequence is 5'- UAGCUUAUC AGACUGAUGUUGA-3 ' (SEQ ID NO: 1). There is also a star miR-21 sequence that is processed from the other strand of the stem loop structure. This star miR-21 sequence (miR-21*) is 5'-

CAACAGCAGUCGAUGGGCUGUC-3' (SEQ ID NO: 2) in the mouse. The human star miR-21 sequence varies by one nucleotide from the mouse sequence and is 5'- CAAC ACC AGUCGAUGGGCUGUC-S' (SEQ ID NO: 17).

At baseline, miR-21 is very broadly expressed with a high expression level in the pancreas and a fairly low expression level in brain and heart. However, upon a variety of pathological cardiac stresses, miR-21 is significantly upregulated, while physiological stress, like voluntary wheel running in mice, fails to induce such an increase. This stress-dependent increase is very likely dependent on a highly conserved region upstream of the primary transcript containing MEF2, NFAT, API, and SRF binding sites, all of which are known to be involved in stress-related gene expression. MiR-21 has no family members, and cardiac overexpression of miR-21 is sufficient to drive cardiac pathology. Therefore, miR-21 modulation, removal, or inhibition may result in cardiac protection against stress-induced remodeling.

In one embodiment, the invention provides a method of treating pathologic cardiac hypertrophy, heart failure, or myocardial infarction in a subject in need thereof comprising administering to the subject an inhibitor of miR-21. In another embodiment, the method comprises identifying a subject at risk of developing pathologic cardiac hypertrophy or heart failure and inhibiting expression or activity of miR-21 in heart cells of the subject. "Heart cells" as used herein include cardiomyocytes, cardiac fibroblasts, and cardiac endothelial cells. The subject at risk of developing pathologic cardiac hypertrophy or heart failure may exhibit one or more risk factors including, for example, long standing uncontrolled hypertension, uncorrected valvular disease, chronic angina, recent myocardial infarction, congenital predisposition to heart disease or pathological hypertrophy. In certain embodiments, the subject at risk may be diagnosed as having a genetic predisposition to cardiac hypertrophy. In some embodiments of the invention, the subject at risk may have a familial history of cardiac hypertrophy. In another embodiment, the present invention provides a method of preventing cardiac hypertrophy and dilated cardiomyopathy in a subject in need thereof comprising inhibiting expression or activity of miR-21 in heart cells of the subject. In yet a further embodiment, the present invention provides a method of inhibiting progression of cardiac hypertrophy in a subject in need thereof comprising inhibiting expression or activity of miR- 21 in heart cells of the subject. In further embodiments, the present invention provides a method of increasing exercise tolerance, reducing hospitalization, improving quality of life, decreasing morbidity, and/or decreasing mortality in a subject with heart failure or cardiac hypertrophy comprising inhibiting expression or activity of miR-21 in heart cells of the subject.

Thus, the present invention provides methods for the treatment of cardiac hypertrophy, heart failure, or myocardial infarction utilizing inhibitors of miR-21. Preferably, administration of a miR-21 inhibitor results in the improvement of one or more symptoms of cardiac hypertrophy, heart failure, or myocardial infarction in the subject, or in the delay in the transition from cardiac hypertrophy to heart failure. The one or more improved symptoms may be, for example, increased exercise capacity, increased cardiac ejection volume, decreased left ventricular end diastolic pressure, decreased pulmonary capillary wedge pressure, increased cardiac output, increased cardiac index, lowered pulmonary artery pressures, decreased left ventricular end systolic and diastolic dimensions, decreased cardiac fibrosis, decreased collagen deposition in cardiac muscle, decreased left and right ventricular wall stress, decreased wall tension, increased quality of life, and decreased disease related morbidity or mortality. In addition, use of inhibitors of miR-21 may prevent cardiac hypertrophy and its associated symptoms from arising.

Inhibition of microRNA function may be achieved by administering antisense oligonucleotides targeting the mature miR-21 sequence. The antisense oligonucleotides may be ribonucleotides or deoxyribonucleotides. Preferably, the antisense oligonucleotides have at least one chemical modification. Antisense oligonucleotides may be comprised of one or more "locked nucleic acids". "Locked nucleic acids" (LNAs) are modified ribonucleotides that contain an extra bridge between the 2' and 4' carbons of the ribose sugar moiety resulting in a "locked" conformation that confers enhanced thermal stability to oligonucleotides containing the LNAs. Alternatively, the antisense oligonucleotides may comprise peptide nucleic acids (PNAs), which contain a peptide-based backbone rather than a sugar-phosphate backbone. Other chemical modifications that the antisense oligonucleotides may contain include, but are not limited to, sugar modifications, such as T- O-alkyl (e.g. 2'-O-methyl, 2'-O-methoxyethyl), 2'-fluoro, and 4' thio modifications, and backbone modifications, such as one or more phosphorothioate, morpholino, or phosphonocarboxylate linkages (see, for example, U.S. Patent Nos. 6,693,187 and 7,067,641, which are herein incorporated by reference in their entireties). In some embodiments, suitable antisense oligonucleotides are 2'-O-methoxyethyl "gapmers" which contain 2'-O- methoxyethyl-modified ribonucleotides on both 5' and 3' ends with at least ten deoxyribonucleotides in the center. These "gapmers" are capable of triggering RNase Independent degradation mechanisms of RNA targets. Other modifications of antisense oligonucleotides to enhance stability and improve efficacy, such as those described in U.S. Patent No. 6,838,283, which is herein incorporated by reference in its entirety, are known in the art and are suitable for use in the methods of the invention. Preferable antisense oligonucleotides useful for inhibiting the activity of micro RNAs are about 19 to about 25 nucleotides in length. Antisense oligonucleotides may comprise a sequence that is at least partially complementary to a mature miRNA sequence, e.g. at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary to a mature miRNA sequence. In some embodiments, the antisense oligonucleotide may be substantially complementary to a mature miRNA sequence, that is at least about 95%, 96%, 97%, 98%, or 99% complementary to a target polynucleotide sequence. In one embodiment, the antisense oligonucleotide comprises a sequence that is 100% complementary to a mature miRNA sequence.

In some embodiments, the antisense oligonucleotides are antagomirs. "Antagomirs" are single-stranded, chemically-modified ribonucleotides that are at least partially complementary to the miRNA sequence. Antagomirs may comprise one or more modified nucleotides, such as 2'-O-methyl-sugar modifications. In some embodiments, antagomirs comprise only modified nucleotides. Antagomirs may also comprise one or more phosphorothioate linkages resulting in a partial or full phosphorothioate backbone. To facilitate in vivo delivery and stability, the antagomir may be linked to a cholesterol or other moiety at its 3' end. Antagomirs suitable for inhibiting miRNAs may be about 15 to about 50 nucleotides in length, more preferably about 18 to about 30 nucleotides in length, and most preferably about 20 to about 25 nucleotides in length. "Partially complementary" refers to a sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary to a target polynucleotide sequence. The antagomirs may be at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary to a mature miRNA sequence. In some embodiments, the antagomir may be substantially complementary to a mature miRNA sequence, that is at least about 95%, 96%, 97%, 98%, or 99% complementary to a target polynucleotide sequence. In other embodiments, the antagomirs are 100% complementary to the mature miRNA sequence.

Another approach for inhibiting the function of miR-21 is administering an inhibitory RNA molecule having a double stranded region that is at least partially identical and partially complementary to the mature miR-21 sequence. The inhibitory RNA molecule may be a double-stranded, small interfering RNA (siRNA) or a short hairpin RNA molecule (shRNA) comprising a stem-loop structure. The double-stranded regions of the inhibitory RNA molecule may comprise a sequence that is at least partially identical and partially complementary, e.g. about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical and complementary, to the mature miRNA sequence. In some embodiments, the double- stranded regions of the inhibitory RNA comprise a sequence that is at least substantially identical and substantially complementary to the mature miRNA sequence. "Substantially identical and substantially complementary" refers to a sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical and complementary to a target polynucleotide sequence. In other embodiments, the double-stranded regions of the inhibitory RNA molecule may contain 100% identity and complementarity to the target miRNA sequence.

The inhibitory nucleotide molecules described herein preferably target a mature sequence of miR-21 (e.g. SEQ ID NO: 1) or a star sequence of miR-21 (e.g. SEQ ID NO: 2 or SEQ ID NO: 17). In some embodiments, inhibitors of miR-21 are antagomirs comprising a sequence that is perfectly complementary to a mature miR-21 sequence. In one embodiment, an inhibitor of miR-21 is an antagomir having a sequence that is partially or perfectly complementary to 5 '-UAGCUUAUCAGACUGAUGUUGA-S' (SEQ ID NO: 1). In another embodiment, an inhibitor of miR-21 is an antagomir having a sequence that is partially or perfectly complementary to 5 ' -CAAC AGC AGUCGAUGGGCUGUC-3 ' (SEQ ID NO: 2). In another embodiment, an inhibitor of miR-21 is an antagomir having a sequence that is partially or perfectly complementary to 5 '-CAACACCAGUCGAUGGGCUGUC-S' (SEQ ID NO: 17). In another embodiment, an inhibitor of miR-21 is an antagomir having the sequence of SEQ ID NO: 15.

In some embodiments, inhibitors of miR-21 are chemically-modified antisense oligonucleotides. In one embodiment, an inhibitor of miR-21 is a chemically-modified antisense oligonucleotide comprising a sequence substantially complementary to 5'- UAGCUUAUCAGACUGAUGUUGA-S' (SEQ ID NO: 1). In another embodiment, an inhibitor of miR-21 is a chemically-modified antisense oligonucleotide comprising a sequence substantially complementary to 5'-CAACAGCAGUCGAUGGGCUGUC-S ' (SEQ ID NO: 2). In another embodiment, an inhibitor of miR-21 is a chemically- modified antisense oligonucleotide comprising a sequence substantially complementary to 5'- CAAC ACC AGUCGAUGGGCUGUC-3' (SEQ ID NO: 17). As used herein "substantially complementary" refers to a sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% complementary to a target polynucleotide sequence (e.g. mature or precursor miRNA sequence).

Antisense oligonucleotides may comprise a sequence that is substantially complementary to a precursor miRNA sequence (pre-miRNA) for miR-21. In some embodiments, the antisense oligonucleotide comprises a sequence that is substantially complementary to a sequence located outside the stem- loop region of the pre-miR-21 sequence. In one embodiment, an inhibitor of miR-21 function is an antisense oligonucleotide having a sequence that is substantially complementary to a pre-miR-21 sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8.

In other embodiments of the invention, inhibitors of miR-21 may be inhibitory RNA molecules, such as ribozymes, siRNAs, or shRNAs. In one embodiment, an inhibitor of miR-21 is an inhibitory RNA molecule comprising a double-stranded region, wherein the double-stranded region comprises a sequence having 100% identity and complementarity to a mature miR-21 sequence (e.g. SEQ ID NO: 1). In another embodiment, an inhibitor of miR- 21 is an inhibitory RNA molecule comprising a double-stranded region, wherein the double- stranded region comprises a sequence having 100% identity and complementarity to a star miR-21 sequence (e.g. SEQ ID NO: 2 or SEQ ID NO: 17). In some embodiments, inhibitors of miR-21 function are inhibitory RNA molecules which comprise a double-stranded region, wherein said double-stranded region comprises a sequence of at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity and complementarity to a mature miR-21 sequence. In another embodiment, an expression vector may be used to deliver an inhibitor of miR-21 to a cell or subject. 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. 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.

In one embodiment, an expression vector for expressing an inhibitor of miR-21 comprises a promoter operably linked to a polynucleotide encoding an antisense oligonucleotide, wherein the sequence of the expressed antisense oligonucleotide is partially or perfectly complementary to the mature miR-21 sequence. 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. In another embodiment, an expression vector for expressing an inhibitor of miR-21 comprises one or more promoters operably linked to a polynucleotide encoding a shRNA or siRNA, wherein the expressed shRNA or siRNA comprises a double stranded region that is identical and complementarty or partially identical and partially complementary to the mature miR-21. "Partially identical and partially complementary" refers to a sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical and complementary to a target polynucleotide sequence. In certain embodiments, the nucleic acid encoding a polynucleotide of interest is under transcriptional control of a promoter. 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. The term promoter will be used here to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase I, II, or III. In some embodiments, the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, the Rous sarcoma virus long terminal repeat, rat insulin promoter and glyceraldehyde-3-phosphate dehydrogenase can be used to obtain high-level expression of the coding sequence of interest. The use of other viral or mammalian cellular or bacterial phage promoters which are well-known in the art to achieve expression of a coding sequence of interest is contemplated as well, provided that the levels of expression are sufficient for a given purpose.

By employing a promoter with well-known properties, the level and pattern of expression of the protein of interest following transfection or transformation can be optimized. Further, selection of a promoter that is regulated in response to specific physiologic signals can permit inducible expression of the gene product. Tables 1 and 2 list several regulatory elements that may be employed, in the context of the present invention, to regulate the expression of the polynucleotide of interest (e.g. miR-21 inhibitor). This list is not intended to be exhaustive of all the possible elements involved in the promotion of gene expression but, merely, to be exemplary thereof. Below is a list of viral promoters, cellular promoters/enhancers and inducible promoters/enhancers that could be used in combination with the polynucleotide of interest in an expression construct (Table 1 and Table 2). Additionally, any promoter/enhancer combination (as per the Eukaryotic Promoter Data Base EPDB) could also be used to drive expression of the gene. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional genetic expression construct.

TABLE 1 Promoter and/or Enhancer

Promoter/Enhancer References

Of particular interest are muscle specific promoters (e.g. muscle creatine kinase), and more particularly, cardiac specific promoters. These include the myosin light chain-2 promoter (Franz et ah, 1994; Kelly et ah, 1995), the α actin promoter (Moss et ah, 1996), the troponin 1 promoter (Bhavsar et ah, 1996); the Na⁺/Ca²⁺ exchanger promoter (Barnes et ah, 1997), the dystrophin promoter (Kimura et ah, 1997), the α7 integrin promoter (Ziober and Kramer, 1996), the brain natriuretic peptide promoter (LaPointe et ah, 1996) and the α B- crystallin/small heat shock protein promoter (Gopal-Srivastava, 1995), α myosin heavy chain promoter (Yamauchi-Takihara et ah, 1989) and the ANF promoter (LaPointe et ah, 1988). A polyadenylation signal may be included to effect proper polyadenylation of the gene transcript where desired. The nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and any such sequence may be employed such as human growth hormone and SV40 polyadenylation signals. Also contemplated as an element of the expression cassette is a terminator. These elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.

In certain embodiments, the cells containing nucleic acid constructs of the present invention may be identified in vitro or in vivo by including a marker in the expression construct. Such markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression construct. Usually the inclusion of a drug selection marker aids in cloning and in the selection of transformants, for example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selectable markers. Alternatively, enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be employed. Immunologic markers also can be employed. The selectable marker employed is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding a gene product. Further examples of selectable markers are well known to one of skill in the art.

There are a number of ways in which expression vectors may introduced into cells. In certain embodiments of the invention, the expression construct comprises a virus or engineered construct derived from a viral genome. The ability of certain viruses to enter cells via receptor-mediated endocytosis, to integrate into host cell genome and express viral genes stably and efficiently have made them attractive candidates for the transfer of foreign genes into mammalian cells (Ridgeway, 1988; Nicolas and Rubenstein, 1988; Baichwal and Sugden, 1986; Temin, 1986).

One of the preferred methods for in vivo delivery involves the use of an adenovirus expression vector. "Adenovirus expression vector" is meant to include those constructs containing adenovirus sequences sufficient to (a) support packaging of the construct and (b) to express an antisense polynucleotide (or other inhibitory polynucleotide) that has been cloned therein. The expression vector comprises a genetically engineered form of adenovirus. Knowledge of the genetic organization of adenovirus, a 36 kB, linear, double-stranded DNA virus, allows substitution of large pieces of adenoviral DNA with foreign sequences up to 7 kB (Grunhaus and Horwitz, 1992). In contrast to retrovirus, the adenoviral infection of host cells does not result in chromosomal integration because adenoviral DNA can replicate in an episomal manner without potential genotoxicity. Also, adenoviruses are structurally stable, and no genome rearrangement has been detected after extensive amplification. Adenovirus can infect virtually all epithelial cells regardless of their cell cycle stage.

Adenovirus is particularly suitable for use as a gene transfer vector because of its mid-sized genome, ease of manipulation, high titer, wide target cell range and high infectivity. Both ends of the viral genome contain 100-200 base pair inverted repeats (ITRs), which are cis elements necessary for viral DNA replication and packaging.

Other than the requirement that the adenovirus vector be replication defective, or at least conditionally defective, the nature of the adenovirus vector is not believed to be crucial to the successful practice of the invention. The adenovirus may be of any of the 42 different known serotypes or subgroups A-F. Adenovirus type 5 of subgroup C is the preferred starting material in order to obtain the conditional replication-defective adenovirus vector for use in the present invention. This is because Adenovirus type 5 is a human adenovirus about which a great deal of biochemical and genetic information is known, and it has historically been used for most constructions employing adenovirus as a vector.

The typical vector according to the present invention is replication defective and will not have an adenovirus El region. Thus, it will be most convenient to introduce the polynucleotide encoding the gene of interest at the position from which the El -coding sequences have been removed. However, the position of insertion of the construct within the adenovirus sequences is not critical to the invention. The polynucleotide encoding the gene of interest may also be inserted in lieu of the deleted E3 region in E3 replacement vectors, as described by Karlsson et al (1986), or in the E4 region where a helper cell line or helper virus complements the E4 defect.

Adenovirus vectors have been used in eukaryotic gene expression (Levrero et al, 1991; Gomez-Foix et al, 1992) and vaccine development (Grunhaus and Horwitz, 1992; Graham and Prevec, 1991). Recently, animal studies suggested that recombinant adenovirus could be used for gene therapy (Stratford-Perricaudet and Perricaudet, 1991; Stratford- Perricaudet et al, 1990; Rich et al., 1993). Studies in administering recombinant adenovirus to different tissues include trachea instillation (Rosenfeld et al., 1991; Rosenfeld et al., 1992), muscle injection (Ragot et al, 1993), peripheral intravenous injections (Herz and Gerard, 1993) and stereotactic inoculation into the brain (Le Gal La Salle et al, 1993). Retroviral vectors are also suitable for expressing miR-21 inhibitors of the invention in cells. The retroviruses are a group of single-stranded RNA viruses characterized by an ability to convert their RNA to double-stranded DNA in infected cells by a process of reverse-transcription (Coffin, 1990). The resulting DNA then stably integrates into cellular chromosomes as a pro virus and directs synthesis of viral proteins. The integration results in the retention of the viral gene sequences in the recipient cell and its descendants. The retroviral genome contains three genes, gag, pol, and env that code for capsid proteins, polymerase enzyme, and envelope components, respectively. A sequence found upstream from the gag gene contains a signal for packaging of the genome into virions. Two long terminal repeat (LTR) sequences are present at the 5' and 3' ends of the viral genome. These contain strong promoter and enhancer sequences and are also required for integration in the host cell genome (Coffin, 1990). In order to construct a retroviral vector, an antisense polynucleotide or other inhibitory nucleotide (e.g. siRNA or shRNA) is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective. In order to produce virions, a packaging cell line containing the gag, pol, and env genes but without the LTR and packaging components is constructed (Mann et al, 1983). When a recombinant plasmid containing a cDNA, together with the retroviral LTR and packaging sequences is introduced into this cell line (by calcium phosphate precipitation for example), the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media (Nicolas and Rubenstein, 1988; Temin, 1986; Mann et al, 1983). The media containing the recombinant retroviruses is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors are able to infect a broad variety of cell types. However, integration and stable expression require the division of host cells (Paskind et al, 1975).

Other viral vectors may be employed as expression constructs in the present invention. Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al, 1988) adeno-associated virus (AAV) (Ridgeway, 1988; Baichwal and Sugden, 1986; Hermonat and Muzycska, 1984) and herpesviruses may be employed. These vectors offer several attractive features for various mammalian cells (Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al, 1988; Horwich et al, 1990).

In order to effect expression of sense or antisense gene constructs, the expression construct must be delivered into a cell. This delivery may be accomplished in vitro, as in laboratory procedures for transforming cells lines, or in vivo or ex vivo, as in the treatment of certain disease states. One mechanism for delivery is via viral infection where the expression construct is encapsidated in an infectious viral particle.

Several non-viral methods for the transfer of expression constructs into cultured mammalian cells also are contemplated by the present invention. These include calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al, 1990) DEAE-dextran (Gopal, 1985), electroporation (Tur-Kaspa et al, 1986; Potter et al, 1984), direct microinjection (Harland and Weintraub, 1985), DNA-loaded liposomes (Nicolau and Sene, 1982; Fraley et al, 1979) and lipofectamine-DNA complexes, cell sonication (Fechheimer et al., 1987), gene bombardment using high velocity microprojectiles (Yang et al., 1990), and receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu, 1988). Some of these techniques may be successfully adapted for in vivo or ex vivo use.

Once the expression construct has been delivered into the cell the nucleic acid encoding the gene of interest may be positioned and expressed at different sites. In certain embodiments, the nucleic acid encoding the gene may be stably integrated into the genome of the cell. This integration may be in the cognate location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non-specific location (gene augmentation). In yet further embodiments, the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA. Such nucleic acid segments or "episomes" encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. The method by which the expression construct is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression construct employed. In another embodiment of the invention, the expression construct may simply consist of naked recombinant DNA or plasmids. Transfer of the construct may be performed by any of the methods mentioned above which physically or chemically permeabilize the cell membrane. This is particularly applicable for transfer in vitro but it may be applied to in vivo use as well. Dubensky et al. (1984) successfully injected polyomavirus DNA in the form of calcium phosphate precipitates into liver and spleen of adult and newborn mice demonstrating active viral replication and acute infection. Benvenisty and Neshif (1986) also demonstrated that direct intraperitoneal injection of calcium phosphate -precipitated plasmids results in expression of the transfected genes. It is envisioned that DNA encoding a polynucleotide of interest may also be transferred in a similar manner in vivo and express the gene product.

In still another embodiment of the invention for transferring a naked DNA expression construct into cells may involve particle bombardment. This method depends on the ability to accelerate DNA-coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them (Klein et al., 1987). Several devices for accelerating small particles have been developed. One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force (Yang et al., 1990). The microprojectiles used have consisted of biologically inert substances such as tungsten or gold beads.

Selected organs including the liver, skin, and muscle tissue of rats and mice have been bombarded in vivo (Yang et al., 1990; Zelenin et al., 1991). This may require surgical exposure of the tissue or cells, to eliminate any intervening tissue between the gun and the target organ, i.e., ex vivo treatment. Again, DNA encoding a particular polynucleotide of interest may be delivered via this method and still be incorporated by the present invention.

In a further embodiment of the invention, the expression construct may be entrapped in a liposome. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991). Also contemplated are lipofectamine-DNA complexes.

In certain embodiments of the invention, the liposome may be complexed with a hemagglutinating virus (HVJ). This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA (Kaneda et al., 1989). In other embodiments, the liposome may be complexed or employed in conjunction with nuclear non-histone chromosomal proteins (HMG-I) (Kato et al., 1991). In yet further embodiments, the liposome may be complexed or employed in conjunction with both HVJ and HMG- 1. In that such expression constructs have been successfully employed in transfer and expression of nucleic acid in vitro and in vivo, then they are applicable for the present invention. Where a bacterial promoter is employed in the DNA construct, it also will be desirable to include within the liposome an appropriate bacterial polymerase.

Other expression constructs which can be employed to deliver a nucleic acid encoding a particular polynucleotide into cells are receptor-mediated delivery vehicles. These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis in almost all eukaryotic cells. Because of the cell type-specific distribution of various receptors, the delivery can be highly specific (Wu and Wu, 1993). Receptor-mediated gene targeting vehicles generally consist of two components: a cell receptor-specific ligand and a DNA-binding agent. Several ligands have been used for receptor-mediated gene transfer. The most extensively characterized ligands are asialoorosomucoid (ASOR) (Wu and Wu, 1987) and transferrin (Wagner et al., 1990). Recently, a synthetic neoglycoprotein, which recognizes the same receptor as ASOR, has been used as a gene delivery vehicle (Ferkol et al, 1993; Perales et al, 1994) and epidermal growth factor (EGF) has also been used to deliver genes to squamous carcinoma cells (Myers, EPO 0273085).

In other embodiments, the delivery vehicle may comprise a ligand and a liposome. For example, Nicolau et al. (1987) employed lactosyl-ceramide, a galactose-terminal asialganglioside, incorporated into liposomes and observed an increase in the uptake of the insulin gene by hepatocytes. Thus, it is feasible that a nucleic acid encoding a particular gene also may be specifically delivered into a cell type {e.g. cardiac cell) by any number of receptor-ligand systems with or without liposomes. For example, epidermal growth factor (EGF) may be used as the receptor for mediated delivery of a nucleic acid into cells that exhibit upregulation of EGF receptor. Mannose can be used to target the mannose receptor on liver cells. Also, antibodies to CD5 (CLL), CD22 (lymphoma), CD25 (T-cell leukemia) and MAA (melanoma) can similarly be used as targeting moieties.

In a particular example, the polynucleotide may be administered in combination with a cationic lipid. Examples of cationic lipids include, but are not limited to, lipofectin, DOTMA, DOPE, and DOTAP. The publication of WO0071096, which is specifically incorporated by reference, describes different formulations, such as a DOTAPxholesterol or cholesterol derivative formulation that can effectively be used for gene therapy. Other disclosures also discuss different lipid or liposomal formulations including nanoparticles and methods of administration; these include, but are not limited to, U.S. Patent Publication Nos. 20030203865, 20020150626, 20030032615, and 20040048787, which are specifically incorporated by reference to the extent they disclose formulations and other related aspects of administration and delivery of nucleic acids. Methods used for forming particles are also disclosed in U.S. Patents 5,844,107, 5,877,302, 6,008,336, 6,077,835, 5,972,901, 6,200,801, and 5,972,900, which are incorporated by reference for those aspects. In certain embodiments, gene transfer may more easily be performed under ex vivo conditions. Ex vivo gene therapy refers to the isolation of cells from an animal, the delivery of a nucleic acid into the cells in vitro, and then the return of the modified cells back into an animal. This may involve the surgical removal of tissue/organs from an animal or the primary culture of cells and tissues.

Stress response factor (SRF) has been shown to bind to the regulatory region of miR- 21 and activate transcription of miR-21 (see Example 4). Accordingly, the present invention includes methods of regulating SRF function as a means of regulating miR-21 expression. In one embodiment, inhibition of miR-21 expression or function comprises administering an inhibitor of SRF activity. Inhibitors of SRF activity may include agents that interfere with the binding of SRF to its binding site (serum response element; SRE) in the regulatory region of miR-21 and agents that prevent activation of signaling proteins upstream of SRF activation, such as mitogen activated protein kinases.

The present invention also contemplates methods for scavenging or clearing miR-21 inhibitors following treatment. The method may comprise overexpressing binding sites for the miR-21 inhibitors in cardiac tissue. In another embodiment, the present invention provides a method for scavenging or clearing miR-21 following treatment. In one embodiment, the method comprises overexpression of binding site regions for miR-21 in cardiac muscle using a heart muscle specific promoter {e.g. α-MHC) or a fibroblast specific promoter. The binding site regions preferably contain a sequence of the seed region for miR- 21. In some embodiments, the binding site may contain a sequence from the 3'UTR of one or more targets of miR-21, such as PP ARa, Sproutyl, Sprouty2, or PTEN.

In another embodiment of the invention, an inhibitor of miR-21 is administered to the subject in combination with other therapeutic modalities. Current medical management of cardiac hypertrophy in the setting of a cardiovascular disorder includes the use of at least two types of drugs: inhibitors of the rennin-angiotensin system, and β-adrenergic blocking agents (Bristow, 1999). Therapeutic agents to treat pathologic hypertrophy in the setting of heart failure include angiotensin II converting enzyme (ACE) inhibitors and β-adrenergic receptor blocking agents (Eichhorn and Bristow, 1996). Other pharmaceutical agents that have been disclosed for treatment of cardiac hypertrophy include angiotensin II receptor antagonists (U.S. Patent 5,604,251) and neuropeptide Y antagonists (WO 98/33791). Non-pharmacological treatment is primarily used as an adjunct to pharmacological treatment. One means of non-pharmacological treatment involves reducing the sodium in the diet. In addition, non-pharmacological treatment also entails the elimination of certain precipitating drugs, including negative inotropic agents (e.g., certain calcium channel blockers and antiarrhythmic drugs like disopyramide), cardiotoxins (e.g., amphetamines), and plasma volume expanders (e.g., nonsteroidal anti-inflammatory agents and glucocorticoids).

Thus, in addition to the therapies described above, one may also provide to the subject more "standard" pharmaceutical cardiac therapies with the inhibitor of miR-21. Examples of other therapies include, without limitation, so-called "β blockers," anti- hypertensives, cardiotonics, anti-thrombotics, vasodilators, hormone antagonists, iontropes, diuretics, endothelin receptor antagonists, calcium channel blockers, phosphodiesterase inhibitors, ACE inhibitors, angiotensin type 2 antagonists and cytokine blockers/inhibitors, and HDAC inhibitors. The combination therapy also may involve inhibiting the expression or activity of additional miRNAs involved in cardiac remodeling such as miR-499, miR-208, miR-208b and miR-195 (miR-15 family members). Combination therapy may also include overexpression of particular microRNAs, such as miR-29.

Combinations may be achieved by contacting cardiac cells with a single composition or pharmacological formulation that includes an inhibitor of miR-21 and a standard pharmaceutical agent, or by contacting the cell with two distinct compositions or formulations, at the same time, wherein one composition includes an inhibitor of miR-21 and the other includes the standard pharmaceutical agent. Alternatively, the therapy using an inhibitor of miR-21 may precede or follow administration of the other agent(s) by intervals ranging from minutes to weeks. In embodiments where the standard pharmaceutical agent and miR-21 inhibitor 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 pharmaceutical agent and miR-21 inhibitor 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 and, more preferably, within about 6-12 hours of each other, with a delay time of only about 12 hours being most preferred. 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.

It also is conceivable that more than one administration of either an inhibitor of miR-

21, or the other pharmaceutical agent will be desired. In this regard, various combinations may be employed. By way of illustration, where the inhibitor of miR-21 is "A" and the other agent is "B", the following permutations based on 3 and 4 total administrations are exemplary:

A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B 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/A/B B/A/A/A A/B/A/A A/A/B/A A/B/B/B B/A/B/B B/B/A/B

Other combinations are likewise contemplated.

Treatment regimens would vary depending on the clinical situation. However, long- term maintenance would appear to be appropriate in most circumstances. It also may be desirable to treat hypertrophy with inhibitors of miR-21 intermittently, such as within a brief window during disease progression.

Pharmacological therapeutic agents and methods of administration, dosages, etc., are well known to those of skill in the art (see for example, the "Physicians Desk Reference", Klaassen's "The Pharmacological Basis of Therapeutics", "Remington's Pharmaceutical Sciences", and "The Merck Index, Eleventh Edition", incorporated herein by reference in relevant parts), and may be combined with the invention in light of the disclosures herein. 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, and such individual determinations are within the skill of those of ordinary skill in the art. Non-limiting examples of a pharmacological therapeutic agent that may be used in the present invention include an antihyperlipoproteinemic agent, an antiarteriosclerotic agent, an antithrombotic/fibrinolytic agent, a blood coagulant, an antiarrhythmic agent, an antihypertensive agent, a vasopressor, a treatment agent for congestive heart failure, an antianginal agent, an antibacterial agent or a combination thereof. In addition, it should be noted that any of the following may be used to develop new sets of cardiac therapy target genes as β-blockers were used in the present examples (see below). While it is expected that many of these genes may overlap, new gene targets likely can be developed.

In certain embodiments, administration of an agent that lowers the concentration of one of more blood lipids and/or lipoproteins, known herein as an "antihyperlipoproteinemic," may be combined with a cardiovascular therapy according to the present invention, particularly in treatment of athersclerosis and thickenings or blockages of vascular tissues. In certain embodiments, an antihyperlipoproteinemic agent may comprise an aryloxyalkanoic/fibric acid derivative, a resin/bile acid sequesterant, a HMG CoA reductase inhibitor, a nicotinic acid derivative, a thyroid hormone or thyroid hormone analog, a miscellaneous agent or a combination thereof. Non-limiting examples of aryloxyalkanoic/fibric acid derivatives include beclobrate, enzafibrate, binifibrate, ciprofibrate, clinofibrate, clofibrate (atromide-S), clofibric acid, etofibrate, fenofibrate, gemfibrozil (lobid), nicofibrate, pirifibrate, ronifibrate, simfibrate and theofibrate. Non- limiting examples of resins/bile acid sequesterants include cholestyramine (cholybar, questran), colestipol (colestid) and polidexide. Non- limiting examples of HMG CoA reductase inhibitors include lovastatin (mevacor), pravastatin (pravochol) or simvastatin (zocor). Non-limiting examples of nicotinic acid derivatives include nicotinate, acepimox, niceritrol, nicoclonate, nicomol and oxiniacic acid. Non-limiting examples of thyroid hormones and analogs thereof include etoroxate, thyropropic acid and thyroxine. Non-limiting examples of miscellaneous antihyperlipoproteinemics include acifran, azacosterol, benfluorex, β-benzalbutyramide, carnitine, chondroitin sulfate, clomestrone, detaxtran, dextran sulfate sodium, 5,8, 11, 14, 17-eicosapentaenoic acid, eritadenine, furazabol, meglutol, melinamide, mytatrienediol, ornithine, γ-oryzanol, pantethine, pentaerythritol tetraacetate, α-phenylbutyramide, pirozadil, probucol (lorelco), β-sitosterol, sultosilic acid-piperazine salt, tiadenol, triparanol and xenbucin. A non-limiting example of an antiarteriosclerotic includes pyridinol carbamate.

In certain embodiments, administration of an agent that aids in the removal or prevention of blood clots may be combined with administration of a modulator, particularly in treatment of athersclerosis and vasculature (e.g., arterial) blockages. Non-limiting examples of antithrombotic and/or fibrinolytic agents include anticoagulants, anticoagulant antagonists, antiplatelet agents, thrombolytic agents, thrombolytic agent antagonists or combinations thereof. In certain embodiments, antithrombotic agents that can be administered orally, such as, for example, aspirin and wafarin (Coumadin), are preferred.

Non-limiting examples of anticoagulants include acenocoumarol, ancrod, anisindione, bromindione, clorindione, coumetarol, cyclocumarol, dextran sulfate sodium, dicumarol, diphenadione, ethyl biscoumacetate, ethylidene dicoumarol, fluindione, heparin, hirudin, lyapolate sodium, oxazidione, pentosan polysulfate, phenindione, phenprocoumon, phosvitin, picotamide, tioclomarol and warfarin.

Non-limiting examples of antiplatelet agents include aspirin, a dextran, dipyridamole (persantin), heparin, sulfinpyranone (anturane) and ticlopidine (ticlid). No n- limiting examples of thrombolytic agents include tissue plaminogen activator

(activase), plasmin, pro-urokinase, urokinase (abbokinase) streptokinase (streptase), anistreplase/ APSAC (eminase).

In certain embodiments wherein a patient is suffering from a hemorrhage or an increased likelihood of hemorrhaging, an agent that may enhance blood coagulation may be used. Non- limiting examples of a blood coagulation promoting agents include thrombolytic agent antagonists and anticoagulant antagonists. Non-limiting examples of anticoagulant antagonists include protamine and vitamine Kl .

Non-limiting examples of thrombolytic agent antagonists include amiocaproic acid (amicar) and tranexamic acid (amstat). Non-limiting examples of antithrombotics include anagrelide, argatroban, cilstazol, daltroban, defibrotide, enoxaparin, fraxiparine, indobufen, lamoparan, ozagrel, picotamide, plafibride, tedelparin, ticlopidine and triflusal.

Non- limiting examples of antiarrhythmic agents include Class I antiarrhythmic agents (sodium channel blockers), Class II antiarrhythmic agents (β-adrenergic blockers), Class III antiarrhythmic agents (repolarization prolonging drugs), Class IV antiarrhythmic agents (calcium channel blockers) and miscellaneous antiarrhythmic agents.

Non-limiting examples of sodium channel blockers include Class IA, Class IB and Class IC antiarrhythmic agents. Non-limiting examples of Class IA antiarrhythmic agents include disppyramide (norpace), procainamide (pronestyl) and quinidine (quinidex). Non- limiting examples of Class IB antiarrhythmic agents include lidocaine (xylocaine), tocainide (tonocard) and mexiletine (mexitil). Non-limiting examples of Class IC antiarrhythmic agents include encainide (enkaid) and fiecainide (tambocor). Non-limiting examples of a β blocker, otherwise known as a β-adrenergic blocker, a β-adrenergic antagonist or a Class II antiarrhythmic agent, include acebutolol (sectral), alprenolol, amosulalol, arotinolol, atenolol, befunolol, betaxolol, bevantolol, bisoprolol, bopindolol, bucumolol, bufetolol, bufuralol, bunitrolol, bupranolol, butidrine hydrochloride, butofilolol, carazolol, carteolol, carvedilol, celiprolol, cetamolol, cloranolol, dilevalol, epanolol, esmolol (brevibloc), indenolol, labetalol, levobunolol, mepindolol, metipranolol, metoprolol, moprolol, nadolol, nadoxolol, nifenalol, nipradilol, oxprenolol, penbutolol, pindolol, practolol, pronethalol, propanolol (inderal), sotalol (betapace), sulfinalol, talinolol, tertatolol, timolol, toliprolol and xibinolol. In certain embodiments, the β blocker comprises an aryloxypropanolamine derivative. Non-limiting examples of aryloxypropanolamine derivatives include acebutolol, alprenolol, arotinolol, atenolol, betaxolol, bevantolol, bisoprolol, bopindolol, bunitrolol, butofilolol, carazolol, carteolol, carvedilol, celiprolol, cetamolol, epanolol, indenolol, mepindolol, metipranolol, metoprolol, moprolol, nadolol, nipradilol, oxprenolol, penbutolol, pindolol, propanolol, talinolol, tertatolol, timolol and toliprolol.

Non- limiting examples of an agent that prolong repolarization, also known as a Class III antiarrhythmic agent, include amiodarone (cordarone) and sotalol (betapace).

Non-limiting examples of a calcium channel blocker, otherwise known as a Class IV antiarrhythmic agent, include an arylalkylamine (e.g., bepridile, diltiazem, fendiline, gallopamil, prenylamine, terodiline, verapamil), a dihydropyridine derivative (felodipine, isradipine, nicardipine, nifedipine, nimodipine, nisoldipine, nitrendipine) a piperazinde derivative (e.g., cinnarizine, flunarizine, lidoflazine) or a micellaneous calcium channel blocker such as bencyclane, etafenone, magnesium, mibefradil or perhexiline. In certain embodiments a calcium channel blocker comprises a long-acting dihydropyridine (nifedipine-type) calcium antagonist.

Non-limiting examples of miscellaneous antiarrhythmic agents include adenosine (adenocard), digoxin (lanoxin), acecainide, ajmaline, amoproxan, aprindine, bretylium tosylate, bunaftine, butobendine, capobenic acid, cifenline, disopyranide, hydro quinidine, indecainide, ipatropium bromide, lidocaine, lorajmine, lorcainide, meobentine, moricizine, pirmenol, prajmaline, propafenone, pyrinoline, quinidine polygalacturonate, quinidine sulfate and viquidil. Non-limiting examples of antihypertensive agents include sympatholytic, α/β blockers, α blockers, anti-angiotensin II agents, β blockers, calcium channel blockers, vasodilators and miscellaneous antihypertensives.

Non-limiting examples of an α blocker, also known as an α-adrenergic blocker or an α-adrenergic antagonist, include amosulalol, arotinolol, dapiprazole, doxazosin, ergoloid mesylates, fenspiride, indoramin, labetalol, nicergoline, prazosin, terazosin, tolazoline, trimazosin and yohimbine. In certain embodiments, an α blocker may comprise a quinazoline derivative. Non-limiting examples of quinazoline derivatives include alfuzosin, bunazosin, doxazosin, prazosin, terazosin and trimazosin. In certain embodiments, an antihypertensive agent is both an α and β adrenergic antagonist. Non- limiting examples of an α/β blocker comprise labetalol (normodyne, trandate).

Non-limiting examples of anti-angiotensin II agents include include angiotensin converting enzyme inhibitors and angiotensin II receptor antagonists. Non-limiting examples of angiotensin converting enzyme inhibitors (ACE inhibitors) include alacepril, enalapril (vasotec), captopril, cilazapril, delapril, enalaprilat, fosinopril, lisinopril, moveltopril, perindopril, quinapril and ramipril.. Non-limiting examples of an angiotensin II receptor blocker, also known as an angiotensin II receptor antagonist, an ANG receptor blocker or an ANG-II type-1 receptor blocker (ARBS), include angiocandesartan, eprosartan, irbesartan, losartan and valsartan. Non-limiting examples of a sympatholytic include a centrally acting sympatholytic or a peripherially acting sympatholytic. Non-limiting examples of a centrally acting sympatholytic, also known as an central nervous system (CNS) sympatholytic, include clonidine (catapres), guanabenz (wytensin) guanfacine (tenex) and methyldopa (aldomet). Non-limiting examples of a peripherally acting sympatholytic include a ganglion blocking agent, an adrenergic neuron blocking agent, a β-adrenergic blocking agent or a αl -adrenergic blocking agent. Non-limiting examples of a ganglion blocking agent include mecamylamine (inversine) and trimethaphan (arfonad). Non-limiting examples of an adrenergic neuron blocking agent include guanethidine (ismelin) and reserpine (serpasil). Non-limiting examples of a β-adrenergic blocker include acenitolol (sectral), atenolol (tenormin), betaxolol (kerlone), carteolol (cartrol), labetalol (normodyne, trandate), metoprolol (lopressor), nadanol (corgard), penbutolol (levatol), pindolol (visken), propranolol (inderal) and timolol (blocadren). Non-limiting examples of αl -adrenergic blocker include prazosin (minipress), doxazocin (cardura) and terazosin (hytrin).

In certain embodiments a cardiovasculator therapeutic agent may comprise a vasodilator (e.g., a cerebral vasodilator, a coronary vasodilator or a peripheral vasodilator). In certain preferred embodiments, a vasodilator comprises a coronary vasodilator. Non- limiting examples of a coronary vasodilator include amotriphene, bendazol, benfurodil hemisuccinate, benziodarone, chloracizine, chromonar, clobenfurol, clonitrate, dilazep, dipyridamole, droprenilamine, efloxate, erythrityl tetranitrane, etafenone, fendiline, floredil, ganglefene, herestrol bis(β-diethylaminoethyl ether), hexobendine, itramin tosylate, khellin, lidoflanine, mannitol hexanitrane, medibazine, nicorglycerin, pentaerythritol tetranitrate, pentrinitrol, perhexiline, pimefylline, trapidil, tricromyl, trimetazidine, trolnitrate phosphate and visnadine.

In certain embodiments, a vasodilator may comprise a chronic therapy vasodilator or a hypertensive emergency vasodilator. Non-limiting examples of a chronic therapy vasodilator include hydralazine (apresoline) and minoxidil (loniten). Non-limiting examples of a hypertensive emergency vasodilator include nitroprusside (nipride), diazoxide (hyperstat IV), hydralazine (apresoline), minoxidil (loniten) and verapamil.

Non-limiting examples of miscellaneous antihypertensives include ajmaline, γ- aminobutyric acid, bufeniode, cicletainine, ciclosidomine, a cryptenamine tannate, fenoldopam, flosequinan, ketanserin, mebutamate, mecamylamine, methyldopa, methyl 4- pyridyl ketone thiosemicarbazone, muzolimine, pargyline, pempidine, pinacidil, piperoxan, primaperone, a protoveratrine, raubasine, rescimetol, rilmenidene, saralasin, sodium nitrorusside, ticrynafen, trimethaphan camsylate, tyrosinase and urapidil.

In certain embodiments, an antihypertensive may comprise an arylethanolamine derivative, a benzothiadiazine derivative, a 7V-carboxyalkyl(peptide/lactam) derivative, a dihydropyridine derivative, a guanidine derivative, a hydrazines/phthalazine, an imidazole derivative, a quanternary ammonium compound, a reserpine derivative or a suflonamide derivative. Non-limiting examples of arylethanolamine derivatives include amosulalol, bufuralol, dilevalol, labetalol, pronethalol, sotalol and sulfinalol. Non-limiting examples of benzothiadiazine derivatives include althizide, bendroflumethiazide, benzthiazide, benzylhydrochlorothiazide, buthiazide, chlorothiazide, chlorthalidone, cyclopenthiazide, cyclothiazide, diazoxide, epithiazide, ethiazide, fenquizone, hydrochlorothizide, hydroflumethizide, methyclothiazide, meticrane, metolazone, paraflutizide, polythizide, tetrachlormethiazide and trichlormethiazide. Non-limiting examples of N- carboxyalkyl(peptide/lactam) derivatives include alacepril, captopril, cilazapril, delapril, enalapril, enalaprilat, fosinopril, lisinopril, moveltipril, perindopril, quinapril and ramipril. Non-limiting examples of dihydropyridine derivatives include amlodipine, felodipine, isradipine, nicardipine, nifedipine, nilvadipine, nisoldipine and nitrendipine. Non-limiting examples of guanidine derivatives include bethanidine, debrisoquin, guanabenz, guanacline, guanadrel, guanazodine, guanethidine, guanfacine, guanochlor, guanoxabenz and guanoxan. Non- limiting examples of hydrazines/phthalazines include budralazine, cadralazine, dihydralazine, endralazine, hydracarbazine, hydralazine, pheniprazine, pildralazine and todralazine. Non-limiting examples of imidazole derivatives include clonidine, lofexidine, phentolamine, tiamenidine and tolonidine. Non-limiting examples of quanternary ammonium compounds include azamethonium bromide, chlorisondamine chloride, hexamethonium, pentacynium bis(methylsulfate), pentamethonium bromide, pentolinium tartrate, phenactropinium chloride and trimethidinium methosulfate. Non-limiting examples of reserpine derivatives include bietaserpine, deserpidine, rescinnamine, reserpine and syrosingopine. Non-limiting examples of sulfonamide derivatives include ambuside, clopamide, furosemide, indapamide, quinethazone, tripamide and xipamide. Vasopressors generally are used to increase blood pressure during shock, which may occur during a surgical procedure. Non-limiting examples of a vasopressor, also known as an antihypotensive, include amezinium methyl sulfate, angiotensin amide, dimetofrine, dopamine, etifelmin, etilefrin, gepefrine, metaraminol, midodrine, norepinephrine, pholedrine and synephrine. Non-limiting examples of agents for the treatment of congestive heart failure include anti-angiotensin II agents, afterload-preload reduction treatment, diuretics and inotropic agents.

In certain embodiments, an animal patient that can not tolerate an angiotensin antagonist may be treated with a combination therapy. Such therapy may combine adminstration of hydralazine (apresoline) and isosorbide dinitrate (isordil, sorbitrate). Non-limiting examples of a diuretic include a thiazide or benzothiadiazine derivative (e.g., althiazide, bendroflumethazide, benzthiazide, benzylhydrochlorothiazide, buthiazide, chlorothiazide, chlorothiazide, chlorthalidone, cyclopenthiazide, epithiazide, ethiazide, ethiazide, fenquizone, hydrochlorothiazide, hydroflumethiazide, methyclothiazide, meticrane, metolazone, paraflutizide, polythizide, tetrachloromethiazide, trichlormethiazide), an organomercurial (e.g., chlormerodrin, meralluride, mercamphamide, mercaptomerin sodium, mercumallylic acid, mercumatilin dodium, mercurous chloride, mersalyl), a pteridine (e.g., furterene, triamterene), purines (e.g., acefylline, 7- morpholinomethyltheophylline, pamobrom, protheobromine, theobromine), steroids including aldosterone antagonists (e.g., canrenone, oleandrin, spironolactone), a sulfonamide derivative (e.g., acetazolamide, ambuside, azosemide, bumetanide, butazolamide, chloraminophenamide, clofenamide, clopamide, clorexolone, diphenylmethane-4,4'- disulfonamide, disulfamide, ethoxzolamide, furosemide, indapamide, mefruside, methazolamide, piretanide, quinethazone, torasemide, tripamide, xipamide), a uracil (e.g., aminometradine, amisometradine), a potassium sparing antagonist (e.g., amiloride, triamterene)or a miscellaneous diuretic such as aminozine, arbutin, chlorazanil, ethacrynic acid, etozolin, hydracarbazine, isosorbide, mannitol, metochalcone, muzolimine, perhexiline, ticrnafen and urea.

Non-limiting examples of a positive inotropic agent, also known as a cardiotonic, include acefylline, an acetyldigitoxin, 2-amino-4-picoline, amrinone, benfurodil hemisuccinate, bucladesine, cerberosine, camphotamide, convallatoxin, cymarin, denopamine, deslanoside, digitalin, digitalis, digitoxin, digoxin, dobutamine, dopamine, dopexamine, enoximone, erythrophleine, fenalcomine, gitalin, gitoxin, glycocyamine, heptaminol, hydrastinine, ibopamine, a lanatoside, metamivam, milrinone, nerifolin, oleandrin, ouabain, oxyfedrine, prenalterol, proscillaridine, resibufogenin, scillaren, scillarenin, strphanthin, sulmazole, theobromine and xamoterol.

In particular embodiments, an intropic agent is a cardiac glycoside, a β-adrenergic agonist or a phosphodiesterase inhibitor. Non-limiting examples of a cardiac glycoside includes digoxin (lanoxin) and digitoxin (crystodigin). Non-limiting examples of a β- adrenergic agonist include albuterol, bambuterol, bitolterol, carbuterol, clenbuterol, clorprenaline, denopamine, dioxethedrine, dobutamine (dobutrex), dopamine (intropin), dopexamine, ephedrine, etafedrine, ethy norepinephrine, fenoterol, formoterol, hexoprenaline, ibopamine, isoetharine, isoproterenol, mabuterol, metaproterenol, methoxyphenamine, oxyfedrine, pirbuterol, procaterol, protokylol, reproterol, rimiterol, ritodrine, soterenol, terbutaline, tretoquinol, tulobuterol and xamoterol. Non-limiting examples of a phosphodiesterase inhibitor include amrinone (inocor).

Antianginal agents may comprise organonitrates, calcium channel blockers, β blockers and combinations thereof. Non-limiting examples of organonitrates, also known as nitrovasodilators, include nitroglycerin (nitro-bid, nitrostat), isosorbide dinitrate (isordil, sorbitrate) and amyl nitrate (aspirol, vaporole). Endothelin (ET) is a 21 -amino acid peptide that has potent physiologic and pathophysiologic effects that appear to be involved in the development of heart failure. The effects of ET are mediated through interaction with two classes of cell surface receptors. The type A receptor (ET-A) is associated with vasoconstriction and cell growth while the type B receptor (ET-B) is associated with endothelial-cell mediated vasodilation and with the release of other neurohormones, such as aldosterone. Pharmacologic agents that can inhibit either the production of ET or its ability to stimulate relevant cells are known in the art. Inhibiting the production of ET involves the use of agents that block an enzyme termed endothelin- converting enzyme that is involved in the processing of the active peptide from its precursor. Inhibiting the ability of ET to stimulate cells involves the use of agents that block the interaction of ET with its receptors. Non-limiting examples of endothelin receptor antagonists (ERA) include Bosentan, Enrasentan, Ambrisentan, Darusentan, Tezosentan, Atrasentan, Avosentan, Clazosentan, Edonentan, sitaxsentan, TBC 3711, BQ 123, and BQ 788.

In certain embodiments, the secondary therapeutic agent may comprise a surgery of some type, which includes, for example, preventative, diagnostic or staging, curative and palliative surgery. Surgery, and in particular a curative surgery, may be used in conjunction with other therapies, such as the present invention and one or more other agents.

Such surgical therapeutic agents for vascular and cardiovascular diseases and disorders are well known to those of skill in the art, and may comprise, but are not limited to, performing surgery on an organism, providing a cardiovascular mechanical prostheses, angioplasty, coronary artery reperfusion, catheter ablation, providing an implantable cardioverter defibrillator to the subject, mechanical circulatory support or a combination thereof. Non-limiting examples of a mechanical circulatory support that may be used in the present invention comprise an intra-aortic balloon counterpulsation, left ventricular assist device or combination thereof. The present invention also provides a method of treating cancer in a subject in need thereof. MiRNA-21 is upregulated in several types of human cancer, like human hepatocellular cancer (HCC) and breast cancer. Aberrant expression of miR-21 can contribute to HCC growth and spread by mediating phenotypic characteristics of cancer cells such as cell growth, migration, and invasion (Meng et ah, 2006; 2007). Overexpression of anti-miR-21 oligonucleotides suppressed both cell growth in vitro and tumor growth in the xenograft mouse model (Si et ah, 2007).

Interestingly, a near-universal property of primary and metastatic cancers is upregulation of glycolysis, resulting in increased glucose consumption (Warburg Effect). The upregulation of glycolysis leads to microenvironmental acidosis requiring evolution to pheno types resistant to acid- induced cell toxicity. Subsequent cell populations with upregulated glycolysis and acid resistance have a powerful growth advantage, which promotes unconstrained proliferation and invasion (Gillies et ah, 2007).

Upregulation of miR-21 in the heart induces a shift towards glucose metabolism (see Example 6). Therefore, inhibition of miR-21 in cancer cells may prevent an increase in glycolysis and block unconstrained cell proliferation. Thus, in one embodiment of the invention, the method of treating cancer in a subject in need thereof comprises contacting a cancer cell in the subject with an inhibitor of miR-21. An inhibitor of miR-21 can be an inhibitory polynucleotide, such as an antagomir, antisense, siRNA, or shRNA, as described herein. Cancers that may be treated with the methods of the invention include, but are not limited to, brain cancer, head & neck cancer, lung cancer, esophageal cancer, liver cancer, pancreatic cancer, stomach cancer, colon cancer, rectal cancer, prostate cancer, bladder cancer, ovarian cancer, uterine cancer, cervical cancer, breast cancer, testicular cancer, and skin cancer.

MiR-21 regulates the expression of PTEN (phosphatase and tensin ho mo log), which is a known tumor suppressor gene (See Example 3). Aberrant PTEN function results in unrestrained cell proliferation and is associated with many forms of human cancers. Thus, in another embodiment, the present invention provides a method for increasing the expression of PTEN in a cell comprising contacting the cell with a miR-21 inhibitor. The miR-21 inhibitor can be an antisense, an antagomir, or an inhibitory RNA molecule as described herein. In one embodiment, the cell is a cancer cell. In another embodiment, the cell is a fibroblast and the method prevents fibroblast proliferation and the development of fibrosis. In another embodiment, the cell is in vivo.

In another embodiment of the invention, the miR-21 inhibitor is administered with a second therapy, such as a standard cancer therapy. The standard cancer therapy may include chemotherapy, radiotherapy, immunotherapy, toxin therapy or gene therapy. In one embodiment, the second therapy impedes glycolytic oxidation in a cancer cell. The second therapy may be administered at the same time as the miR-21 inhibitor or the second therapy may be administered either before or after administration of the miR-21 inhibitor.

Agents or factors suitable for use in a combined therapy are any chemical compound or treatment method that induces DNA damage when applied to a cell. Such agents and factors include radiation and waves that induce DNA damage such as, γ-irradiation, X-rays, UV-irradiation, microwaves, electronic emissions, and the like. A variety of chemical compounds, also described as "chemotherapeutic agents," function to induce DNA damage, all of which are intended to be of use in the combined treatment methods disclosed herein. Chemotherapeutic agents contemplated to be of use, include, e.g., adriamycin, 5-fluorouracil (5FU), etoposide (VP-16), camptothecin, actinomycin-D, mitomycin C, cisplatin (CDDP) and even hydrogen peroxide. The invention also encompasses the use of a combination of one or more DNA damaging agents, whether radiation-based or actual compounds, such as the use of X-rays with cisplatin or the use of cisplatin with etoposide.

In treating cancer according to the invention, one would contact the tumor cells with a standard cancer therapeutic agent in addition to the miR-21 inhibitors. This may be achieved by irradiating the localized tumor site with radiation such as X-rays, UV-light, γ-rays or even microwaves. Alternatively, the tumor cells may be contacted with the agent by administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a miR-21 inhibitor and a compound such as, adriamycin, 5-fluorouracil, etoposide, camptothecin, actinomycin-D, mitomycin C, or more preferably, cisplatin. Another particular class of drugs that can be combined advantageously with a miR-21 inhibitor are inhibitors of the Akt pathway. Given the implications for miR-21 in the Warburg effect and glycolysis, and the role that the Akt pathway plays in the latter, it is believed that a particularly effective treatment may result from the combination of a miR-21 inhibitior and an inhibitor of the Akt pathway. The following patents and patent applications, dealing with Akt inhibitors, are hereby incorporated by reference: U.S. Patent Publications 2007/0238745, 2007/0161665, 2007/0054330, 2006/0229241, 2006/0104951, 2006/0030536, 2004/0229944, 2004/0110684, 2004/0102360 and 2003/0018003, and U.S. Patent 7,041,687.

In addition to combining miR-21 inhibitors with chemo- and radiotherapies, it also is contemplated that combination with gene therapies would be advantageous. For example, any tumor-related gene conceivably can be targeted in combination with the miR-21 inhibitor, for example, p21, Rb, APC, DCC, NF-I, NF-2, BCRA2, pl6, FHIT, WT-I, MEN-

I, MEN-II, BRCAl, VHL, FCC, MCC, ras, myc, neu, rafi erb, src, fins, jun, trk, ret, gsp, hst, bcl and abl. The present invention also encompasses a pharmaceutical composition comprising an inhibitor of miR-21. Where clinical applications are contemplated, pharmaceutical compositions will be prepared in a form appropriate for the intended application. Generally, this will entail preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals. 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 microRNA function or constructs expressing inhibitory nucleotides. Commercially available fat emulsions that are suitable for delivering the nucleic acids of the invention to tissues, such as cardiac muscle tissue, 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 US 5,981,505; US 6,217,900; US 6,383,512; US 5,783,565; US 7,202,227; US 6,379,965; US 6,127,170; US 5,837,533; US 6,747,014; and WO03/093449, which are herein incorporated by reference in their entireties. One will generally desire to employ appropriate salts and buffers to render delivery vehicles stable and allow for uptake by target cells. Buffers also will be employed when recombinant cells are introduced into a subject. Aqueous compositions of the present invention comprise an effective amount of the delivery vehicle comprising the inhibitor polynucleotides (e.g. liposomes or other complexes or expression vectors) or cells, 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 carrier" 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 vectors or cells of the compositions.

The active compositions of the present invention may 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 or intravenous injection, or by direct injection into cardiac tissue. Pharmaceutical compositions comprising miRNA inhibitors or expression constructs encoding inhibitory polynucleotides may also be administered by catheter systems or systems that isolate coronary circulation for delivering therapeutic agents to the heart. 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. 6,716,196; 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 in their entireties. Such compositions would normally be administered as pharmaceutically acceptable compositions, as described supra.

The active compounds may also be administered parenterally or intraperitoneally. By way of illustration, solutions of the active compounds 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 ordinary conditions of storage and use, these preparations generally contain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use or catheter delivery include, for example, sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Generally, these preparations are sterile and fluid to the extent that easy injectability 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, chlorobutanol, 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. 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.

The compositions of the present invention generally may be formulated in a neutral or salt form. Pharmaceutically-acceptable 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, mandelic, 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, trimethylamine, histidine, procaine and the like.

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 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 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" 15th 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, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.

Any of the compositions described herein may be comprised in a kit. In a non- limiting example, a miR-21 inhibitor, such as an antagomir, is included in a kit. The kit may further include water and/or buffers to stabilize the inhibitory polynucleotides. The kit may also include one or more transfection reagent(s) to facilitate delivery of the miR inhibitors to cells.

The components of the kits may be packaged either in aqueous media or in lyophilized form. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there is more than one component in the kit (labeling reagent and label may be packaged together), the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial. The kits of the present invention also will typically include a means for containing the nucleic acids, and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.

When the components of the kit are provided in one and/or more liquid solutions, the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly preferred.

However, the components of the kit may be provided as dried powder(s). When reagents and/or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means.

The container means will generally include at least one vial, test tube, flask, bottle, syringe and/or other container means, into which the nucleic acid formulations are placed, preferably, suitably allocated. The kits may also comprise a second container means for containing a sterile, pharmaceutically acceptable buffer and/or other diluent. Such kits may also include components that preserve or maintain the miR inhibitors or that protect against their degradation. Such components may be RNAse-free or protect against RNAses. Such kits generally will comprise, in suitable means, distinct containers for each individual reagent or solution.

A kit will also include instructions for employing the kit components as well the use of any other reagent not included in the kit. Instructions may include variations that can be implemented. A kit may also include utensils or devices for administering the miRNA inhibitor by various administration routes, such as parenteral or catheter administration.

It is contemplated that such reagents are embodiments of kits of the invention. Such kits, however, are not limited to the particular items identified above and may include any reagent used for the manipulation or characterization of miRNA.

The present invention further comprises methods for identifying modulators of miR- 21. Identified inhibitors of miR-21 are useful in the prevention or treatment or reversal of cardiac hypertrophy or heart failure. Inhibitors of miR-21 function are also useful in the treatment of cancer. Modulators (e.g. inhibitors) of miR-21 may be included in pharmaceutical compositions for the treatment of cardiac disorders and/or cancer according to the methods of the present invention.

These assays may comprise random screening of large libraries of candidate compounds; alternatively, the assays may be used to focus on particular classes of compounds selected with an eye towards structural attributes that are believed to make them more likely to inhibit the expression and/or function of miR-21.

To identify a modulator of miR-21, one generally will determine the function of a miR-21 in the presence and absence of the candidate compound. For example, a method generally comprises:

(a) providing a candidate compound; (b) admixing the candidate compound with miR-21 ;

(c) measuring miR-21 activity; and

(d) comparing the activity in step (c) with the activity in the absence of the candidate compound, wherein a difference between the measured activities indicates that the candidate compound is a modulator of miR-21.

Assays also may be conducted in isolated cells, organs, or in living organisms.

Assessing the miR-21 activity or expression may comprise assessing the expression level of miR-21. Those in the art will be familiar with a variety of methods for assessing

RNA expression levels including, for example, northern blotting or RT-PCR. Assessing the miR-21 activity or expression may comprise assessing the activity of miR-21. In some embodiments, assessing the activity of miR-21 comprises assessing expression or activity of a gene regulated by miR-21. Genes regulated by miR-21 include, for example, PTEN, PP ARa, Sproutyl, and Sprouty2. Those in the art will be familiar with a variety of methods for assessing the activity or expression of genes regulated by miR-21. Such methods include, for example, northern blotting, RT-PCR, ELISA, or western blotting. It will, of course, be understood that all the screening methods of the present invention are useful in themselves notwithstanding the fact that effective candidates may not be found. The invention provides methods for screening for such candidates, not solely methods of finding them.

As used herein the term "candidate compound" refers to any molecule that may potentially modulate the function of miR-21. One will typically acquire, from various commercial sources, molecular libraries that are believed to meet the basic criteria for useful drugs in an effort to "brute force" the identification of useful compounds. Screening of such libraries, including combinatorially-generated libraries (e.g., antagomir libraries), is a rapid and efficient way to screen large number of related (and unrelated) compounds for activity. Combinatorial approaches also lend themselves to rapid evolution of potential drugs by the creation of second, third, and fourth generation compounds modeled on active, but otherwise undesirable compounds. Non-limiting examples of candidate compounds that may be screened according to the methods of the present invention are proteins, peptides, polypeptides, polynucleotides, oligonucleotides or small molecules. Modulators of miR-21 may also be agonists or inhibitors of upstream regulators of miR-21 , such as serum response factor (SRF).

A quick, inexpensive and easy assay to run is an in vitro assay. Such assays generally use isolated molecules, can be run quickly and in large numbers, thereby increasing the amount of information obtainable in a short period of time. A variety of vessels may be used to run the assays, including test tubes, plates, dishes and other surfaces such as dipsticks or beads.

A technique for high throughput screening of compounds is described in WO 84/03564, which is herein incorporated by reference in its entirety. Large numbers of small antogomir compounds may be synthesized on a solid substrate, such as plastic pins or some other surface. Such molecules can be rapidly screening for their ability to hybridize to miR- 21. The present invention also contemplates the screening of compounds for their ability to modulate miR-21 expression and function in cells. Various cell lines, including those derived from skeletal muscle cells, can be utilized for such screening assays, including cells specifically engineered for this purpose. Primary cardiac cells also may be used, as can the H9C2 cell line.

In vivo assays involve the use of various animal models of heart disease or cancer, including transgenic animals, that have been engineered to have specific defects, or carry markers that can be used to measure the ability of a candidate compound to reach and affect different cells within the organism. Due to their size, ease of handling, and information on their physiology and genetic make-up, mice are a preferred embodiment, especially for transgenics. However, other animals are suitable as well, including rats, rabbits, hamsters, guinea pigs, gerbils, woodchucks, cats, dogs, sheep, goats, pigs, cows, horses and monkeys (including chimps, gibbons and baboons). Assays for inhibitors may be conducted using an animal model derived from any of these species. Treatment of animals with test compounds will involve the administration of the compound, in an appropriate form, to the animal. Administration will be by any route that could be utilized for clinical purposes. Determining the effectiveness of a compound in vivo may involve a variety of different criteria, including but not limited to alteration of hypertrophic signaling pathways in the heart, and physical symptoms of cardiac hypertrophy or cancer. Also, measuring toxicity and dose response can be performed in animals in a more meaningful fashion than in in vitro or in cyto assays.

In one embodiment, the present invention provides a method of regulating cardiac energy metabolism comprising administering a modulator of miR-21 to heart muscle cells. In another embodiment, the modulator is an agonist of miR-21 expression or activity. In another embodiment, cardiac energy metabolism is shifted to glucose metabolism following administration of a miR-21 agonist. In another embodiment, the modulator of miR-21 is an inhibitor of miR-21 expression or activity. In still another embodiment, cardiac energy metabolism is shifted to fatty acid metabolism following administration of a miR-21 inhibitor. In some embodiments, the expression of PP ARa, PTEN, Sproutyl, and Sprouty2 are increased in a cell by contacting the cell with a miR-21 inhibitor. In other embodiments, expression of PP ARa, PTEN, Sproutyl, and Sprouty2 are decreased in a cell by contacting the cell with a miR-21 agonist.

Thus, the present invention includes a method of regulating expression of PP ARa in a cell comprising contacting the cell with a modulator of miR-21. In one embodiment, the expression of PP ARa is decreased in the cell following administration of a miR-21 agonist. In another embodiment, the expression of PP ARa is increased in the cell following administration of a miR-21 inhibitor. In another embodiment, the present invention provides a method of regulating expression of Sprouty 1 and/or Sprouty 2 in a cell comprising contacting the cell with a modulator of miR-21. In another embodiment, the expression of Sprouty 1 and/or Sprouty 2 is decreased in the cell following administration of a miR-21 agonist. In another embodiment, the expression of Sprouty 1 and/or Sprouty 2 is increased in the cell following administration of a miR-21 inhibitor.

An agonist of miR-21 may be a polynucleotide comprising a mature miR-21 or a star miR-21 sequence. In one embodiment, the polynucleotide comprises the sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 17. In another embodiment, the agonist of miR-21 may be a polynucleotide comprising the pri-miRNA or pre-miRNA sequence for miR-21. The polynucleotide comprising the mature miR-21 sequence may be single stranded or double stranded. The polynucleotides may contain one or more chemical modifications, such as locked nucleic acids, peptide nucleic acids, sugar modifications, such as 2'-O-alkyl (e.g. 2'-O-methyl, 2'-O-methoxyethyl), 2'-fiuoro, and 4' thio modifications, and backbone modifications, such as one or more phosphorothioate, morpholino, or phosphonocarboxylate linkages. In one embodiment, the polynucleotide comprising a miR-21 sequence is conjugated to cholesterol. In another embodiment, the agonist of miR-21 may be an agent distinct from miR-21 that acts to increase, supplement, or replace the function of miR-21, for example, stress response factor (SRF) or an activator thereof. In another embodiment, the agonist of miR-21 may be expressed in vivo from a vector.

In one embodiment, the present invention provides a method for treating pathologic cardiac hypertrophy, heart failure, or myocardial infarction in a subject in need thereof comprising: identifying a subject having cardiac hypertrophy, heart failure, or myocardial infarction; and administering a miR-21 inhibitor to the subject. In certain embodiments of the invention the miR-21 inhibitor may be identified by a method comprising: (a) contacting a cell with a candidate compound; (b) assessing miR-21 activity or expression; and (c) comparing the activity or expression in step (b) with the activity or expression in the absence of the candidate compound, wherein a reduction in the activity or expression of miR-21 in the cell contacted with the candidate compound compared to the activity or expression in the cell in the absence of the candidate compound indicates that the candidate compound is an inhibitor of miR-21.

In another embodiment, the present invention provides a method for treating cancer in a subject in need thereof comprising: contacting a cancer cell in the subject with an inhibitor of miR-21 expression or function. In certain embodiments of the invention, the miR-21 inhibitor may be identified by a method comprising: (a) contacting a cell with a candidate compound; (b) assessing miR-21 activity or expression; and (c) comparing the activity or expression in step (b) with the activity or expression in the absence of the candidate compound, wherein a decrease in the activity or expression of miR-21 in the cell contacted with the candidate compound compared to the activity or expression in the cell in the absence of the candidate compound indicates that the candidate substance is an inhibitor of miR-21.

A particular embodiment of the present invention provides transgenic animals that lack one or both functional miR-21 alleles. Also, transgenic animals that express miR-21 under the control of an inducible, tissue selective or a constitutive promoter, recombinant cell lines derived from such animals, and transgenic embryos may be useful in determining the exact role that miR-21 plays in the development and differentiation of cardiomyocytes and in the development of pathologic cardiac hypertrophy and heart failure. Furthermore, these transgenic animals may provide an insight into heart development. The use of a constitutively expressed miR-21 encoding nucleic acid provides a model for over- or unregulated expression. Also, transgenic animals that are "knocked out" for miR-21, in one or both alleles, are contemplated.

In a general embodiment, a transgenic animal is produced by the integration of a given transgene into the genome in a manner that permits the expression of the transgene. Methods for producing transgenic animals are generally described by Wagner and Hoppe (U.S. Patent 4,873,191; incorporated herein by reference), and Brinster et al. (1985; incorporated herein by reference). Typically, a gene flanked by genomic sequences is transferred by microinjection into a fertilized egg. The microinjected eggs are implanted into a host female, and the progeny are screened for the expression of the transgene. Transgenic animals may be produced from the fertilized eggs from a number of animals including, but not limited to reptiles, amphibians, birds, mammals, and fish.

DNA clones for microinjection can be prepared by any means known in the art. For example, DNA clones for microinjection can be cleaved with enzymes appropriate for removing the bacterial plasmid sequences, and the DNA fragments electrophoresed on 1% agarose gels in TBE buffer, using standard techniques. The DNA bands are visualized by staining with ethidium bromide, and the band containing the expression sequences is excised. The excised band is then placed in dialysis bags containing 0.3 M sodium acetate, pH 7.0. DNA is electroeluted into the dialysis bags, extracted with a 1 :1 phenol: chloroform solution and precipitated by two volumes of ethanol. The DNA is redissolved in 1 ml of low salt buffer (0.2 M NaCl, 20 mM Tris, pH 7.4, and 1 mM EDTA) and purified on an Elutip-D™ column. The column is first primed with 3 ml of high salt buffer (1 M NaCl, 20 mM Tris, pH 7.4, and 1 mM EDTA) followed by washing with 5 ml of low salt buffer. The DNA solutions are passed through the column three times to bind DNA to the column matrix. After one wash with 3 ml of low salt buffer, the DNA is eluted with 0.4 ml high salt buffer and precipitated by two volumes of ethanol. DNA concentrations are measured by absorption at 260 nm in a UV spectrophotometer. For microinjection, DNA concentrations are adjusted to 3 μg/ml in 5 mM Tris, pH 7.4 and 0.1 mM EDTA. Other methods for purification of DNA for microinjection are described in in Palmiter et al. (1982); and in Sambrook eϊ α/. (2001).

In an exemplary microinjection procedure, female mice six weeks of age are induced to superovulate with a 5 IU injection (0.1 cc, ip) of pregnant mare serum gonadotropin (PMSG; Sigma) followed 48 hours later by a 5 IU injection (0.1 cc, ip) of human chorionic gonadotropin (hCG; Sigma). Females are placed with males immediately after hCG injection. Twenty-one hours after hCG injection, the mated females are sacrificed by C02 asphyxiation or cervical dislocation and embryos are recovered from excised oviducts and placed in Dulbecco's phosphate buffered saline with 0.5% bovine serum albumin (BSA; Sigma). Surrounding cumulus cells are removed with hyaluronidase (1 mg/ml). Pronuclear embryos are then washed and placed in Earle's balanced salt solution containing 0.5 % BSA (EBSS) in a 37.5°C incubator with a humidified atmosphere at 5% CO₂, 95% air until the time of injection. Embryos can be implanted at the two-cell stage.

Randomly cycling adult female mice are paired with vasectomized males. C57BL/6 or Swiss mice or other comparable strains can be used for this purpose. Recipient females are mated at the same time as donor females. At the time of embryo transfer, the recipient females are anesthetized with an intraperitoneal injection of 0.015 ml of 2.5 % avertin per gram of body weight. The oviducts are exposed by a single midline dorsal incision. An incision is then made through the body wall directly over the oviduct. The ovarian bursa is then torn with watchmakers forceps. Embryos to be transferred are placed in DPBS (Dulbecco's phosphate buffered saline) and in the tip of a transfer pipet (about 10 to 12 embryos). The pipet tip is inserted into the infundibulum and the embryos transferred. After the transfer, the incision is closed by two sutures.

As used herein, the term "heart failure" is broadly used to mean any condition that reduces the ability of the heart to pump blood. As a result, congestion and edema develop in the tissues. Most frequently, heart failure is caused by decreased contractility of the myocardium, resulting from reduced coronary blood flow; however, many other factors may result in heart failure, including damage to the heart valves, vitamin deficiency, and primary cardiac muscle disease. Though the precise physiological mechanisms of heart failure are not entirely understood, heart failure is generally believed to involve disorders in several cardiac autonomic properties, including sympathetic, parasympathetic, and baroreceptor responses. The phrase "manifestations of heart failure" is used broadly to encompass all of the sequelae associated with heart failure, such as shortness of breath, pitting edema, an enlarged tender liver, engorged neck veins, pulmonary rales and the like including laboratory findings associated with heart failure.

The term "treatment" or grammatical equivalents encompasses the improvement and/or reversal of the symptoms of heart failure (i.e., the ability of the heart to pump blood). "Improvement in the physiologic function" of the heart may be assessed using any of the measurements described herein (e.g., measurement of ejection fraction, fractional shortening, left ventricular internal dimension, heart rate, etc.), as well as any effect upon the animal's survival. In use of animal models, the response of treated transgenic animals and untreated transgenic animals is compared using any of the assays described herein (in addition, treated and untreated non-transgenic animals may be included as controls). A compound which causes an improvement in any parameter associated with heart failure used in the screening methods of the instant invention may thereby be identified as a therapeutic compound. The term "dilated cardiomyopathy" refers to a type of heart failure characterized by the presence of a symmetrically dilated left ventricle with poor systolic contractile function and, in addition, frequently involves the right ventricle.

The term "compound" refers to any chemical entity, pharmaceutical, drug, and the like that can be used to treat or prevent a disease, illness, sickness, or disorder of bodily function. Compounds comprise both known and potential therapeutic compounds. A compound can be determined to be therapeutic by screening using the screening methods of the present invention. A "known therapeutic compound" refers to a therapeutic compound that has been shown (e.g., through animal trials or prior experience with administration to humans) to be effective in such treatment. In other words, a known therapeutic compound is not limited to a compound efficacious in the treatment of heart failure.

As used herein, the term "cardiac hypertrophy" refers to the process in which adult cardiac myocytes respond to stress through hypertrophic growth. Such growth is characterized by cell size increases without cell division, assembling of additional sarcomeres within the cell to maximize force generation, and an activation of a fetal cardiac gene program. Cardiac hypertrophy is often associated with increased risk of morbidity and mortality, and thus studies aimed at understanding the molecular mechanisms of cardiac hypertrophy could have a significant impact on human health.

As used herein, the term "modulate" refers to a change or an alteration in a biological activity. Modulation may be an increase or a decrease in protein activity, a change in kinase activity, a change in binding characteristics, or any other change in the biological, functional, or immunological properties associated with the activity of a protein or other structure of interest. The term "modulator" refers to any molecule or compound which is capable of changing or altering biological activity as described above.

The term "β-adrenergic receptor antagonist" refers to a chemical compound or entity that is capable of blocking, either partially or completely, the beta (β) type of adreno receptors (i.e., receptors of the adrenergic system that respond to catecholamines, especially norepinephrine). Some β-adrenergic receptor antagonists exhibit a degree of specificity for one receptor subtype (generally βi); such antagonists are termed "βi-specific adrenergic receptor antagonists" and "β2-specific adrenergic receptor antagonists." The term β- adrenergic receptor antagonist" refers to chemical compounds that are selective and non- selective antagonists. Examples of β-adrenergic receptor antagonists include, but are not limited to, acebutolol, atenolol, butoxamine, carteolol, esmolol, labetolol, metoprolol, nadolol, penbutolol, propanolol, and timolol. The use of derivatives of known β-adrenergic receptor antagonists is encompassed by the methods of the present invention. Indeed any compound, which functionally behaves as a β-adrenergic receptor antagonist is encompassed by the methods of the present invention.

The terms "angiotensin-converting enzyme inhibitor" or "ACE inhibitor" refer to a chemical compound or entity that is capable of inhibiting, either partially or completely, the enzyme involved in the conversion of the relatively inactive angiotensin I to the active angiotensin II in the rennin-angiotensin system. In addition, the ACE inhibitors concomitantly inhibit the degradation of bradykinin, which likely significantly enhances the antihypertensive effect of the ACE inhibitors. Examples of ACE inhibitors include, but are not limited to, benazepril, captopril, enalopril, fosinopril, lisinopril, quiapril and ramipril. The use of derivatives of known ACE inhibitors is encompassed by the methods of the present invention. Indeed any compound, which functionally behaves as an ACE inhibitor, is encompassed by the methods of the present invention.

As used herein, the term "genotypes" refers to the actual genetic make-up of an organism, while "phenotype" refers to physical traits displayed by an individual. In addition, the "phenotype" is the result of selective expression of the genome (i.e., it is an expression of the cell history and its response to the extracellular environment). Indeed, the human genome contains an estimated 30,000-35,000 genes. In each cell type, only a small (i.e., 10- 15%) fraction of these genes are expressed.

The use of the word "a" or "an" when used in conjunction with the term " comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." It is contemplated that any embodiment discussed herein can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, compositions and kits of the invention can be used to achieve methods of the invention.

Throughout this application, the term "about" is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or." As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. The following examples are included to further illustrate various aspects of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques and/or compositions discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

EXAMPLES

Example 1. Regulation of cardiac hypertrophy and heart failure by stress-responsive miRNAs

In light of their involvement in modulating cellular phenotypes, the inventors hypothesized that miRNAs might play a role in regulating the response of the heart to cardiac stress, which is known to result in transcriptional and translational changes in gene expression. To investigate the potential involvement of miRNAs in cardiac hypertrophy, the inventors performed a side -by-side miRNA microarray analysis in 2 established mouse models of cardiac hypertrophy, using a microarray that represented 186 different miRNAs (Figures IA-D) (van Rooij et al, 2006). Mice that were subjected to thoracic aortic banding (TAB), which induces hypertrophy by increased afterload on the heart (Hill et al, 2000), were compared to sham operated animals. In a second model, transgenic mice expressing activated calcineurin (CnA) in the heart, which results in a severe, well-characterized form of hypertrophy (Molkentin et al, 1998), were compared to wild-type littermates (Figure IA). RNA isolated from hearts of mice subjected to TAB showed increased expression of 27 miRNAs compared to sham-operated controls, and CnA Tg mice showed increased expression of 33 miRNAs compared with non-transgenic littermate controls, of which 21 were up-regulated in both models. Similarly, TAB and CnA-induced hypertrophy were accompanied by reduced expression of 15 and 14 miRNAs, respectively, of which 7 miRNAs were down-regulated in common (Figure IA). Northern analysis of these miRNAs and previous microarray analyses (Barad et al, 2004; Sempere et al, 2004; Shingara et al, 2005; Babak et al, 2004) indicate that they are expressed in a wide range of tissues. Based on their relative expression levels, conservation across species, and levels of expression during hypertrophy, the inventors focused on 11 up- and 5 down-regulated miRNAs.

Total RNA was isolated from mouse cardiac tissue samples using Trizol reagent (Gibco/BRL), and Northern blots were performed to detect microRNAs as described previously (Bartel, 2004). A U6 probe served as a loading control (U6 forward: 5- GTGCTCGCTTCGGCAGC-3 (SEQ ID NO: 13), U6 reverse: 5- AAAATATGGAACGCTTCACGAATTTGCG-3 (SEQ ID NO: 14)).

Northern blot analysis of cardiac RNA from WT and CnA Tg animals confirmed an increased expression of miRs -21, -23, -24, -125b, -195, -199a, and -214, and decreased expression of miRs -29c, -93, -150 and -181b (Figure IB). Mir-21 was the most dramatically up-regulated of all microRNAs during hypertrophy (Figures 1C and ID). Collectively, these data indicate that distinct miRNAs are regulated during cardiac hypertrophy, suggesting that they may function as modulators of this process. Example 2. MiR-21 expression in response to diverse cardiac stresses

MiR-21 is located right outside of the 3'UTR of the TMEM49 gene on human chromosome 17 and is expressed as a separate 3.4 kb transcript (Figure 2A). Processing of the pre-miR-21 sequence results in a mature sequence (miR-21; SEQ ID NO: 1) and a star sequence (miR-21*; SEQ ID NO: 2). Both the mature and star miR-21 sequences are conserved among species (Figure 2A). Northern blot analysis demonstrates that miR-21 is expressed in various tissues, with high expression in the pancreas and lungs (Figure 2B).

Ventricular hypertrophy develops in response to numerous forms of cardiac stress and often leads to heart failure in humans. To determine if miR-21 is upregulated in cardiac tissue in response to stress signals, Northern blot analysis of cardiac tissue from animals of different stress models (e.g thoracic aortic banding, constitutively active calcineurin, myocardial infarction, and in vivo administration of isoproterenol or angiotensin II) was performed. As shown in Figure 3 A, miR-21 is strongly upregulated in five different models of cardiac stress. MiR-21 was not upregulated in the cardiac tissue in an exercise-induced model of hypertrophy, suggesting that miR-21 plays a specific role in stress-induced cardiac hypertrophy.

In a second experiment, primary cardiomyocytes were treated in vitro with serum free medium (SF) or phenylephrine (PE), a potent hypertrophic agonist. The expression of miR- 21 was strongly induced by phenylephrine (PE), demonstrating that miR-21 is upregulated in response to stress signals (Figure 3A) Note that the baseline expression of miR-21 in fibroblasts is much higher as compared to cardiomyocytes (Figure 3A).

In addition, miR-21 expression is increased with age in a hypertensive rat model (Figure 3B). This increased expression correlates with the level of cardiac hypertrophy as indicated by heart weight and left ventricular weight to body weight ratios (Figure 3C). MEF2 is a transcription factor that, upon stress, induces gene expression that mediates cardiac hypertrophy and remodeling. To determine whether miR-21 expression is dependent on MEF2, electrophoretic mobility assays were performed with oligonucleotides containing the putative MEF2 binding site in the regulatory region of miR-21. Oligonucleotides containing the conserved MEF2 binding site from the miR-21 regulatory region as well as oligonucleotide containing a mutated MEF2 binding site were synthesized (Integrated DNA Technology). Annealed oligonucleotides were radiolabeled with [³²PJdCTP using the Klenow fragment of DNA polymerase and purified using G50 spin columns (Roche). Nuclear cell extracts were isolated from Cos-1 cells that were transfected with pcDNAMYC-MEF2C. Reaction conditions of the gel mobility-shift assays were the same as those previously described (McFadden et ah, 2000). Unlabeled oligonucleotides used as competitors were annealed as described above and added to the reactions at the indicated concentrations. DNA-protein complexes were resolved on 5% polyacrylamide native gels and the gels were exposed to BioMax X-ray film (Kodak). The results of the mobility shift assays are shown in Figure 4 and demonstrate that MEF2 does interact with the binding site in the miR-21 regulatory region suggesting that miR-21 transcription is dependent on a MEF2.

This series of experiments demonstrates that miR-21 is induced in cardiac tissue in response to stress signals, and miR-21 expression correlates with cardiac hypertrophy. These results suggest that miR-21 is likely to play a role in the development of cardiac hypertrophy.

Example 3. MiR-21 knockout mice are protected from pressure overload

As discussed in Example 2, miR-21 is located adjacent to the TMEM49 gene. To examine whether miR-21 is transcribed independently or with TMEM49, Northern blot and RT-PCR analyses were performed on various tissues to determine whether the expression levels of miR-21 correlated with those of TMEM49. As shown by the results in Figure 5A, miR-21 exhibited a different expression pattern from that of TMEM49 indicating that miR- 21 and TMEM49 are expressed from different transcripts. The expression of TMEM49 in cardiac tissues after thoracic aortic banding (TAB) and myocardial infarction was also examined. Although both miR-21 and TMEM49 appeared to be upregulated in heart tissue after TAB, only miR-21 was induced after myocardial infarction (Figure 5B). These results further suggest that miR-21 is transcribed independently from TMEM49.

To more carefully examine the role of miR-21 in pathological cardiac hypertrophy, mice lacking one or both miR-21 alleles were generated. The targeting strategy used to generate the knockout animals is shown in Figure 6A. To generate the miR-21 targeting vector, a 4.8 Kb fragment (5' arm) extending upstream of the miR-21 coding region was digested with SacII and Notl and ligated into the pGKneoF2L2dta targeting plasmid upstream of the loxP sites and the Frt-flanked neomycin cassette. A 2.2 kb fragment (3' arm) was digested with Sail and HindIII and ligated into the vector between the neomycin resistance and Dta negative selection cassettes. A 246 bp fragment containing the pre-miR-21 was inserted with Sma. Targeted ES-cells carrying the disrupted allele were identified by Southern blot analysis with a 3' probe (Figure 6B). Three miR-21 targeted ES clones were identified and used for blastocyst injection. The resulting chimeric mice were bred to C57BL/6 to obtain germline transmission of the mutant allele.

Genomic deletion of miR-21 resulted in viable and fertile animals. Realtime PCR analysis demonstrated a dose-dependent decrease in miR-21 expression in heterozygote and homozygote animals (Figure 7A). Expression of miR-21 in the lung also exhibited the expected dose-dependent decrease in heterozygote and homozygote animals (Figure 7B). TMEM49 expression was not affected by deletion of one or both miR-21 alleles (Figure 7C), supporting the earlier findings that miR-21 is produced from a separate transcript.

A microarray analysis was performed on cardiac tissue isolated from wild-type mice and miR-21 knockout mice. Total RNA from cardiac tissue was isolated using Trizol (Invitrogen). Microarray analysis was performed using Mouse Genome 430 2.0 array (Affymetrix). Several genes were found to be significantly upregulated and downregulated in the knockout animals as compared to their wild-type litter mates (Figure 8).

One particular gene of interest that was significantly upregulated in miR-21 knockout animals was PTEN (phosphatase and tensin homolog). PTEN is a phosphatase that terminates AKT signaling by dephosphorylating phosphatidylinositol (3,4,5)-trisphosphate (PIP3). Termination of this signaling cascade prevents cells from proliferating and can cause cells to undergo apoptosis. PTEN is a tumor suppressor gene and mutation or loss of the gene is associated with many forms of human cancer. Western blot analysis of cardiac tissue isolated from miR-21 knockout animals revealed a significant increase in expression of PTEN (Figure 9). Expression of Sproutyl, a previously identified miR-21 target, was not affected by deletion of miR-21. However, cardiomyocyte specific overexpression decreases Sproutyl in myocytes (data not shown), indicating a cell type specific targeting effect of miR-21.

Thoracic aortic banding (TAB) was performed in both wild-type mice and miR-21 knockout mice to determine the effect of miR-21 deletion on cardiac hypertrophy. Northern blot analysis on cardiac tissue showed increased expression of miR-21 in wild- type animals after stress (Figure 10A). MiR-21 expression is not evident in knockout animals in either the sham condition or the stress condition. Cardiac hypertrophy was measured by calculating the ratio between heart weight and body weight. The ratio between heart weight and body weight under sham (unstressed) conditions appeared to be comparable for both wild-type and knockout animals. However, in response to stress the wild-type hearts exhibited significantly more hypertrophy than the knockout hearts (Figure 10B). The expression of βMHC, a stress responsive gene, is strongly induced in wild-type hearts after stress (TAB). The induction of βMHC is virtually absent in the knockout animals (Figure 10C). The expression of thioredoxin-interacting protein (Txnip), which was shown by the microarray analysis to be upregulated in miR-21 knockout animals (Figure 8), was increased in the miR-21 knockout animals as measured by Real-time PCR analysis (Figure 10D). These results suggest that miR-21 is necessary for stress-induced cardiac hypertrophy. Strategies to decrease miR-21 expression or activity may be a viable therapeutic approach to prevent the development of cardiac hypertrophy.

Example 4. MiR-21 is regulated by stress response factor (SRF)

In addition to the MEF2 binding site (see Example 2), there is a conserved binding site sequence for stress response factor (SRF) in the regulatory region of miR-21 (Figure HA). To determine if miR-21 expression is regulated by SRF, eletrophoretic mobility shift assays were performed. Oligonucleotides containing the conserved SRF-binding site in the miR-21 regulatory region, and oligonucleotides containing mutated SRF-binding sites were synthesized (Integrated DNA Technology). Annealed oligonucleotides were radiolabeled with [32P]dCTP using the Klenow fragment of DNA polymerase and purified using G50 spin columns (Roche). Nuclear cell extracts were isolated from Cos-1 cells that were transfected with pcDNAMYC-SRF. Unlabeled oligonucleotides used as competitors were annealed as described above and added to the reactions. DNA-protein complexes were resolved on 5% polyacrylamide native gels and the gels were exposed to BioMax X-ray film (Kodak). Gelshift analysis showed that SRF avidly binds the sequence located upstream of miR-21, while a mutated form of the binding sequence eliminates SRF binding (Figure HB). This result indicates that SRF can specifically bind to the DNA sequence located upstream of the pri-miRNA-21 suggesting that SRF may regulate miR-21 expression. To test whether SRF regulates transcription of miR-21, luciferase assays, in which a luciferase reporter gene was under the control of the upstream regulatory sequence of miR- 21, were performed. A mouse genomic DNA fragment covering the region upstream of the transcriptional start site of miR-21 was cloned into pGL2 luciferase vector. Mutations of the SRF site were introduced by PCR-based site-directed mutagenesis. COS cells were trans fected with Fugeneό (Stratagene) according to the manufacturer's instructions. The total amount of DNA was kept constant by adding the corresponding amount of expression vector without a cDNA insert. Forty eight hours after transfection, cells were treated with varying concentrations of myocardin, an activator of SRF, and cell extracts were assayed for luciferase expression using the luciferase assay kit (Promega). Relative promoter activities were expressed as luminescence relative units normalized for β-galactosidase expression in the cell extracts. As shown in Figure 12A, myocardin induced expression of the luciferase gene when the reporter was under the control of the miR-21 regulatory region. Mutations in the SRF binding site abolished luciferase expression. These results demonstrate SRF can bind to the regulatory region of miR-21 and activate transcription.

MiR-21 may regulate the cardiac stress response by reducing fibroblast proliferation (Figure 12B). When stressed, the heart secretes pro-fibrotic cytokines/hormones, which stimulate the activity of SRF. SRF activates the transcription of miR-21 resulting in increased levels of miR-21. PTEN, a target of miR-21 , normally inhibits the proliferation and migration of fibroblasts. The increase in miR-21 expression reduces the level of PTEN, resulting in the proliferation of fibroblasts. Fibroblast proliferation during cardiac stress induces the onset of cardiac fibrosis through the expression of collagens. The expression of βMHC has been shown to be directly related to the regional presence of interstitial fibrosis. Therefore, inhibition of miR-21 leads to the increased expression of PTEN, which in turn blocks the proliferation of fibroblasts thereby partially preventing the onset of fibrosis. This indirectly reduces the level of the stress responsive βMHC.

Example 5. MiR-21 antagomirs effectively knockdown miR-21 expression

To determine if miR-21 could be modulated in vivo, antagomirs designed to inhibit miR-21 activity were administered to mice. Chemically modified oligonucleotides comprising a sequence complementary to the mature miR-21 (anti miR-21; SEQ ID NO: 15) were used to inhibit miR-21 activity (Figure 13A). All nucleosides were 2'-0Me modified, and the 5' terminal two and 3' terminal four bases contained a phosphorothioate internucleoside. Cholesterol was attached to the 3' end of the passenger strand through a hydroxyprolinol linker. Eight week old C57BL/6 male mice received either anti miR-21 (SEQ ID NO: 15) or mismatch miR-21 (SEQ ID NO: 16) at a dose of 80 mg/kg body weight or a comparable volume of saline through tail vein injection. Tissues were collected 3 days after treatment. Intravenous injection (IV) of 80 mg/kg anti miR-21 effectively reduced the level of miR-21 in most tissues. Administration of the mismatched miR-21, which contained four base mismatches as compared to the anti miR-21 sequence, did not significantly affect the expression of miR-21 (Figure 13B). Synthetic oligonucleotides containing a sequence that is complementary to the mature miR-21 sequence can be used to downregulate miR-21 expression in vivo.

Example 6. Cardiac over-expression of miR-21 causes cardiac dysfunction. To further define the role of miR-21 in stress-induced cardiac hypertrophy, transgenic mice that overexpressed miR-21 specifically in the heart were generated. A mouse genomic fragment flanking miR-21 was subcloned into a cardiac-specific expression plasmid containing the α-MHC and human GH poly(A)+ signal (Kiriazis and Kranias, 2000). Genomic DNA was isolated from mouse tail biopsies and analyzed by PCR using primers specific for the human GH poly(A)+ signal. These transgenic mice were healthy and fertile, but died at 4-5 months of age. Northern blot analysis revealed that the transgenic mice efficiently overexpressed miR-21 under the control of the α- myosin heavy chain (MHC) promoter in cardiac tissue (Figure 14A). Significant cardiac remodeling occurred in the miR- 21 transgenic mice as evidence by histological analysis (Figure 14B). Overexpression of miR-21 resulted in cardiac hypertrophy and fibrosis. The heart weight (HW) to body weight (BW) ratio was higher in transgenic animals as compared to wild-type animals (Figure 14C), and this increased ratio correlated with increases in expression of stress responsive genes, atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP). Thus, miR-21 is sufficient to induce cardiac remodeling. Microarray analysis was conducted on cardiac tissue from miR-21 transgenic animals at 6 weeks of age to determine changes in gene expression patterns. Overexpression of miR- 21 in cardiomyocytes produces downregulation of several genes involved in cardiac metabolism, including the regulatory subunit of phosphatidylinositol 3-kinase (PI-3K) and PP ARa, which encodes a transcription factor that regulates fatty acid metabolism (Figure 15). Pathological cardiac remodeling is accompanied by an unfavorable switch in energy substrate preference from fatty acids to glucose, which is primarily regulated by PP ARa (Neubauer, 2007). In both animal and human hypertrophic and failing hearts, the expression of PP ARa is decreased in proportion to the depression of fatty acid utilization, which is suggested to play a major role in the transition from hypertrophy to heart failure (Luptak et al, 2005). To determine if PP ARa is a direct target of miR-21, real-time PCR analysis was performed with cardiac tissue isolated from miR-21 transgenic mice. A binding site for miR- 21 is found in the 3' UTR of the PP ARa gene, and this binding site region is conserved across species (Figure 16A). The results of the real-time PCR analysis show that PP ARa is downregulated in response to miR-21 overexpression, while PGC lα, an upstream regulator of PP ARa, is unaffected (Figure 16B). These results suggest that PP ARa is a target of miR- 21.

MiR-21 transgenic mice also exhibit gross mitochondrial abnormalities as compared to wild- type animals (Figure 17). These abnormalities are likely to be a result of the metabolic disruption induced by miR-21 overexpression. In contrast to the dramatic effects of miR-21 on cardiac structure, function, and gene expression, cardiac over-expression of miR-214 at levels comparable to those of miR-21 had no phenotypic effect. Thus, the cardiac lethality in the miR-21 transgenic animals is specifically due to functional effects of this miRNA rather than a general non-specific effect resulting from miRNA over-expression. Upregulation of miR-21 during cardiac stress decreases expression of PPARα and induces a deleterious alteration in cardiac energy metabolism (Figure 18). In addition to PPARα, two other potential miR-21 targets that may be involved in the remodeling process are Sproutyl and 2 (Figure 19). Strategies to suppress expression of miR-21 or its association with target mRNAs to prevent a shift from oxidative to glycolytic metabolism may have beneficial effects on the heart in the settings of pathological cardiac remodeling. All publications, patents, and patent applications discussed and cited herein are incorporated herein by reference in their entireties. All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods, and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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Claims

WHAT IS CLAIMED IS:

1. A method of treating pathologic cardiac hypertrophy, heart failure, or myocardial infarction in a subject in need thereof comprising:

(a) identifying a subject having cardiac hypertrophy, heart failure, or myocardial infarction; and

(b) inhibiting expression or activity of miR-21 in heart cells of said subject.

2. The method of claim 1, wherein inhibiting comprises administering to said subject an inhibitor of miR-21.

3. The method of claim 2, wherein the inhibitor of miR-21 is an antagomir, an antisense oligonucleotide, or an inhibitory RNA molecule.

4. The method of claim 3, wherein the antagomir or antisense oligonucleotide comprises a sequence that is complementary to a mature miR-21 sequence.

5. The method of claim 4, wherein the antagomir or antisense oligonucleotide comprises a sequence that is complementary to SEQ ID NO: 1 or SEQ ID NO: 17.

6. The method of claim 3, wherein the inhibitory RNA molecule comprises a double- stranded region, wherein the double-stranded region comprises a sequence that is substantially identical and substantially complementary to a mature miR-21 sequence.

7. The method of claim 6, wherein the double-stranded region comprises a sequence that is substantially identical and substantially complementary to SEQ ID NO: 1 or SEQ ID NO: 17.

8. The method of claim 2, wherein the inhibitor of miR-21 is administered by parenteral administration or direct injection into cardiac tissue.

9. The method of claim 8, wherein the parenteral administration is intravenous or subcutaneous.

10. The method of claim 2, wherein the inhibitor of miR-21 is administered by oral, transdermal, sustained release, controlled release, delayed release, suppository, catheter, or sublingual administration.

11. The method of claim 1 , further comprising administering to said subject a second cardiac hypertrophic therapy.

13. The method of claim 11, wherein said second therapy is selected from the group consisting of a β blocker, an ionotrope, a diuretic, ACE-I, All antagonist, BNP, a Ca⁺⁺-blocker, an endothelin receptor antagonist, and an HDAC inhibitor.

14. The method of claim 11, wherein said second therapy is administered at the same time as the inhibitor of miR-21.

15. The method of claim 11, wherein said second therapy is administered either before or after the inhibitor of miR-21.

16. The method of claim 1, wherein one or more symptoms of pathologic cardiac hypertrophy, heart failure, or myocardial infarction is improved in the subject following administration of the inhibitor of miR-21.

17. The method of claim 16, wherein said one or more symptoms is increased exercise capacity, increased cardiac ejection volume, decreased left ventricular end diastolic pressure, decreased pulmonary capillary wedge pressure, increased cardiac output, increased cardiac index, lowered pulmonary artery pressures, decreased left ventricular end systolic and diastolic dimensions, decreased left and right ventricular wall stress, decreased wall tension, increased quality of life, decreased disease related morbidity or mortality, or combinations thereof.

18. The method of claim 2, wherein administration of the inhibitor of miR-21 delays the transition from cardiac hypertrophy to heart failure in the subject.

19. A method of preventing pathologic hypertrophy or heart failure in a subject in need thereof comprising:

(a) identifying a subject at risk of developing pathologic cardiac hypertrophy or heart failure; and

(b) inhibiting expression or activity of miR-21 in heart cells of said subject.

20. The method of claim 19, wherein inhibiting comprises delivering to the heart cells an inhibitor of miR-21.

21. The method of claim 20, wherein the inhibitor of miR-21 is an antagomir, an antisense oligonucleotide, or an inhibitory RNA molecule.

22. The method of claim 21, wherein the antagomir or antisense oligonucleotide comprises a sequence that is complementary to a mature miR-21 sequence.

23. The method of claim 22, wherein the antagomir or antisense oligonucleotide comprises a sequence that is complementary to SEQ ID NO: 1 or SEQ ID NO: 17.

24. The method of claim 21, wherein the inhibitory RNA molecule comprises a double- stranded region, wherein the double-stranded region comprises a sequence that is substantially identical and substantially complementary to a mature miR-21 sequence.

25. The method of claim 24, wherein the double-stranded region comprises a sequence that is substantially identical and substantially complementary to SEQ ID NO: 1 or SEQ ID NO: 17.

26. The method of claim 19, wherein the subject at risk may exhibit one or more risk factors selected from the group consisting of long standing uncontrolled hypertension, uncorrected valvular disease, chronic angina, recent myocardial infarction, congenital predisposition to heart disease, and pathological hypertrophy.

27. The method of claim 19, wherein the subject at risk has been diagnosed as having a genetic predisposition to cardiac hypertrophy.

28. The method of claim 19, wherein the subject at risk has a familial history of cardiac hypertrophy.

29. A transgenic, non-human mammal, the cells of which fail to express a functional miR-21.

30. The transgenic mammal of claim 29, wherein said mammal is a mouse.

31. A transgenic, non-human mammal, the cells of which comprise a miR-21 coding region under the control of a heterologous promoter active in the cells of said non- human mammal.

32. The transgenic mammal of claim 31 , wherein said mammal is a mouse.

33. The transgenic mammal of claim 31, wherein said promoter is a tissue specific promoter.

34. The transgenic mammal of claim 33, wherein the tissue specific promoter is a muscle specific promoter.

35. The transgenic mammal of claim 33, wherein the tissue specific promoter is a heart muscle specific promoter.

36. A transgenic, non-human mammalian cell lacking one or both native miR-21 alleles.

37. The cell of claim 36, wherein said cell lacks both of said native miR-21 alleles.

38. A method of preventing cardiac hypertrophy and dilated cardiomyopathy in a subject in need thereof comprising inhibiting expression or activity of miR-21 in heart cells of said subject.

39. A method of inhibiting progression of cardiac hypertrophy in a subject in need thereof comprising inhibiting expression or activity of miR-21 in heart cells of said subject.

40. A method for identifying a modulator of miR-21 comprising:

(a) contacting a cell with a candidate compound;

(b) assessing miR-21 activity or expression; and

(c) comparing the activity or expression in step (b) with the activity or expression in the absence of the candidate compound,

wherein a difference between the measured activities or expression indicates that the candidate compound is a modulator of miR-21.

41. The method of claim 40, wherein the cell is contacted with the candidate compound in vitro.

42. The method of claim 40, wherein the cell is contacted with the candidate compound in vivo.

43. The method of claim 40, wherein the modulator of miR-21 is an agonist of miR-21.

44. The method of claim 40, wherein the modulator of miR-21 is an inhibitor of miR-21.

45. The method of claim 40, wherein the candidate compound is a protein, a peptide, polypeptide, polynucleotide, an oligonucleotide, or small molecule.

46. The method of claim 40, wherein assessing the miR-21 activity or expression comprises assessing the expression of miR-21.

47. The method of claim 46, wherein assessing the expression of miR-21 comprises Northern blotting or RT-PCR.

48. The method of claim 40, wherein assessing the miR-21 activity or expression comprises assessing the activity of miR-21.

49. The method of claim 48, wherein assessing the activity of miR-21 comprises assessing expression or activity of gene regulated by miR-21.

50. The method of claim 49, wherein the gene regulated by miR-21 is PPARα or PTEN.

51. A pharmaceutical composition comprising an inhibitor of miR-21.

52. The pharmaceutical composition of claim 51, wherein said inhibitor is an antagomir, an antisense oligonucleotide, or an inhibitory RNA molecule.

53. The pharmaceutical composition of claim 52, wherein the antagomir or antisense oligonucleotide comprises a sequence that is complementary to a mature miR-21 sequence.

54. The pharmaceutical composition of claim 53, wherein the antagomir or antisense oligonucleotide comprises a sequence that is complementary to SEQ ID NO: 1 or SEQ ID NO: 17.

55. The pharmaceutical composition of claim 52, wherein the inhibitory RNA molecule comprises a double-stranded region, wherein the double-stranded region comprises a sequence that is substantially identical and substantially complementary to a mature miR-21 sequence.

56. The pharmaceutical composition of claim 55, wherein the double-stranded region comprises a sequence that is substantially identical and substantially complementary to SEQ ID NO: 1 or SEQ ID NO: 17.

57. The pharmaceutical composition of claim 51 , wherein the composition is formulated for injection.

58. The pharmaceutical composition of claim 51 in combination with a kit for parenteral administration.

59. The pharmaceutical composition of claim 58, wherein parenteral administration is intravenous or subcutaneous.

60. The pharmaceutical composition of claim 51 in combination with a kit for catheter administration.