HK1134042B - IDENTIFICATION OF A MICRO-RNA THAT ACTIVATES EXPRESSION OF β-MYOSIN HEAVY CHAIN - Google Patents

Description

Identification of micro-RNAs that activate expression of the beta-myosin heavy chain

Background

The invention was made with financial support from the national institutes of health, U.S. under the funding number HL 53351-06. The government has certain rights in the invention.

The present application claims U.S. provisional application serial No. 60/834,667 filed on 8/1/2006; 60/952,911 filed on 31/7/2007; and 60/952,917 priority filed on 31/7/2007, the entire contents of which are incorporated herein by reference.

1. Field of the invention

The present invention relates generally to the fields of developmental biology and molecular biology. More specifically, the present invention concerns gene regulation and cellular physiology in cardiac and skeletal muscle cells. Specifically, the present invention relates to the inhibition of mirnas, resulting in reduced expression of β -myosin heavy chain (β -MHC), thereby treating cardiac hypertrophy and heart failure. The invention also encompasses the upregulation of this miRNA for the treatment of musculoskeletal diseases.

2. Description of the related Art

Cardiac hypertrophy in response to an increase in workload imposed on the heart is a fundamental adaptive mechanism. It is a specialized process that reflects the quantitative increase in cell size and mass (not cell number) that occurs as a result of any one or a combination of neural, endocrine, or mechanical stimuli. Hypertension, another factor of cardiac hypertrophy, is often a precursor to congestive heart failure. When heart failure occurs, the left ventricle is often hypertrophied and dilated, and indices of systolic function, such as ejection fraction, are reduced. Clearly, the cardiac hypertrophy response is a complex syndrome and elucidation of the mechanisms leading to cardiac hypertrophy would be beneficial for the treatment of heart disease caused by various stimuli.

Pathologic myocardial hypertrophy is characterized by an increase in cardiomyocyte proteins and a recurring (reminiscence) gene expression profile for early embryonic development. In particular, the first and second (c) substrates,increased expression of beta-myosin heavy chain (beta-MHC), skeletal alpha-actin (sACT), and atrial and cerebral natriuretic peptides (ANP and BNP, respectively), and adult cardiac muscle specific gene, alpha-myosin heavy chain (alpha-MHC), and sarcoplasmic reticulum Ca²⁺-reduced expression of ATPase (SERCA). More specifically, there is compelling evidence to indicate a role for changes in MHC isoform (isoform) expression in the pathogenesis of heart failure in humans. Indeed, α -MHCmRNA and protein levels are significantly reduced in the weakened heart, and improvement of left ventricular ejection fraction by β -blocker therapy is associated with normalization of α -MHC expression. In addition, a mutation in the human α -MHC gene was identified in association with hypertrophic cardiomyopathy, demonstrating that α -MHC expression levels are critical for normal cardiac function despite its low abundance. Thus, it is clear that both MHC, α and β, play a role in cardiac hypertrophy development, but the exact characteristics of the role of these products in the development and/or maintenance of pathological states remain unknown.

Summary of The Invention

Thus, in accordance with the present invention, there is provided in one embodiment a method of modulating cardiac contractility and remodeling (remodelling) comprising administering to cardiac cells a modulator of miR-208 expression or activity. In one embodiment, a method of modulating cardiac contractile protein gene expression is provided, comprising administering to cardiac cells a modulator of miR-208 expression or activity. The modulator can be an agonist or antagonist of miR-208 expression or activity. In certain aspects of the invention, methods of reducing beta-MHC expression in cardiac cells are provided, comprising administering to the cardiac cells an inhibitor of miR-208 expression or activity. In other aspects of the invention, methods are provided for increasing β -MHC expression in cardiac cells, comprising increasing endogenous miR-208 expression or activity or administering exogenous miR-208 to the cardiac cells. In one aspect of the invention, there is provided a method of increasing expression of a rapid skeletal muscle contraction protein gene in a cardiac cell, comprising administering to the cardiac cell an inhibitor of miR-208 expression or activity. In another aspect of the invention, methods are provided for reducing the expression of a rapid skeletal muscle contraction protein gene in a cardiac cell, comprising increasing endogenous miR-208 expression or activity or administering exogenous miR-208 to the cardiac cell. Examples of fast skeletal muscle contraction protein genes that can be increased or decreased according to the methods of the present invention include: skeletal troponin I, troponin T3, myosin light chain, or alpha-skeletal actin.

In one embodiment, the present invention provides a method of treating pathologic cardiac hypertrophy or heart failure comprising: identifying a patient with cardiac hypertrophy, heart failure, or post myocardial infarction remodeling; and inhibiting miR-208 expression or activity in heart cells of the patient. In another embodiment, there is provided a method of preventing pathologic hypertrophy or heart failure comprising: identifying a patient at risk of developing pathologic cardiac hypertrophy or heart failure; and inhibiting miR-208 expression or activity in heart cells of the patient.

In one embodiment, the invention provides a method of treating myocardial infarction comprising inhibiting miR-208 expression or activity in heart cells of the subject. In another embodiment, the invention provides a method of preventing cardiac hypertrophy and dilated cardiomyopathy comprising inhibiting miR-208 expression or activity in cardiac cells of a subject. In yet another embodiment, the invention provides a method of inhibiting progression of cardiac hypertrophy comprising inhibiting miR-208 expression or activity in cardiac cells of a subject. In certain embodiments, the invention provides methods of increasing exercise tolerance, reducing hospitalization, improving quality of life, reducing morbidity, and/or reducing mortality in a subject having heart failure or cardiac hypertrophy comprising inhibiting miR-208 expression or activity in heart cells of the subject. In other aspects of the invention, methods are provided for increasing alpha-MHC expression in cardiac cells under pathological conditions by administering a miR-208 inhibitor.

In certain aspects of the invention, inhibiting expression or activity of miR-208 comprises administering an antagomir of miR-208. In one embodiment, the present invention provides miR-208 antagomir. administration of antagomir or other modulators of miR-208 expression or activity can be by any method known to those skilled in the art suitable for delivery to the target organ, tissue, or cell type. For example, in certain aspects of the invention, a modulator of miR-208 can be administered by intravenous injection, intra-arterial injection, intra-pericardial injection, or direct injection into a tissue (e.g., cardiac tissue, skeletal muscle tissue). In some aspects, administration comprises oral, transdermal, intraperitoneal, subcutaneous, sustained release, controlled release, delayed release, suppository, or sublingual administration of miR-208.

In certain aspects of the invention, treating or preventing pathologic cardiac hypertrophy or heart failure in a patient further comprises administering a second cardiac hypertrophy therapy to the patient. The second therapy can be, for example, a beta blocker, an ionotrope, a diuretic, an ACE-I, AII antagonist, BNP, Ca⁺⁺-a blocking agent, and ERA, or an HDAC inhibitor. The second therapy can be administered prior to, concurrently with, or subsequent to inhibition of miR-208.

Treatment of pathologic cardiac hypertrophy or heart failure may be defined as an improvement in one or more symptoms of pathologic cardiac hypertrophy or heart failure. The symptoms that are improved may be, for example, increased exercise capacity (exercise capacity), increased cardiac ejection volume, decreased left ventricular end diastolic pressure, decreased pulmonary capillary wedge pressure, increased cardiac output or index, decreased pulmonary artery pressure, decreased left ventricular end systolic and diastolic diameters (end systole and diastole dimensions), decreased left and right ventricular wall pressures (wall stress), decreased wall tension (wall tension), increased quality of life, and decreased disease related morbidity or mortality. The treatment of pathologic cardiac hypertrophy may also be defined as delaying the transition from cardiac hypertrophy to heart failure.

In certain embodiments of the present invention, methods of preventing pathologic hypertrophy or heart failure in a patient at risk of developing pathologic cardiac hypertrophy or heart failure are provided. Patients at risk of developing pathologic cardiac hypertrophy or heart failure may exhibit one or more risk factors including, for example, long-term uncontrolled hypertension, uncorrected valvular disease, chronic angina, recent myocardial infarction, congenital predisposition to heart disease, or pathologic hypertrophy. In certain aspects, the patient at risk may be diagnosed as having a genetic predisposition to cardiac hypertrophy. In certain aspects of the invention, a patient at risk may have a family history of cardiac hypertrophy.

In one embodiment, the invention provides a method of reducing rapid skeletal muscle contraction protein gene expression or activity in a skeletal muscle cell, comprising administering miR-208 to a skeletal muscle cell. In one embodiment, the present invention provides a method of treating or preventing a musculoskeletal disorder in a subject, comprising: identifying a patient having or at risk of developing a musculoskeletal disorder; and increasing miR-208 expression and/or activity in skeletal muscle cells of the patient. A musculoskeletal disorder may be, for example, disuse atrophy, muscle wasting in response to microgravity, or denervation. In certain aspects of the invention, the method of treating or preventing a musculoskeletal disorder further comprises administering a second non-miR-208 therapy.

Increasing the expression and/or activity of miR-208 can include administering miR-208 to the subject or administering an expression vector that expresses miR-208 to the subject. The expression vector is a viral expression vector. The viral expression vector may be, for example, an adenoviral or retroviral expression vector. In certain aspects, the expression vector is a non-viral expression vector. In certain aspects of the invention, miR-208 or an expression vector encoding miR-208 is associated with a lipid mediator. Alternatively, only a separate miR-208 may be provided, optionally included within a delivery vehicle, such as a liposome or nanoparticle. miR-208 is heart-specific, which prevents unwanted side effects in other organs.

In one embodiment, the invention provides a method of identifying a modulator of miR-208, comprising: (a) contacting a cell with a candidate compound; (b) assessing miR-208 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 activity or expression measured indicates that the candidate compound is a modulator of miR-208. In certain aspects of the invention, a cell is contacted with a candidate compound in vitro. In other aspects of the invention, the cell is contacted with the candidate compound in vivo. The modulator of miR-208 can be an agonist or antagonist of miR-208. Non-limiting examples of candidate compounds that can be screened according to the methods of the invention are peptides, polypeptides, polynucleotides, or small molecules.

Assessing miR-208 activity or expression can include assessing the expression level of miR-208. Those skilled in the art are familiar with a variety of methods for assessing RNA expression levels, including, for example, Northern blotting or RT-PCR. Assessing miR-208 activity or expression can include assessing miR-208 activity. In some embodiments, assessing the activity of miR-208 comprises assessing the expression or activity of a gene regulated by miR-208. Genes regulated by miR-208 include, for example, the alpha and beta-myosin heavy chain and fast skeletal muscle protein genes, such as fast skeletal troponin I, troponin T3, myosin light chain, and alpha skeletal actin. In certain aspects of the invention, assessing the activity of miR-208 comprises assessing the ratio of alpha-myosin heavy chain expression level to beta-myosin heavy chain expression level. Those skilled in the art are familiar with various methods for assessing the activity or expression of genes regulated by miR-208. Such methods include, for example, Northern blotting, RT-PCR, ELISA, or Western blotting.

In one embodiment, the invention provides a miR-208 modulator identified by a method comprising: (a) contacting a cell with a candidate compound; (b) assessing miR-208 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 activity or expression measured indicates that the candidate compound is a modulator of miR-208. modulators of miR-208 can be included in pharmaceutical compositions for treating cardiac and/or musculoskeletal disorders according to the methods of the invention.

In another embodiment, the invention provides a miR-208 inhibitor identified by a method comprising: (a) contacting a cell with a candidate compound; (b) assessing miR-208 activity or expression; and (c) comparing the activity or expression in step (b) to the activity or expression in the absence of the candidate compound, wherein a decrease in activity or expression in a cell contacted with the candidate compound compared to the activity or expression in a cell in the absence of the candidate compound indicates that the candidate compound is an inhibitor of miR-208.

In one embodiment, the present invention provides a method of treating pathologic cardiac hypertrophy or heart failure comprising: identifying a patient with cardiac hypertrophy or heart failure; and administering the miR-208 inhibitor to the patient. In certain aspects of the invention, the miR-208 inhibitor can be identified by a method comprising: (a) contacting a cell with a candidate compound; (b) assessing miR-208 activity or expression; and (c) comparing the activity or expression in step (b) to the activity or expression in the absence of the candidate compound, wherein a decrease in miR-208 activity or expression in cells contacted with the candidate compound as compared to the activity or expression in cells in the absence of the candidate compound is indicative of the candidate compound being an inhibitor of miR-208.

In another embodiment, the invention provides a method of treating a musculoskeletal disorder, comprising: identifying a patient having or at risk of developing a musculoskeletal disorder; and administering a miR-208 agonist to the patient. In certain aspects of the invention, the miR-208 agonist can be identified by a method comprising: (a) contacting a cell with a candidate compound; (b) assessing miR-208 activity or expression; and (c) comparing the activity or expression in step (b) to the activity or expression in the absence of the candidate compound, wherein an increase in miR-208 activity or expression in cells contacted with the candidate compound compared to the activity or expression in cells in the absence of the candidate compound is indicative of the candidate compound being an agonist of miR-208.

In one embodiment, the invention provides a transgenic non-human mammal whose cells fail to express a functional miR-208. In another embodiment, the invention provides a transgenic non-human mammal, the cells of which comprise a miR-208 coding region under the control of a heterologous promoter that is active in the cells of the non-human mammal. The transgenic mammal may be, for example, a mouse or rat. The promoter may be a tissue-specific promoter, such as, for example, a skeletal muscle-specific promoter or a cardiac muscle-specific promoter. In certain embodiments, the invention provides a transgenic non-human mammalian cell that lacks one or both native miR-208 alleles.

The use of the words "a" or "an" when used in the claims/specification in conjunction with the terms "comprising", "including" or "containing" may mean "a" or "an", but is also consistent with the meaning of "one or more", "at least one", or "one or more" or "more than one".

For any method or composition of the invention, any embodiment discussed herein may be implemented, and vice versa. In addition, the compositions and kits of the invention can be used to practice the methods of the invention.

Throughout this application, the term "about" is used to indicate that a value includes the standard deviation of the device or method used 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 only one or the options are mutually exclusive, although the disclosure supports the definition of only one and "and/or".

As used in this specification and claims, the terms "comprises" (and any form of comprising, such as "comprises" and "comprising"), "has" (and any form of having, such as "has" and "with"), "includes" (and any form of including, such as "includes" and "containing"), or "contains" (and any form of containing, such as "contains") mean either inclusive or open-ended, and do not exclude additional, unrecited elements or method steps.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

Brief Description of 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.

FIG. 1-miR-208 is contained in cardiac alpha-MHC gene. miR-208 is encoded by intron 27 of the alpha MHC gene. Asterisks indicate sequence conservation.

FIG. 2-miR-208 has the same expression pattern as alpha-MHC. miR-208 transcript detection by Northern analysis of adult mouse tissues. U6mRNA served as loading control.

FIGS. 3A-C-Modulation of miR-208 expression by thyroid hormone. (FIG. 3A) schematic representation of PTU/T3 regulation of alpha-and beta-MHC. (FIG. 3B) rats were treated with PTU for 1 week with and without T3 and examined for α MHC and β MHC mRNA by real-time PCR. (FIG. 3C) rats were treated with PTU or PTU + T3 for 1 week as indicated and miR-208 expression was detected by Northern blotting. Hearts from 4 animals under each condition were analyzed.

FIGS. 4A-C-alpha-MHC expression inhibition leads to decreased miR-208 levels. (FIG. 4A-B) relative expression levels of α -and β -MHC transcripts at days 0, 3, 6, 9, 12, 15, 18 and 21. (FIG. 4C) Northern blot analysis of miR-208 in rat heart tissue at the time points indicated during PTU treatment.

FIGS. 5A-B-miR-208 gene knockout. FIG. 5A strategy for generating miR-208 mutant mice by homologous recombinationBut not shown. The pre-miRNA (pre-miRNA) sequence was replaced with a neomycin resistance cassette (Neo) flanked by loxP sites. The neomycin cassette in the mouse germline was eliminated by mating heterozygous mice with transgenic mice containing the CAG-Cre transgene. DTA, diphtheria toxin a. (FIG. 5B) miR-208 transcript detection by Northern analysis of hearts from wild-type (WT) and miR-208 mutant (KO) mice.

FIGS. 6A-B-Deletion of miR-208 does not alter alpha-MHC expression. (FIG. 6A) analysis of alpha MHC transcripts by RT-PCR of RNA from hearts of mice of the indicated genotypes. The position of the primers relative to the alpha MHC exon is shown and the primer pairs are shown above each set of samples. (FIG. 6B) Western analysis of aMHC and bMHC protein levels in the hearts of neonatal mice of the genotype indicated. 2 mice of each genotype were analyzed. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was detected as a loading control.

FIG. 7-Up-regulation of fast skeletal genes in miR-208 knockout。

FIG. 8-MiR-208 knock-out of cardiac stress response (stress response) gene dysregulation in mice。

FIG. 9-model of the role of miR-208 in cardiac gene regulation. The alpha MHC gene encodes miR-208, which down-regulates expression of thap 1 and the skeletal muscle gene (and possibly other targets). The α -and β -MHC genes are linked (linked), and miR-208 is required for β MHC upregulation and PTU blockade of T3 signaling, which occurs in response to stress signaling. Alpha-and beta-MHC promote rapid and slow contractions, respectively.

FIG. 10-Human heart samples: non-failing (failing)。

FIG. 11-THRAP1 as a predicted target for miR-208. Sequence alignment of the putative miR-208 binding site in the 3' UTR of THRAP1 shows a high level of complementarity and sequence conservation.

FIG. 12-3' UTR THRAP1 luciferase assay。

FIG. 13-Heart of miR-208 mutant mouseRapid skeletal muscle gene upregulation in the zang organs。

FIGS. 14A-E-Wild type and miR-208 ^-/- Analysis of animals after TAB. (FIG. 14A) wild type and miR-208 after sham surgery or TAB21 days^-/-Alpha MHC mRNA expression was detected in mice by real-time PCR. (FIG. 14B) by from wild type and miR-208^-/-Northern blotting of cardiac tissue from mice detects miR-208. (FIG. 14C) echocardiographic analysis indicated a decrease in FS due to miR-208^-/-And littermate wild type mice in response to TAB. The anterior and posterior wall (AW and PW) thickness of post-TAB systole(s) or diastole (d) shows miR-208 in comparison to wild-type animals^-/-Blunted (blunted) hypertrophic responses in animals (n-5-7 per group).^*p < 0.05, compared to the corresponding wild type group. (FIG. 14D) miR-208 transcripts from hearts of wild-type and miR-208 transgenic mice were detected by Northern analysis. (FIG. 14E) transcripts of α MHC, β MHC, ANF and BNP were detected by real-time PCR in hearts from the indicated genotypes. Values are expressed as fold (± SEM) increase in expression compared to wild type mice (n ═ 3).

FIGS. 15A-G-miR-208 ^-/- Mice show a reduction in cardiac hypertrophy in response to pressure overload. (FIG. 15A) wild type and miR-208^-/-Masson trichrome stained histological sections of mouse hearts. Deletion of miR-208 attenuated the hypertrophy and fibrosis seen in wild-type mice subjected to 21-day TAB. The scale is equal to 2mm in the top and 20 μm in the bottom. (FIG. 15B) detection of wild type and miR-208 after sham or TAB surgery by real-time PCR^-/-Transcripts of β MHC, ANF and BNP in the heart of mice. Values are expressed as fold (± SEM) increase in expression compared to sham operated wild type mice (n ═ 3). (FIG. 15C) Western analysis of α MHC and β MHC protein levels in adult wild type and miR-208 mutant mice 21 days post-TAB and sham surgery. FIG. 15D 6 weeks of expression of the calcineurin (calceneurin) transgene (CnA-Tg)Heart and miR-208 of aged mice^-/-Histological sections of the heart; masson trichrome staining was performed on CnA-Tg. Deletion of miR-208 attenuates hypertrophy and fibrosis seen in CnA-Tg mice. The scale is equal to 2mm in the top and 20 μm in the bottom. (FIG. 15E) transcripts of β MHC, ANF and BNP in hearts from the indicated genotypes were detected by real-time PCR. Values are expressed as fold (± SEM) increase in expression compared to wild type mice (n ═ 3). (FIG. 15F) Western analysis of alpha and beta MHC protein levels in adult wild type and miR-208 mutant mice with and without CnA transgene. (FIG. 15G) Western analysis of α MHC and β MHC protein levels in adult wild type and miR-208 transgenic animals.

FIGS. 16A-C-Wild type and miR-208 after PTU treatment ^-/- Analysis of animals. (FIG. 16A) detection of wild type and miR-208 from PTU-treated by Northern blot^-/-miR-208 of heart tissue of mice. (FIG. 16B) echocardiography shows wild-type and miR-208^-/-Comparable Heart Rate (HR) and Fractional Shortening (FS) decreases in response to PTU in mice due to an increase in left ventricular dilation (LVID) and thinning of the anterior and posterior left ventricular walls (AW, PW) (n 6) in both diastole (d) and systole(s).^*p < 0.05, compared to the corresponding wild type group. (FIG. 16C) detection of wild type and miR-208 from PTU post-treatment by real-time PCR^-/-Transcripts of ANF and BNP in the heart of mice. Values are expressed as fold (± SEM) increase in expression compared to wild type mice receiving regular food (n ═ 3).

FIGS. 17A-B-modulation of responsiveness of miR-208 to beta MHC gene. (FIG. 17A) Western analysis of alpha and beta MHC expression at baseline and 2 weeks post PTU treatment in wild type and miR-208 mutant mice. (FIG. 17B) detection of wild type and miR-208 from PTU post-treatment by real-time PCR^-/-Alpha and beta MHC transcripts in the hearts of mice. Values are expressed as fold (± SEM) increase in expression compared to wild type mice receiving regular food (n ═ 3).

FIGS. 18A-D-miR-208 targets THRAP1. (FIG. 18A) THRAP1The sequence alignment of the putative miR-208 binding site in the 3' UTR of (A) shows a high level of complementarity and sequence conservation. (FIG. 18B) COS1 cells were transfected with the THRAP 13' UTR luciferase construct and expression plasmids for miR-126 and miR-208. Values are expressed as fold increase in luciferase expression (± SD) compared to the reporter alone. (FIG. 18) COS1 was transfected with HA-MCD-WT UTR or HA-MCD-mutant UTR, and increasing doses of pCMV-miR-208 ranging from 0.1-2. mu.g. HA-levels were detected using immunoblotting. (FIG. 18D) THRAP1 Western blot of cardiac cell lysates immunoprecipitated with THRAP1 using THRAP 1-specific antibody using 400 μ g from wild-type animals or miR-208^-/-Animal protein.

FIG. 19-RT-PCR analysis of THRAP1 transcript. By from wild type and miR-208^-/-THRAP1 transcript analysis by RT-PCR of RNA from mouse hearts. The position of the primer in the mRNA transcript is shown.

FIG. 20-Real-time PCR analysis of thyroid hormone receptor signaling targets. Detection of nucleic acids from wild type and miR-208 by real-time PCR^-/-Transcripts of SERCA2a and PLB, and GLUT4 in the heart of mice. Values are expressed as fold increase (± SEM) in expression compared to wild type mice.

FIG. 21-Shows wild type, miR-208 ^+/- And miR-208 ^-/- Form of miR-499 expression in the heart Northern blotting of. There was a positive correlation between miR-208 expression and miR-499, and Myb7b in wild-type and mutant mice.

FIG. 22-The structure of the Myh7b locus and the location of the miR-499 coding region therein。

FIG. 23-Northern blot showing miR-499 expression in cardiac and soleus muscles. mir-499 is not expressed in fast skeletal muscle fibers such as gastrocnemius/plantar muscle (GP), Tibialis Anterior (TA), or Extensor Digitorum Longus (EDL).

FIG. 24-Northern blot showing miR-499 expression in wild-type mice with Heart diseaseTrace. MI, myocardial infarction. CnA Tg, calcineurin-dependent transgenic mice.

FIG. 25-Schematic of miR-499 regulation by miR-208 in heart muscle。

FIGS. 26A-C-MiRNA expression during cardiac hypertrophy. (FIG. 26A) H from mice after sham surgery and TAB21 days and from representative hearts of CnA Tg mice&E staining the sections. The scale is equal to 2 mm. (FIG. 26B) Venn diagram showing the number of microRNAs expressing changes in each type of heart is shown below. (FIG. 26C) Northern blot of microRNAs with changes in expression during the hypertrophy process. U6RNA was tested as a loading control.

FIG. 27-miR-29 expression downregulation in response to cardiac stress. Hearts from wild type mice (WT) and mice with phosphatase-dependent transgene (CnA) or TAB-induced hypertrophy and fibrosis are shown on the left. The relative expression levels of miR-29 in each type of heart are shown on the right.

FIG. 28-Microarray analysis of hearts from miR-208 knockout mice compared to wild type. Microarray analysis of mRNA isolated from wild-type and miR-208 deficient hearts at 6 weeks of age was performed. The most downregulated miRNA next to miR-208 is miR-499.

FIG. 29-The miR-29 family is significantly up-regulated in miR-208-deficient heart。

FIG. 30-The miR-29 family targets other agents encoding collagen and involved in fibrosis in the extracellular matrix Fractionated mRNA。

FIG. 31-model of miR-208 and miR-29 families for controlling cardiac fibrosis. In normal heart, miR-208 inhibits the expression of miR-29. In the case of miR-208 deletion, miR-29 expression is upregulated, preventing extracellular matrix expression and fibrosis that occurs in response to stress. The functions of miR-208, -499 and-29 are interrelated. The deletion of miR-208 may be cardioprotective, by preventing miR-499 expression and upregulating miR-29 expression, and thus blocking fibrosis.

Detailed description of illustrative embodiments

Heart failure is one of the leading causes of morbidity and mortality in the world. In the united states alone, it is estimated that 300 million people are currently suffering from cardiomyopathy, and another 40 million people are diagnosed each year. Dilated Cardiomyopathy (DCM), also known as "congestive cardiomyopathy", is the most common form of cardiomyopathy, and has an estimated prevalence of approximately 40 out of 10 million people (Durand et al, 1995). Although DCM has other causes, familial dilated cardiomyopathy has been indicated to account for approximately 20% of "idiopathic" DCM. About half of DCM cases are idiopathic, the remainder being associated with known disease processes. For example, certain drugs used in cancer chemotherapy, such as doxorubicin (doxorubicin) and daunorubicin (daunorubicin), can cause severe myocardial injury. In addition, many patients with DCM are chronic alcoholics. Fortunately, for these patients, the progression of myocardial dysfunction can be halted or reversed if drinking is reduced or halted early in the course of the disease. Perinatal cardiomyopathy is another idiopathic form of DCM, as are diseases associated with infectious sequelae. In summary, cardiomyopathy, including DCM, is a significant well-known health problem.

Heart disease and its symptoms, including coronary heart disease, myocardial infarction, congestive heart failure and cardiac hypertrophy, are clearly one of the major health risks facing the united states today. The cost of diagnosing, treating, and maintaining patients with these diseases is as high as billions of dollars. Two particularly severe signs of heart disease are myocardial infarction and cardiac hypertrophy. With regard to myocardial infarction, typically, acute platelet coronary occlusion occurs in coronary arteries due to atherosclerosis and causes myocardial cell death. Because cardiomyocytes, i.e., cardiac muscle cells, are terminally differentiated and generally unable to undergo cell division, they are generally replaced by scar tissue after death during an acute myocardial infarction. Scar tissue is non-contractile, does not contribute to cardiac function, and often dilates or increases the size and effective radius of the ventricle (e.g., becomes hypertrophic) during cardiac contraction to have a deleterious effect on cardiac function. With respect to cardiac hypertrophy, one theory regards it as a disease that resembles abnormal development, thus raising the question of whether developmental signals in the heart contribute to the hypertrophic disease. Cardiac hypertrophy is an adaptive response to almost all forms of heart disease, including those arising from hypertension, mechanical stress, myocardial infarction, arrhythmias, endocrine disorders, and genetic mutations in the cardiac contractile protein genes. Although the hypertrophic response is initially a compensatory mechanism that elevates cardiac output, continued hypertrophy can lead to DCM, heart failure, and sudden death. In the united states, approximately 50 million people are diagnosed with heart failure annually, with a mortality rate of approximately 50%.

There have been many studies on the cause and result of cardiac hypertrophy, but the underlying molecular mechanism has not been elucidated. Understanding these mechanisms is a major concern in the prevention and diagnosis of heart disease and will be of critical importance as a therapeutic modality in designing new drugs that specifically target cardiac hypertrophy and heart failure. Because pathologic cardiac hypertrophy usually does not produce any symptoms until the heart damage is severe enough to produce heart failure, the symptoms of cardiomyopathy are the symptoms associated with heart failure. These symptoms include shortness of breath, exhaustion (fatigue with exhaustion), inability to lie flat without becoming shortness of breath (sitting up breathing), paroxysmal nocturnal dyspnea, enlarged heart dimensions, and/or swollen lower limbs. Patients also often present with elevated blood pressure, additional heart sounds, heart murmurs, pulmonary and systemic emboli, chest pain, pulmonary congestion, and palpitations. In addition, DCM induces a reduced ejection fraction (i.e. a measure of both intrinsic contractile function and remodeling). The disease is further characterized by a marked impairment of contractile function due to ventricular dilatation and decreased myocardial contractility, leading in many patients to dilated heart failure. The diseased heart also undergoes cellular/luminal remodeling as a result of myocyte/myocardial dysfunction, contributing to the development of the "DCM phenotype". As the disease progresses, symptoms also progress. The incidence of life-threatening arrhythmias (including ventricular tachycardia and ventricular fibrillation) in DCM patients is also greatly increased. In these patients, syncope (dizziness) events are considered as precursors to sudden death.

Diagnosis of dilated cardiomyopathy typically relies on the manifestation of enlarged heart chambers, particularly enlarged ventricles. Dilation can usually be observed with chest X-rays, but assessment using echocardiography is more accurate. DCM is often difficult to distinguish from acute myocarditis, valvular heart disease, coronary heart disease, and hypertensive heart disease. Once a diagnosis of dilated cardiomyopathy is made, efforts are made to identify and treat the underlying reversible etiology and prevent further heart damage. For example, coronary heart disease and valvular heart disease must be excluded. Anemia, abnormal tachycardia, nutritional deficiencies, thyroid disorders, and/or other problems need to be treated and controlled.

As mentioned above, treatment with pharmacological agents remains the primary mechanism for reducing or eliminating the signs of heart failure. Diuretics are the first line treatment for mild to moderate heart failure. Unfortunately, many of the commonly used diuretics (e.g., thiazines) have many adverse effects. For example, certain diuretics may increase serum cholesterol and triglycerides. In addition, diuretics are generally ineffective for patients with severe heart failure.

If the diuretic is ineffective, a vasodilator (vasodialator agent) may be used; angiotensin Converting (ACE) inhibitors, such as enalopril (enalopril) and lisinopril (lisinopril), not only provide symptomatic relief, but have also been reported to reduce mortality (Young et al, 1989). However, ACE inhibitors are still associated with adverse effects, leading to their contraindications for patients with certain disease states (e.g. renal artery stenosis). Similarly, inotropic agent therapy (i.e., a drug that improves cardiac output by increasing the strength of myocardial muscle contraction) is associated with various adverse effects, including gastrointestinal problems and central nervous system dysfunction.

It follows that the pharmacological agents currently used have serious drawbacks in a particular patient population. New, safe and effective agents will undoubtedly benefit patients who cannot use the currently available pharmacological modalities or who cannot obtain adequate relief from those modalities. The prognosis for DCM patients is variable and depends on the extent of ventricular dysfunction, with most deaths occurring within five years after diagnosis.

I. The invention

The ratio of alpha-to beta-MHC equivalents in the adult heart is a major determinant of cardiac contractility. beta-MHC is the major myosin isoform in the adult heart, which exhibits relatively low ATPase activity, whereas alpha-MHC has high ATPase activity. In response to various pathological stimuli (such as myocardial infarction, hypertension, and other conditions), β -MHC expression is elevated and α -MHC expression is reduced, with the result that myofibrillar atpase activity is reduced and the rate of shortening of cardiac myofibrils is reduced, leading to eventual systolic dysfunction. It is noteworthy that slight variations in cardiac alpha-MHC content can have profound effects on cardiac performance.

Micrornas (mirs) are small, approximately 22-nucleotide RNAs that are derived from larger pre-mirs (pre-mirs). mirs act as repressors of target mrnas by either promoting target mRNA degradation (when their sequences are fully complementary) or inhibiting target mRNA translation (when their sequences contain mismatches). microRNA-208 (miR-208) is encoded by an intron of the alpha-MHC gene and is specifically expressed in the heart. The inventors created miR-208 knockout mice and found that miR-208 is necessary for activation of β -MHC gene expression in the adult heart as well as activation of several other contractile protein gene expression. In addition, miR-208 inhibition results in a dramatic decrease in cardiac fibrosis. These findings indicate that strategies to modulate miR-208 expression would have profound effects on cardiac contractility in humans, e.g., inhibition of miR-208 in the heart following cardiac injury to prevent β -MHC expression and maintain α -MHC expression.

Another aspect of the invention is the agonism of miR-208 expression or activity by introducing exogenous miR-208 into the heart, either by therapeutically activating the endogenous miR-208 gene or by using an adenoviral vector or other vectors (ectopic expression means of the adenoviral system are not required to increase beta-MHC expression) for the treatment of individuals with mutations in the alpha-MHC gene. Upregulation of several fast skeletal muscle contraction protein genes in the heart of miR-208 mutant mice also suggests that miR-208 typically suppresses the fast skeletal muscle gene program. Activation of these genes in the heart is a potential means of regulating cardiac contractility.

In addition, the present inventors suggest the use of miR-208 to suppress fast fiber genes in skeletal muscle, thereby activating the interactive (transcriptional) expression of slow fiber genes associated with enhanced insulin sensitivity and skeletal muscle endurance. Suppression of slow fiber genes and activation of fast fiber genes in skeletal muscle are associated with a number of musculoskeletal disorders, including disuse atrophy (disuse atrophy), muscle wasting in response to gravity, and denervation.

Thus, the inventors have discovered that miR-208 is a muscle-specific, critical regulator of β -MHC gene expression in the heart, and additionally regulates cardiac fibrosis. The discovery that miR-208 regulates β -MHC expression and rapid skeletal muscle gene expression is entirely new, as is the use of micrornas to control cardiac contractility and skeletal muscle function.

II.miRNA

A. Background of the invention

In 2001, several groups isolated and identified a large class of "microRNAs" (miRNAs) from C.elegans (C.elegans), Drosophila (Drosophila), and humans using a novel cloning method (Lagos-Quintona et al, 2001; Lau et al, 2001; Lee and Ambros, 2001). Hundreds of mirnas were identified in plants and animals, including humans that did not appear to have endogenous sirnas. Thus, mirnas, although similar to sirnas, are distinct.

The mirnas observed to date are approximately 21-22 nucleotides long and they are derived from longer precursors that are transcribed from non-protein coding genes. See review Carrington et al (2003). The precursors form a structure that folds back-to-back with each other in self-complementary regions; they are then processed by Dicer nuclease in animals or DCL1 in plants. miRNA molecules block translation by precise or imprecise base pairing with their target.

mirnas are involved in gene regulation. Some miRNAs, including lin-4 and let-7, inhibit protein synthesis by binding to a partially complementary 3 'untranslated region (3' UTR) in the target mRNA. Other mirnas, including Scarecrow miRNA found in plants, function like sirnas, binding to fully complementary mRNA sequences to disrupt the target transcript (Grishok et al, 2001).

As scientists begin to understand the broad role micro-RNAs play in regulating eukaryotic gene expression, there has been an increasing search for these molecules. The two most well-known miRNAs, lin-4 and let-7, regulate developmental timing in C.elegans by regulating translation of a key mRNA family (reviewed in Pasquinelli, 2002). Hundreds of mirnas have been identified in caenorhabditis elegans, drosophila, mice, and humans. As can be imagined for molecules that regulate gene expression, miRNA levels have been shown to vary between tissues and between developmental states. In addition, one study showed a strong correlation between the reduced expression of both mirnas and chronic lymphocytic leukemia, providing a possible link between mirnas and cancer (cain, 2002). Although this field is still young, mirnas are presumed to be of equal importance in regulating gene expression in higher eukaryotic cells as transcription factors.

Some examples illustrate that mirnas play a crucial role in cell differentiation, early development, and cellular processes like apoptosis and fat metabolism. Both lin-4 and let-7 regulate the transition from one larval state to another during C.elegans development (Ambros, 2003). mir-14 and bantam are drosophila mirnas that regulate cell death, and their regulation is apparently achieved by modulating expression of apoptosis-related genes (Brennecke et al, 2003, Xu et al, 2003). miR14 has also been suggested to be involved in fat metabolism (Xu et al, 2003). Lsy-6 and miR-273 are C.elegans miRNAs that modulate asymmetry in sensory neurons (Chang et al, 2004). Another animal miRNA that regulates cell differentiation is miR-181, which directs hematopoietic cell differentiation (Chen et al, 2004). These molecules represent the entire range of animal mirnas with known functions. No doubt, further understanding of miRNA function will reveal regulatory networks that contribute to normal development, differentiation, inter-and intracellular communication, cell cycle, angiogenesis, apoptosis, and many other cellular processes. Given the important role of mirnas in many biological functions, mirnas have the potential to provide important indications for therapeutic intervention or diagnostic analysis.

Characterization of the function of a biological molecule like miRNA often involves introducing the molecule into a cell or eliminating the molecule from the cell and measuring the result. If introduction of a miRNA into a cell results in apoptosis, the miRNA is undoubtedly involved in the apoptotic pathway. Methods for introducing and eliminating mirnas into and from cells have been described. Two recent publications describe antisense molecules that can be used to inhibit the activity of specific miRNAs (Meister et al, 2004; Hutvagner et al, 2004). Another publication describes the therapeutic use of transcription by endogenous RNA polymerases and the production of specific mirnas after transfection into cells (Zeng et al, 2002). These two reagent combinations have been used to evaluate single mirnas.

B.miR-208

miR-208 is an intronic miRNA, which is located within the 27 th intron of the α -MHC gene. Fig. 1. The coding sequences of human, mouse, rat, and canine miR-208 pre-mirnas are shown in SEQ id nos: 14. SEQ ID NO: 15. SEQ ID NO: 16. SEQ ID NO: 17. the mature miR-208 sequence is shown in SEQ ID NO: 5. like α -MHC, miR-208 is expressed only in the heart. Fig. 2.

Using the PicTar algorithm to identify miRNA targets (Krek et al, 2005), the inventors identified thyroid hormone receptor-related protein 1 (thap 1) as a predicted target for miR-208. The THRAP 13' UTR sequences from human, chimpanzee, mouse, rat, dog, chicken, puffer, and zebrafish are shown in SEQ ID NOs: 6. SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 12. and SEQ ID NO: 13.

inhibitors of miR-208

Generally, inhibitors of mirnas take the form of "antagomirs", i.e. chemically engineered single stranded short oligonucleotides complementary to the mirnas, blocking the function of the mirnas (Kriitzfeldt et al, 2005). Other approaches include inhibition of mirnas with antisense 2 '-O-methyl (2' -OMe) oligonucleotides and small interfering double stranded rnas (sirnas) engineered to have certain "drug-like" properties (chemical modifications for stability; cholesterol coupling for delivery) (Krutzfeldt et al, 2005).

Methods of treating cardiac hypertrophy

A. Treatment regimens

Medical management of cardiac hypertrophy in the context of current cardiovascular disorders includes the use of at least two types of drugs: inhibitors of the rennin-angiotensin system and beta-adrenergic blockers (Bristow, 1999). Therapeutic agents for the treatment of pathologic hypertrophy in the context of heart failure include angiotensin II converting enzyme (ACE) inhibitors and β -adrenergic receptor blockers (Eichhorn and Bristow, 1996). Other pharmaceutical agents that have been disclosed for the treatment of cardiac hypertrophy include angiotensin II receptor antagonists (us patent 5,604,251) and neuropeptide Y antagonists (WO 98/33791). Despite the currently available pharmaceutical compounds, the prevention and treatment of cardiac hypertrophy and subsequent heart failure remains a therapeutic challenge.

Non-pharmacological treatments are mainly used as an adjunct to pharmacological treatments. One approach to non-pharmacological treatment involves reducing dietary sodium. In addition, non-pharmacological treatments also require the elimination of certain pro-drugs (stimulating drugs), including negative inotropic agents (e.g., certain calcium channel blockers and antiarrhythmic agents such as propitamine), cardiotoxins (e.g., amphetamine), and blood (plasma) volume expanders (e.g., non-steroidal anti-inflammatory agents and glucocorticoids).

In one embodiment of the invention, methods of treating cardiac hypertrophy or heart failure utilizing inhibitors of miR-208 are provided. For the purposes of this application, treatment includes reducing one or more symptoms of cardiac hypertrophy, such as reduced exercise capacity, reduced ejection volume, increased left ventricular end diastolic pressure, increased pulmonary capillary wedge pressure, reduced cardiac output, cardiac output index, increased pulmonary artery pressure, increased left ventricular end systolic and diastolic diameters, and increased left ventricular wall pressure, wall tension and wall thickness — as for the right ventricle. In addition, the use of inhibitors of miR-208 can prevent cardiac hypertrophy and the symptoms associated therewith from occurring.

The treatment regimen will vary depending on the clinical condition. In most cases, however, long-term maintenance seems appropriate. It may also be desirable to treat hypertrophy intermittently with inhibitors of miR-208, such as during a short-term window in the progression of the disease.

B. Combination therapy

In another embodiment, an inhibitor of miR-208 is used in combination with other modes of treatment. Thus, in addition to the therapies described above, more "standard" pharmaceutical cardiac therapies may be provided to the patient. Examples of other therapies include, but are not limited to, so-called "beta blockers", antihypertensives, cardiotonics, antithrombotic agents, vasodilators, hormone antagonists, inotropic agents (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 can be achieved by contacting the heart cells with a single composition or pharmacological formulation containing both agents, or by contacting the cells simultaneously with two different compositions or formulations, one containing the expression construct and the other containing the agent. Alternatively, therapy with a miR-208 inhibitor can be performed before or after administration of the other agent, with intervals ranging from minutes to weeks. In embodiments where the further agent and the expression construct are applied separately to the cell, it will generally be ensured that a long period of time does not elapse between the time points of each delivery, so that the agent and the expression construct are still able to exert a favourable combined effect on the cell. In such cases, the cells are typically exposed to the two forms within about 12-24 hours of each other, more preferably within about 6-12 hours of each other, and most preferably with a delay of only about 12 hours. However, in some instances it may be desirable to significantly extend the period of treatment with intervals of days (2, 3, 4, 5,6, or 7 days) to weeks (1, 2, 3, 4, 5,6, 7, or 8 weeks) between administrations.

It is also contemplated that it may be desirable to administer the miR-208 inhibitor or another agent more than once. For this, various combinations may be used. For example, based on a total of 3 and 4 administrations, there are the following exemplary permutations, where "a" is a miR-208 inhibitor and "B" is another agent: A/B/A, B/A/B, B/B/A, A/A/B, B/A/A, A/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/B, B/B/A, A/A/B, B/A/A/A, A/B/A/A, A/A/B/A, A/B/B/B, B/A/B/B, Other combinations are also contemplated by B/B/A/B.

C. Pharmacological therapeutic agents

Pharmacological therapeutics and methods of administration, dosages, and The like, are well known to those skilled in The art (see, e.g., "Physicians Desk Reference", Klaassen, "The Pharmacological Basis of therapeutics", "Remington's Pharmaceutical Sciences", and "The Merck Index, Eleventh Edition", which are incorporated herein by Reference in The relevant section), and may be combined with The present invention in accordance with The disclosure herein. Depending on the condition of the subject being treated, some dose variation may be necessary. In any event, the person responsible for administration will determine the appropriate dosage for the subject individual, and such individual determinations are within the skill of one of ordinary skill in the art.

Non-limiting examples of pharmacological therapeutic agents useful in the present invention include antihyperlipidemic drugs, anti-arteriosclerotic drugs, antithrombotic/fibrinolytic drugs, coagulation drugs, antiarrhythmic drugs, antihypertensive drugs, vasopressors (vesopressers), therapeutic agents for congestive heart failure, antianginal drugs, antibacterial drugs, or combinations thereof.

In addition, it should be noted that any of the following may be used to develop a new set of cardiac therapy target genes, whereas the present examples (see below) use a beta-blocker. Although many of these genes may overlap, new gene targets may be developed.

i. Antihyperlipidemic drugs

In certain embodiments, administration of an agent that reduces the concentration of one or more blood lipids and/or lipoproteins (referred to herein as an "antihyperlipidemic drug") can be combined with cardiovascular therapy according to the invention, particularly in the treatment of atherosclerosis and vascular tissue thickening (thick) or obstruction. In certain aspects, the antihyperlipidemic agent can include an aryloxyalkanoic acid/fibric acid (fibric acid) derivative, a resin/bile acid sequestrant (sequestrant), an HMG CoA reductase inhibitor, a nicotinic acid derivative, a thyroid hormone or thyroid hormone analog, a heteroid agent (miscella neous agent), or a combination thereof.

a. Aryloxy alkanoic/fibric acid derivatives

Non-limiting examples of aryloxyalkanoic acid/fibric acid derivatives include benclofibrate (beclomerate), enzafibrate, binifibrate, ciprofibrate, clinofibrate, clofibrate or clofibrate (clofibrate) (atromid-S), clofibric acid (clofibrate acid), etofibrate (etofibrate), fenofibrate (fenofibrate), gemfibrozil (gemfibrozil) (lobeline (lobid)), nicofibrate (nifibrate), piribrate (pirifibrate), chloronicotinate (ronifibrate), simfibrate (simfibrate), and copolyester (theofibrate).

b. Resin/bile acid sequestrant

Non-limiting examples of resin/bile acid sequestrants include cholestyramine (cholestyramine) (cholebar, questran), colestipol (colestipol), and cholecystamine (polidexide).

HMG CoA reductase inhibitors

Non-limiting examples of HMG CoA reductase inhibitors include lovastatin (mevastatin), pravastatin (pravastatin), or simvastatin (simvastatin) (zocor).

d. Nicotinic acid derivatives

Non-limiting examples of nicotinic acid derivatives include nicotinate (nicotinate), acipimox (acepromox), niceritrol (nicotinate), nicoloester (nicolonate), nicomol (nicomol), and oxynicotinic acid (oxineic acid).

e. Thyroid hormones and analogs

Non-limiting examples of thyroid hormones and their analogs include etoroxate, thyropropionic acid (thyropropic acid), and thyroxine (thyroxine).

f. Miscellaneous antihyperlipidemic agents

Non-limiting examples of miscellaneous anti-hyperlipoproteinemic agents include acifran (acifran), azacholin (azacoterol), benzoflurane (benflurorex), beta-benzylidenebutanamide (benzalbutyramide), carnitine (carnitine), chondroitin sulfate (chondrin sulfate), chlorestrone (clomesterone), dextran (dextran), sodium dextran sulfate (dextran sulfate), 5,8, 11, 14, 17-eicosapentaenoic acid (eicosapentaenoic acid), erythropurines (eritadiene), franazabol (furazabol), meglumine (meglumine), melinamide (melinamide), dimehypoestriol (mycoenediol), ornithine (ornithine), gamma-oryzanol (yoxyverine), pantethyene (pantethyene), pentaerythrityl (pentaerythrityl-piperazine), pentaerythrityl (pyridoxine) (pyraclostrobin), pentaerythrine (pyridoxine) (piperazine), pyributrythrine (pyridoxine) (piperazine), pyriproxyfen (piperazine), pyributine (pyridoxine) (piperazine) (pentanate), pyributine (pyridoxine) (piperazine) (beta-acetate), pyributrythrine (pyridoxine) (piperazine) (piperazinate), pyributrythrine, pyributine (pyridoxine) (pentaline) (piperazinate), pyributine (piperazinone, pyributine (penta-acetate), pyributine (e), pyributhionine (e), pyributine (, Thiodiol (tiadenol), tripananol (tripananol) and biphenylbutyric acid (xenbucin).

Antiatherosclerotic agents

Non-limiting examples of anti-atherosclerotic agents include pyridinol carbamate (pyridinolcarbamate).

Antithrombotic/fibrinolytic agents

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 the treatment of atherosclerosis and blockage of the vascular system (e.g., arteries). Non-limiting examples of antithrombotic and/or fibrinolytic agents include anticoagulants, anticoagulant antagonists, antiplatelet agents, thrombolytic agent antagonists, or combinations thereof.

In certain aspects, antithrombotic agents that can be administered orally, such as, for example, aspirin (aspirin) and warfarin (wafarin) (Coumadin) are preferred.

a. Anticoagulant agent

Non-limiting examples of anticoagulants include acerolarin (acenocoumarol), ancrod (ancrod), anisindione (anisindione), bromoindandione (broninone), clidandione (clindione), kumemantinol (coumetanol), cycloprocyanin (cyclocumarol), sodium sulfate (dextran sulfate), dicoumarol (dicumolol), dianiline (diphenadione), dicoumarin ethyl ester (ethyl biscoumarate), dicoumarin ethylene ester (ethydicoumarine), fluoroindanone (flurondone), heparin (heparin), hirudin (hirudin), sodium apocynate (lyapolonate), oxandione (oxazidione), pentosan polysulfate (pentasanophenylfantate), phenindione (phenformin), coumarine (chromocoumarin), coumarine (phosphocoumarin), coumarine (coumarine), coumarin.

b. Antiplatelet agents

Non-limiting examples of antiplatelet agents include aspirin (aspirin), dextran (dextran), dipyridamole (dipyridamole), heparin (heparin), sulpirenone (antranilone), and ticlopidine (ticlopidine).

c. Thrombolytic agent

Non-limiting examples of thrombolytic agents include tissue plasminogen activator (Activase), plasmin, prourokinase, urokinase (Abbokinase)), streptokinase (Streptase), anistreplase (anistreplase)/APSAC (eminase).

Coagulant agents iv

In certain embodiments where the patient has a hemorrhage or an increased likelihood of bleeding, an agent that enhances blood clotting may be used. Non-limiting examples of blood coagulation promoting agents include thrombolytic and anticoagulant antagonists.

a. Anticoagulant antagonists

Non-limiting examples of anticoagulant antagonists include protamine (protamine) and vitamin K1.

b. Thrombolytic agent antagonist and antithrombotic agent

Non-limiting examples of thrombolytic antagonists include aminocaproic acid (amicar) and tranexamic acid (amstat). Non-limiting examples of antithrombotic agents include anagrelide, argatroban, cilostazol, daltroban, defibrotide, enoxaparin, fraxiparine, indobufen, lamoparan, ozagrel, picotamide, prasugrel, tedipiparin, ticlopidine, and triflusal.

Antiarrhythmic agents

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

a. Sodium channel blockers

Non-limiting examples of sodium channel blockers include class IA, class IB and class IC antiarrhythmic agents. Non-limiting examples of class IA antiarrhythmics include propyramide (noperse), procainamide (procainamide), and quinidine (quinidex). Non-limiting examples of class IB antiarrhythmic agents include lidocaine (lidocaine) (xylocaine), tocainide (tocardine), and mexiletine (mexilitine).

b. Beta blockers

Non-limiting examples of beta blockers (also known as beta-adrenergic blockers, beta-adrenergic antagonists, or class II antiarrhythmics) include acebutolol (sectral), alprenolol (alprenolol), amosulolol (amosulolol), alolol (arotinolol), atenolol (atenolol), benfuralol (befanolol), betaxolol (betaxolol), bevantolol (bevantolol), bisoprolol (bisoprolol), bopindolol (bopindolol), bucumolol (bucumolol), bufenolol (bufenolol), bunnilolol (bunolol), bucindolol (bunranolol), bucindolol (bucindolol), buclizolol (butirolol), bucindolol (bucindolol), bucindolol (buclol), bucindolol (bucindolol), bucindolo, Esmolol (esmolol) (brevibloc), indenolol (indenolol), labetalol (labetalol), levobunolol (levobunolol), mepindolol (mepindolol), metipranolol (metipranolol), metoprolol (metoprolol), moprolol (moprolol), nadolol (nadolol), nafolol (nadolol), nifedilol (nifenalol), nipradilol (nipradilol), oxprenolol (oxprenolol), penbutolol (penbutolol), pindolol (pindolol), practilol (practilol), propranolol (prophalalol), propranolol (panolol) (propranolol (indelol)), sotalol (sotalol) (solalol), thiolol (betaxolol), tarolol (taurololol), timolol (timolol), timolol (lopolol (propiolol), and metoprolol (metoprolol). In certain aspects, the beta blocker includes an aryloxypropanolamine derivative. Non-limiting examples of aryloxypropanolamine derivatives include acebutolol (acebutolol), alprenolol (alprenolol), arotinolol (artolol), atenolol (atenolol), betaxolol (betaxolol), bevantolol (bevantolol), bisoprolol (bisoprolol), bopindolol (bopindolol), bunitrolol (bunitrol), bufalol (butolol), caraolol (carazolol), carteolol (cartepolol), carvedilol (carvedilol), celiprolol (celiprolol), celiprolol (cetamolol), epanolol, indonolol, mepindolol, metipranolol, metoprolol, moprolol, nadolol, nipradilol, oxprenolol, penbutolol, pindolol, propanolol, talinolol, terbatolol, timolol, and toliprolol.

c. Repolarization extender

Non-limiting examples of agents that prolong repolarization (also known as class III antiarrhythmics) include amiodarone and sotalol (betaface).

d. Calcium channel blockers/antagonists

Non-limiting examples of calcium channel blockers (also referred to as class IV antiarrhythmics) include arylalkylamides (e.g., benridil, diltiazem, fendiline (fendiline), gallopamil (gallopamil), prenylamine (prenylamine), terodiline (terodiline), verapamil (verapamil), dihydropyridine derivatives (felodipine), isradipine (isradipine), nicardipine (nicardipine), nifedipine (nifedipine), nimodipine (niipine), nisoldipine (nisoldipine), nitrendipine (nitrendipine), piperazine derivatives (e.g., cinnarizine), flunarizine (flunarizine), lifluzine (lidoflazine), or miscellaneous calcium channel blockers such as e (cyclocarine), fenflurazone (fenfluridone (beaconine), or milbemycin (fenadine). In certain embodiments, the calcium channel blocker comprises a long-acting dihydropyridine (nifedipine-type) calcium antagonist.

e. Miscellaneous anti-arrhythmic drugs

Non-limiting examples of miscellaneous antiarrhythmic agents include adenosine (adenosine), digoxin (lanoxin), acecanine (acecanide), ajmaline (ajmaline), croconium (ampoxan), apraline (aprindine), bromobenzyl tosylate (britylium dysylate), bufaline (bunaftine), butobenidine (butobendine), calcipotic acid (capobenic acid), ciclesonine (ciferine), pyridipropylamine (dispyronane), dihydroquinidine (hydroquinidine), lndicarb (indecainide), ipratropium bromide (iptropiturnb), lidocaine (lidocaine), loramine (loramine), loracarbene (lorafenide), mebendazole (meobentine), moraxezine (moricizine), pirenol (pirmenol), probenazole (prajmaline), propafenone (propafenone), pirroline (pyrinoline), quinidine polygalacturonate (quinidine polygalcturonate), quinidine sulfate (quinidine sulfate), and vequinidil (Viquidil).

Antihypertensive agents

Non-limiting examples of antihypertensive agents include sympathetic blockers, alpha/beta blockers, alpha blockers, angiotensin II agents, beta blockers, calcium channel blockers, vasodilators, and miscellaneous antihypertensive agents.

a. Alpha retarder

Non-limiting examples of alpha blockers (also known as alpha-adrenergic blockers or alpha-adrenergic antagonists) include amosulalol (amosulalol), arotinolol (arotinol), dapiprazole (dapiprazole), doxazosin (doxazosin), mesyl dihydroergotoxine (ergoloid), fenspiride (fenpiride), indoramin (indoramin), labetalol (labetalol), nicergoline (nicergoline), prazosin (prazosin), terazosin (terazosin), tolazoline (tolazoline), trimazosin (trimazosin), and yohimbine (yohimbine). In certain embodiments, the alpha blocker may include a quinazoline derivative. Non-limiting examples of quinazoline derivatives include alfuzosin, bunazosin, doxazosin, prazosin, terazosin, and trimazosin.

b. Alpha/beta blockers

In certain embodiments, the antihypertensive agent is both an alpha adrenergic antagonist and a beta adrenergic antagonist. Non-limiting examples of alpha/beta blockers include labetalol (nonmodyne, rambutal (trandate)).

c. Anti-angiotensin II agents

Non-limiting examples of anti-angiotensin II agents include angiotensin converting enzyme inhibitors and angiotensin II receptor antagonists. Non-limiting examples of angiotensin converting enzyme inhibitors (ACE inhibitors) include alacepril (alacepril), enalapril (enalapril) (vasotec), captopril (captopril), cilazapril (cilazapril), delapril (delapril), enalapril (enalaprilat), fosinopril (fosinopril), lisinopril (lisinopril), mevinpril (moveltopril), perindopril (perindopril), quinapril (quinapril), and ramipril (ramipril). Non-limiting examples of angiotensin II receptor blockers (also known as angiotensin II receptor antagonists, ANG receptor blockers, or ANG-H type-1 receptor blockers (ARBS) include angiocantan, eprosartan (eprosartan), irbesartan (irbessartan), losartan (lossartan), and valsartan (valsartan).

d. Sympathetic nerve blocking agent

Non-limiting examples of sympathetic blocking agents include centrally acting sympathetic blocking agents or peripherally acting sympathetic blocking agents. Non-limiting examples of centrally acting sympatholytic drugs (also known as Central Nervous System (CNS) sympatholytic drugs) include clonidine (clonidine), guanabenz (guanabenz), wytensin, guanfacine (tenex), and methyldopa (aldomet). Non-limiting examples of peripherally acting sympatholytic agents include ganglion blockers, adrenergic neuron blockers, beta-adrenergic blockers, or alpha 1-adrenergic blockers. Non-limiting examples of ganglion blocking agents include mecamylamine (invertsine) and trimethaphan (arfonad). Non-limiting examples of adrenergic neuron blockers include guanethidine (ismelin) and reserpine (serpasil). Non-limiting examples of beta-adrenergic blockers include acebutolol (sectral), atenolol (tenonolol) (tenomin), betaxolol (carolren (kerlone)), carteolol (carteolol) (carteol (cartrol)), labetalol (labetalol) (normodyne, rambutal (trandate)), metoprolol (loperssor), nadolol (nadanol) (congol (corrgard)), penbutolol (pivalol) (levatol), pindolol (visken), propranolol (propranolol) (orange), and timolol (noncalol) (bucandren). Non-limiting examples of α 1-adrenergic blockers include prazosin (minipress), doxazosin (doxazocin), and terazosin (tetralin (hytrin)).

e. Vasodilating agents

In certain embodiments, the vasodilator therapeutic agent may include a vasodilator (e.g., a cerebrovascular vasodilator, a coronary vasodilator, or a peripheral vasodilator). In certain preferred embodiments, the vasodilator comprises a coronary vasodilator. Non-limiting examples of coronary vasodilators include aminooxytriphene (amotriptylene), dibazol (bendazol), benfurethroid (benfurethral hemisuccinate), ethiodarone (benzidol), chlorphenamine (chlorphenazine), chromonar, chlorbenzfurol (clobenfurol), nitroglycerin (clinitrate), dilazep (dilazep), dipyridamole (dipyridamole), hydrabamine (hydrophenamine), ethoxyflavone (efloxate), erythritol tetranitrate (erythrol tetranitrate), ethacrylone (ethrafenone), fendiline (fendilin), floridil (floridil), changelefen (ganlefluxlene), ethacrylene bis (beta-diethylaminoethylether) (beta-dithiophene), nitroglycerin (betaxanthenol), nitroglycerin (pentaerythrine), nitrone (nitrone), nitroglycerin (sodium), nitroglycerin (sodium nitrate), nitroglycerin (sodium benzoate), nitrobetaine (sodium benzoate), nitrobetaine (pentaerythritol), pentaerythritol (pentaerythritol), pentaerythritol (pentaerythritol), pentaerythritol (pentaerythritol), pentaerythritol (pentaerythritol), pentaerythritol, Pentinitrol (penttrinitrol), perhexiline (perhexiline), pimecretol (pimefylline), trapidil (trapidil), tricromyl, trimetazidine (trimetazidine), trinitroethanolamine phosphate (trolnitrate phosphate), and visnadine (visnadine).

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

f. Miscellaneous antihypertensive agents

Non-limiting examples of miscellaneous antihypertensive agents include ajmaline (ajmaline), gamma-aminobutyric acid, butylbenziodoamine (bufenide), cicletanine (cicletanine), verapamine tannate (cryptotamine tannate), fenoldopam (fenoldopam), fluosesamine (flosequinan), ketanserin (ketanserin), mebutamine (mebutamate), mecamylamine (mecamylamine), methyldopamine (methyldopa), methyl 4-pyridylketothiosemicarbazone (methyl 4-pyridol ketothiosemicarbazone), mezzamine (muzolimine), pargyline (pargyline), dipyridamole (pemphidine), pinadil (pinacidil), peroxoxan (piperaoxan), priperone (primaperone), progastrine (protoveratrine), raubasine (raubenine), resisimetone (resinometol), rimenidine (rilmenidene), saralasin (saralasin), sodium nitroprusside (sodiumtrorusiside), tennic acid (tirynafen), trimethaphenamine (trimethaphan camylate), tyrosinase (tyrosine), and urapidil (urapidil).

In certain aspects, the antihypertensive agent can include an arylethanolamine derivative, a phenothiazine derivative, an N-carboxyalkyl (peptide/lactam) derivative, a dihydropyridine derivative, a guanidine derivative, a hydrazine/phthalazine, an imidazole derivative, a quaternary ammonium compound, a reserpine derivative, or a sultamide derivative.

Non-limiting examples of aryl ethanolamine derivatives include amosulalol (amosulalol), bufuralol (bufuralol), dilevalol (dilevalol), labetalol (labetalol), propranolol (propathalol), sotalol (sotalol), and sulfoxolol (sulfinalol).

Non-limiting examples of the phenothiazine derivative include althiazide (alanine), bendroflumethiazide (benzflumethiazide), bendrothiazide (benzthiazide), benzhydrochlorothiazide (benzhydrylchlorothiazide), buthiazide (buthiazide), chlorothiazide (chlorothiazide), chlorthalidone (chlorothalidone), cyclopenthiazide (cycloprothiazide), cyclothiazide (cyclothiazide), diazothiazide (diazoxide), epithizide (ethiazide), ethiazide (ethiazide), fenquinconazole (fenquinazone), hydrochlorothiazide (hydrochlorothiazide), hydrochlorothiazide (hydroflumethiazide), methyclothiazide (hydroflorothiazide), metribuzin (metiramide), metirazone (metathiazide), parathiazide (tetrachlorochlorothiazide), chlorothiazide (tetrachlorochlorothiazide).

Non-limiting examples of N-carboxyalkyl (peptide/lactam) derivatives include alacepril (alacepril), captopril (captopril), cilazapril (cilazapril), delapril (delapril), enalapril (enalapril), enalapril (enalaprilat), fosinopril (fosinopril), lisinopril (lisinopril), moexipril (moveltipril), perindopril (perindopril), quinapril (quinapril) and ramipril (ramipril).

Non-limiting examples of dihydropyridine derivatives include amlodipine (amlodipine), felodipine (felodipine), isradipine (isradipine), nicardipine (nicardipine), nifedipine (nifedipine), nilvadipine (nilvadipine), nisoldipine (nisoldipine), and nitrendipine (nitripine).

Non-limiting examples of guanidine derivatives include betanidine (betanidine), isoquanidine (debrisoquin), guanabenz (guanabenz), guanacline (guanacline), guanadrel (guanadrel), guanazodine (guanazodine), guanethidine (guanethidine), guanfacine (guanfacine), guanchlorophenol (guanochlor), guanoxabenzyl (guanoxaabenz), and guanoxan (guanoxan).

Non-limiting examples of hydrazine/phthalazine include budralazine (budralazine), cadralazine (cadralazine), dihydralazine (dihydralazine), endralazine (endralazine), hydracarbazine (hydrarbazine), hydralazine (hydralazine), phenyliprazine (phenyliprazine), pildralazine (pildralazine), and toldralazine (todralazine).

Non-limiting examples of imidazole derivatives include clonidine (clonidine), lofexidine (lofexidine), phentolamine (phentolamine), timenidine (tiamenine), and tolonidine (tolonidine).

Non-limiting examples of quaternary ammonium compounds include azamethylammonium bromide (azamethiomum bromide), pinedium chloride (chloronium chloride), hexamethonium (hexamethonium), pentacyanomorpholine methionate (methimulate), pentamethylammonium bromide (pentamethylammonium bromide), pentapyrilamine tartrate (pentalinium tartrate), fentolonium chloride (phenotropium chloride), and trimethine methiodidine (trimethine methosulfate).

Non-limiting examples of reserpine derivatives include bitasipine (bietasapine), dessertpine (deserpidine), resisinamine (resinnamine), reserpine (reserpine), and syrosingopine (syringopine).

Non-limiting examples of the sulfophosphoramide derivatives include ambroxide (ambuside), chloramide (cyclopamide), furosemide (furosemide), indapamide (indapamide), quinethazone (quinethazone), tripamide (tripamide), and xipamide (xipamide).

g. Blood vessel pressors (vesopressors)

Vasopressors are commonly used to increase blood pressure during shock that may occur during surgical procedures. Non-limiting examples of vasopressors (also known as anti-hypotensives) include amezinium methyl sulfate (amezinium methyl sulfate), angiotensin amide, metformin (difofrine), dopamine (dopamine), etifenamine (etifelmin), etiefrin (etilefrin), gepefurin (gepefrine), metahydroxylamine (metaraminol), midodrine (middrine), norepinephrine (norepinephrine), fomesamine (pholedrine), and synephrine (synephrine).

Therapeutic agents for congestive heart failure

Non-limiting examples of agents useful in the treatment of congestive heart failure include anti-angiotensin II agents, afterload-preload reduction treatments (reduction treatment), diuretics, and inotropic agents.

a. Afterload-preload reduction

In certain embodiments, an animal patient that is intolerant to angiotensin antagonists may be treated with a combination therapy. Such therapies may be combined with the administration of hydralazine (apresoline) and isosorbide dinitrate (isordil, sorbitate).

b. Diuretic medicine

Non-limiting examples of diuretics include thiazide (thiamide) or benzthiadiazine (benzothiazine) derivatives (e.g., althiazide, bendroflumethiazide, benthiazide (benzthiazide), bendrochlorothiazide, buthiazide, chlorothiazide, chlorthalidone (chlorothalidone), cyclopenthiazide, epithizide, ethiazide, fenquinconazole (fenquinconazole), hydrochlorothiazide (hydrochlorothiazide), hydroflumethiazide (fluthiamide), mechlorethazine (methysticide), mechlorethazine (trimethiazide), trimethiazide (mechlorethazine), mechlorethazine (mechlorethazine), trimethachiazide (mechlorethazine), mechlorethazine (mechlorethamine), mechlorethazine (mechlorethazine), mechlorethazine (mechlorethamine), mechlorethazine), mechlorethamine (mechlorethamine), mechlorethamine (mechlorethazine), mechlorethamine (mechlorethamine), mechlorethamine (mechlorethamine), thimide (mechlorethamine), thimide (thiurazine), thi, Mercurous chloride (mercurous chloride), mersalyl (mercurolide)), pteridine (e.g. furterene (furterene), triamterene (triamterene)), purine (e.g. theophylline acetate piperazine (acefylline), 7-morpholinomethyltheophylline (7-morpholinomethyltheophylline), pamabrine (pamobromone), theobromine (prophylline), theobromine (theobromine), steroids (steroid) including aldosterone antagonists (e.g. canrenone (canrenone), oleandrin (oleandrin), spironolactone (spironolactone)), sulfonamide derivatives (e.g. acetazolamide), ambuside (ambuside), azosulamide (e.g. butosulamide), bumetamide (butamide), butocyclamide (4 ' -chlorolactam), chlorofenamide (4 ' -chloro-sulfonamide (4-chloro-amidoamine), sulfimide (4 ' -chloro-4-methyl-sulfimide (4-chloro-amino-4-dimethyl-methyl-amide (4-chloro-amino, 4-chloro-dimethyl-methyl-sulfamethoxide, 4-amino-dimethyl-one, sulfimide (methamide, sulfimide (4-chloro-amino-dimethyl-one, sulfimide (methamide, sulfimide (methamide, sulfimide, sulf, Esozolamide (ethoxzolamide), furosemide (furosemide), indapamide (indapamide), mefruside (mefruside), methazolamide (methazolamide), piretanide (piretanide), quinethazone (quinethazone), torasemide (toramide), tripamide (tripamide), xipamide (xipamide), uracils (e.g. amimetidine), amimetidine (amimetradine)), potassium-spartate antagonists (e.g. amiloride, triamterene) or miscellaneous diuretics such as chlorpromazine (amiozine), arbutin (arbutin), lorazanil (lorfluzanide), ethacrynic acid (aminocrytacrine), hemihydramine (isocratide), octopamine (ketonurone), octopamine (clavine), ketonurine (mannitol), and mannitol (octopamide).

c. Muscle strength medicine

Non-limiting examples of inotropic drugs (also known as cardiotonics) include theophylline acetic acid (acefyline), acexydinin (acefydigitoxin), 2-amino-4-picoline (2-amino-4-picoline), amrinone (amyrinone), benfuradane (benfuradil hemisuccininate), bravadine (butladesine), vincristine B (cerberosine), laurylamine (camostate), convallastin (convallatoxin), cymarin (cymarin), dinopamine (denopamine), deslanoside (deslanoside), digitoxin (digitalin), digitalisine (digitalis), digitoxin (digoxin), dopamine (doxylamine), polyvidone (pyridoxine), pyridoxine (pyridoxine), pyridoxine (pyridoxine), pyridoxine (pyridoxine), pyridoxine (pyridoxine) (, Goldenseal (hydrastinine), isopbopamine (ibopamine), digitoxin (lantoside), methoxyphenamide (metamivam), milrinone (milrinone), neriifolin (B) (nerifolin), oleandrin (oleandrin), ouabain (ouabain), oxicarbine (oxyperiine), protuberol (prenol), proscillaridin (proscarindine), bufonis (resibufogenin), scilin (scillaren), scillarin ligand (scillarin), curbospirin (strphanthin), thiamazole (sulprazole), theobromine (theobromamine) and zamoterol (xamoterol).

In particular aspects, the positive inotropic agent is a cardiac glycoside, a β -adrenergic agonist, or a phosphodiesterase inhibitor. Non-limiting examples of cardiac glycosides include digoxin (lanoxin) and digitoxin (crystodigin). Non-limiting examples of beta-adrenergic agonists include salbutamol (albuterol), bambuterol (bambuterol), bitolterol (bitolterol), carbbuterol (carburtol), clenbuterol (clenbuterol), clononaline (clorprenaline), dinopramine (denopamine), clenbuterol (dioxephedrine), dobutamine (dobutamine), dopamine (dopamine) (intropin), polypaxamine (dopaxamine), ephedrine (ephrine), diethylfearine (ethephrine), ethylnorepinephrine (ethephrine), fenoterol (fenoterol), formoterol (formoterol), hexoprenaline (xoprenaline), isopipelamine (isoproterenol), isoprenol (ipratropine), propiterol (propinebutaline), salbutamol (ipratropium), propiterol (propineb), propineb (oxyprenol), procaterol (procaterol), meprobrine (procaterol), meprenol (procaryterol), procaryterol (procaryterol), procaryterol (prochloramine (prochlor, Reproterol (reproterol), rimiterol (rimiterol), ritodrine (ritodrine), soterel (solerenol), terbutaline (terbutaline), troquinol (tretoquinol), tulobuterol (tulobuterol), and zamoterol (xamoterol). Non-limiting examples of phosphodiesterase inhibitors include amrinone (inocor).

d. Anti-angina pectoris medicine

Anti-anginal agents may include organic nitrates (organonitrates), calcium channel blockers, beta blockers, and combinations thereof.

Non-limiting examples of organic nitrates (also known as nitrovasodilators) include nitroglycerin (nitroglycerin) (nitro-bid, nitrostat), isosorbide dinitrate (isordil, sorbtrate), and amyl nitrate (aspirin).

Endothelin receptor antagonists

Endothelin (ET) is a 21 amino acid peptide with strong physiological and pathophysiological effects that appear to be associated with the development of heart failure. The effects of ET are mediated via interactions with two classes of cell surface receptors. Type A receptors (ET-A) are associated with vasoconstriction and cell growth, while type B receptors (ET-B) are associated with endothelial cell mediated vasodilation and with the release of other neurohormones, such as aldosterone. Pharmacological agents that can inhibit the production of ET or inhibit its ability to stimulate the relevant cells are known in the art. Inhibition of ET production involves the use of agents that block an enzyme, known as endothelin converting enzyme, involved in the processing of the peptide precursor to the active form. Inhibition of 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 (Bosentan), enrastan (Enrasentan), Ambrisentan (Ambrisentan), darussentan (Darusentan), Tezosentan (Tezosentan), Atrasentan (Atrasetan), Avosetan, Clazosentan, Edonentan, stasentan (sitaxsentan), TBC 3711, BQ 123, and BQ 788.

D. Surgical treatment (therapeutic agent)

In certain aspects, secondary treatment modalities may include some type of surgery, including, for example, prophylactic, diagnostic, or for staging or classification, curative and palliative surgery. Surgery, particularly curative surgery, may be used in combination with other therapies, such as the present invention and one or more other agents.

Such surgical treatment modalities for vascular and cardiovascular diseases and conditions are well known to those skilled in the art, and may include, but are not limited to, performing surgery on an organism, providing cardiovascular mechanical prostheses (cardiovascular mechanical prostheses), angioplasty, coronary reperfusion, catheter ablation (catheter ablation), providing an implantable cardioverter defibrillator (cardioverter defibrillator) to a subject, mechanical circulatory support (mechanical circulatory support), or a combination thereof. Non-limiting examples of mechanical circulatory support that may be used in the present invention include intra-aortic balloon counterpulsation (intra-aortic balloon pulsation), left ventricular assist device (left ventricular assist device), or combinations thereof.

E. Pharmaceutical formulations and routes of administration to patients

In clinical applications, the pharmaceutical compositions are prepared in a form suitable for the intended use. Typically, this requires the preparation of a composition that is substantially free of pyrogens and other impurities that may be harmful to humans or animals.

It will often be desirable to employ suitable salts and buffers to render the delivery vehicle stable and susceptible to uptake by the target cells. Buffers are also employed when introducing recombinant cells into a patient. The aqueous compositions of the present invention contain an effective amount of a carrier or cells dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. The phrases "pharmaceutically acceptable" or "pharmacologically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal or 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, which are acceptable for use in formulating pharmaceutical products, such as those suitable for administration to humans. The use of such media and agents for pharmaceutically active substances is well known in the art. The present invention contemplates the use of any conventional vehicle or agent in the therapeutic compositions unless incompatible with the active ingredients of the present invention. Supplementary active ingredients may also be incorporated into the compositions, provided they do not inactivate the carrier or cells of the composition.

The active compositions of the present invention may include pharmaceutical preparations. Administration of these compositions according to the invention can be via any common route, as long as the target tissue is accessible via that route. This includes oral (oral), nasal, or buccal (buccal). Alternatively, administration may be by intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection, or by direct injection into cardiac tissue. Such compositions will typically be administered as pharmaceutically acceptable compositions, as described above.

The active compounds can also be administered parenterally or intraperitoneally. For example, solutions of the active compound as a free base or pharmacologically acceptable salt 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 the usual conditions of storage and use, these preparations generally contain a preservative to prevent the growth of microorganisms.

Pharmaceutical forms suitable for injectable use include, for example, sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Typically, these preparations are sterile and fluid to the extent that they are easily injectable. The preparation should be stable under the conditions of manufacture and storage and should have a protective measure against the contaminating action of microorganisms, such as bacteria and fungi. Suitable solvents or dispersion media can include, for example, water, ethanol, polyols (e.g., glycerol, polypropylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. For example, proper fluidity can be maintained by the use of a coating (such as lecithin), by the maintenance of the required particle size (in the case of dispersions), and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens (parabens), chlorobutanol (chlorobutanol), phenol, sorbic acid, thimerosal (thimerosal), and the like. In many cases, it is preferred 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 can be prepared by incorporating the active compound in the appropriate amount in the solvent with any other ingredient desired (e.g., as enumerated above) 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, for example those enumerated above. For 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 containing the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

For oral administration (oral administration), the polypeptides of the invention are generally incorporated with excipients and used in the form of non-ingestible mouthwashes and dentifrices. Mouthwashes may be prepared by incorporating the active ingredient in the required amount in a suitable solvent, such as a sodium borate solution (Dobell's solution). Alternatively, the active ingredient may be incorporated into a preservative wash (antiseptic wash) containing sodium borate, glycerol and potassium bicarbonate. Active ingredients may also be dispersed in the dentifrice, including: gels, pastes, powders and ointments. The active ingredients may be added to the paste dentifrice in a therapeutically effective amount, which may include water, binders, abrasives, fragrances, foaming agents, and humectants.

The compositions of the present invention may generally be formulated in neutral or salt form. Pharmaceutically acceptable salts include, for example, the acid addition salts (formed with the free amino groups of the protein) which are derived from sterile acids (e.g., hydrochloric or phosphoric acids) or organic acids (e.g., acetic, oxalic, tartaric, mandelic, and the like). Salts formed with the free carboxyl groups of proteins can also be derived from inorganic bases (e.g., sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide) or organic bases (e.g., isopropylamine, trimethylamine, histidine, procaine, and the like).

Once formulated, the solution is preferably administered in a manner compatible with the dosage form (dosage formulation) and in a therapeutically effective amount. Formulations can be readily administered in a variety of dosage forms (dosage form), such as injectable solutions, drug-releasing capsules, and the like. For example, for parenteral administration of an aqueous solution, the solution is typically suitably buffered and the liquid diluent first rendered isotonic (e.g., with sufficient saline or glucose). Such aqueous solutions may be used, for example, for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. Preferably, a sterile aqueous medium is employed, as known to those skilled in the art, particularly in light of the present disclosure. For example, a single dose can be dissolved in 1ml of isotonic NaCl solution and added either to 1000ml of subcutaneous infusion fluid or injected at the proposed infusion site (see Remington's Pharmaceutical Sciences 15 th edition, pp 1035-1038 and 1570-1580). The dosage will necessarily vary somewhat depending on the condition of the subject being treated. In any event, the person responsible for administration will determine the appropriate dosage for the subject individual. In addition, for human administration, the preparations should meet sterility, pyrogenicity, overall safety and purity standards as required by the FDA Office of biologicals standards.

Methods of treating musculoskeletal and fibrotic diseases

Upregulation of several rapid skeletal muscle contraction protein genes was observed in the heart of miR-208 mutant mice. This up-regulation of the fast skeletal muscle contraction protein gene in the heart of miR-208 mutant mice indicates that miR-208 suppresses the fast skeletal muscle gene program (program). In skeletal muscle, suppression of slow fiber genes and activation of fast fiber genes are associated with numerous musculoskeletal disorders, including disuse atrophy, muscle wasting in response to gravity-free, and denervation. As such, expression of miR-208 in skeletal muscle cells may be useful in suppressing the reciprocal expression of the fast fiber gene, and thus activating the slow fiber gene. Thus, in certain embodiments, the invention provides methods for treating a musculoskeletal disorder by administering miR-208 to skeletal muscle of a subject having or at risk of developing a musculoskeletal disorder.

Adult skeletal muscle fibers can be divided into fast twitch (fastwitch) and slow twitch (slow twitch) subtypes based on specialized contractile and metabolic properties. These properties reflect the expression of a specific set of rapid and slow contractile protein isoforms for myosin heavy and light chains, tropomyosin, and troponin, as well as myoglobin (Naya et al, 2000). Slow twitch muscles are used primarily in chronic activities such as postural maintenance and prolonged motor activity. Fast twitch fibers are used primarily for vigorous explosive activity. The adult skeletal muscle phenotype is not static, but maintains the ability to adapt to changes in load bearing and contractile use patterns, resulting in adaptation of morphology, phenotype, and contractile properties. For example, for rodents and humans, elimination of body load in the microgravity environment of space flight results in a significant degree of muscle atrophy and changes in protein phenotype associated with slow to fast changes in contractile and metabolic properties (Tsika et al, 2002; Baldwin and Haddad, 2001; Edgerton and Roy, 2000; Fitts et al, 2000). Thus, in certain embodiments, the invention provides methods for treating or preventing muscle wasting in response to a reduced gravitational environment by administering miR-208 to skeletal muscle.

Disuse atrophy is muscle atrophy due to lack of use of muscle. Disuse atrophy is commonly seen in bedridden people, plaster of limbs, or people who are inactive for other reasons. In addition, disruption of electrical activity of muscle fibers, including denervation, can lead to muscle atrophy. Muscle atrophy is reversible after a short period of disuse. However, extreme muscle disuse can result in permanent loss of skeletal muscle fibers and replacement with connective tissue. The inventors contemplate that by suppressing fast fiber genes in skeletal muscle, thereby activating the interactive expression of slow fiber genes, the symptoms of muscle atrophy may be alleviated or prevented. Thus, in certain embodiments, the invention provides methods of treating or preventing muscle atrophy by administering miR-208 to skeletal muscle.

In addition to playing an important role in controlling fibrosis in the heart, ubiquitous expression of the family of miR-29 molecules means that it can also play a role in other fibrotic signs, such as those affecting the kidneys, liver and lungs. Fibrosis secondary to diabetes was also observed. Patients with type 1 and type 2 diabetes have an increased risk of cardiomyopathy. Cardiomyopathy in diabetes is accompanied by a group of features including reduced diastolic compliance, interstitial fibrosis, and myocyte hypertrophy. Since miR-208 inhibits miR-29, inhibition of miR-208 can be used to block both cardiac fibrosis as well as non-cardiac fibrosis.

Congenital liver fibrosis (CHF) is a rare disease that affects both the liver and the kidney. The patient inherits autosomal recessive trait. Liver abnormalities are hepatomegaly, elevated pressure in the venous system that transports blood from different organs to the liver (portal hypertension), and fibroid connective tissue that spreads the entire liver (liver fibrosis), often referred to as liver damage. Renal function is also impaired in affected individuals, often caused by Autosomal Recessive Polycystic Kidney Disease (ARPKD). The impaired renal function associated with CHF in adults is caused by Autosomal Dominant Polycystic Kidney Disease (ADPKD).

Progressive loss of kidney function is not only accompanied by the development of glomerulosclerosis, but also by interstitial fibrosis. Interstitial fibrosis is characterized by the destruction of renal tubules and interstitial capillaries and the accumulation of extracellular matrix proteins. The severity of tubulointerstitial fibrosis has long been recognized as a determinant of progressive renal injury in both human and experimental glomerulonephritis.

Pulmonary fibrosis, or scarring of the lung, is the result of the gradual replacement of the normal lung balloon by fibrotic tissue. As scarring progresses, the tissue thickens, causing an irreversible loss of the tissue's ability to transfer oxygen into the blood stream. Symptoms include shortness of breath (particularly with exertion), chronic frequent dry cough, fatigue and weakness, chest discomfort, loss of appetite, and rapid weight loss.

It is hypothesized that pulmonary fibrosis may be an autoimmune disorder, or a post-effect of viral infection. However, more and more people believe that genetic predisposition is a key factor. Mutations in the SP-C protein have been found to be present in families with a history of pulmonary fibrosis. It has recently been assumed that the fibrotic process is a (genetically biased) response to microscopic damage to the lung. Although the exact cause is still unknown, it has been linked to inhaled environmental and occupational pollutants, smoking, diseases (such as scleroderma, rheumatoid arthritis, lupus and sarcoidosis), certain medicines and therapeutic radiation.

Diabetic cardiomyopathy in patients is characterized by myocardial hypertrophy, interstitial fibrosis, capillary endothelial changes, and capillary substrate thickening, which is secondary to changes in collagen structure. Increased collagen accumulation is seen primarily in the epicardial and perivascular regions, where it induces impaired LV diastolic function, often leading to heart failure.

V. kit

Any of the compositions described herein can be included in a kit. In one non-limiting example, the individual miRNA is included in a kit. The kit may further comprise water and hybridization buffer to facilitate hybridization of the two strands of the miRNA. The kit may also include one or more transfection reagents to facilitate delivery of the miRNA to the cell.

The components of the kit may be packaged in aqueous media or in lyophilized form. The means for holding the kit will generally comprise at least one vial, test tube, flask, bottle, syringe or other holding means in which the components may be contained, preferably by suitable aliquoting. If there is more than one component of the kit (the labeling reagent and label may be packaged together), the kit will generally also include a second, third or other additional container for holding the additional components separately. However, the vial may contain various combinations of ingredients. The kits of the invention will also generally include means for holding the nucleic acid, and any other reagent containers, which are sealed and secured for commercial sale. Such containers may include injection or blow molded plastic containers in which the desired vial is placed.

If the components of the kit are provided in one and/or more liquid solutions, the liquid solutions are aqueous solutions, with sterile aqueous solutions being particularly preferred.

However, the components of the kit may be provided as a dry powder. If the reagents and/or ingredients are provided as dry powders, the powders may be reconstituted by the addition of a suitable solvent. The solvent may also be provided in another holding means.

The containing means will generally comprise at least one vial, test tube, flask, bottle, syringe or other container means, containing the nucleic acid formulation, preferably suitably dispensed. The kit may further comprise a second container means for holding sterile, pharmaceutically acceptable buffers and/or other diluents.

The kit of the invention will also generally comprise means for containing vials which are fixed closed for commercial sale, such as, for example, injection/or blow-moulded plastic containers, in which the desired vials are placed.

Such kits may also include components that retain or maintain the miRNA or protect it from degradation. Such components may be rnase-free, or provide protection against rnase. Such kits will generally provide suitable separate containers for each reagent or solution.

Kits will also include instructions for using the kit components and using any other reagents not included in the kit. The description may include variations that may be implemented.

Such reagents are embodiments of the kits of the invention. However, such kits are not limited to the specific items listed above, but may include any reagents for manipulating or characterizing mirnas.

Screening method

The invention further includes methods for identifying miR-208 inhibitors useful in preventing or treating or reversing cardiac hypertrophy or heart failure. These assays may include random screening of large libraries of candidate substances; alternatively, the assays can be used for focused screening of compounds that have been selected for structural attributes that make the compounds more likely to inhibit expression and/or function of miR-208.

To identify modulators of miR-208, miR-208 function will typically be determined in the presence and absence of a candidate agent. For example, the method generally comprises:

(a) providing a candidate modulator;

(b) mixing a candidate modulator with miR-208;

(c) measuring miR-208 activity; and are

(d) Comparing the activity in step (c) with the activity in the absence of the candidate modulator,

wherein a difference between the measured activities indicates that the candidate modulator is indeed a modulator of miR-208.

The assay may also be performed in an isolated cell, organ, or living organism.

It will of course be appreciated that all screening methods of the invention are useful per se, even if it is possible that no valid candidate can be found. The present invention provides methods for screening such candidates, not just for finding them.

A. Regulating agent

As used herein, the term "candidate agent" refers to any molecule that may modulate the beta-MHC-inducing function of miR-208. Libraries of molecules believed to meet the basic criteria for useful drugs are often obtained from a variety of commercial sources to "brute force" the identification of useful compounds. Screening of such libraries, including combinatorially generated libraries, such as antagomir libraries, is a fast and efficient way to perform activity screening of large numbers of related (and unrelated) compounds. Combinatorial approaches can also be used to create second, third and fourth generation compounds based on compounds that are active but otherwise undesirable, thereby facilitating rapid evolution of potential drugs.

B. In vitro assay

An in vitro assay is a fast, inexpensive and easy to run assay. Such assays typically use isolated molecules that can be run rapidly in large quantities, thereby increasing the amount of information available in a short time. Multiple vessels may be used to run assays, including test tubes, plates, trays, and other surfaces, such as dipsticks or beads.

A high throughput compound screening technique is described in WO 84/03564. A large number of small anti-omomir compounds can be synthesized on a solid substrate, such as a plastic needle or some other surface. Such molecules can be rapidly screened for their ability to hybridize to miR-208.

C. Intracellular (In cyto) assay

The invention also encompasses screening compounds for their ability to modulate miR-208 expression and function in a cell. Such screening assays can be performed using a variety of cell lines, including those derived from skeletal muscle cells, including cells specifically engineered for this purpose. Primary heart cells may also be used, as may the H9C2 cell line.

D. In vivo assay

In vivo assays involve the use of various animal models of heart or musculoskeletal diseases, including transgenic animals, which are engineered to have specific deficiencies or carry markers that can be used to measure the ability of a candidate substance to reach and affect different cells within an organism. Mice are a preferred embodiment, especially for transgenics, due to their size, ease of manipulation, and information about their physiological and genetic makeup. However, other animals are also suitable, including rats, rabbits, hamsters, guinea pigs, gerbils, woodchucks, cats, dogs, sheep, goats, pigs, cows, horses, and monkeys (including chimpanzees, gibbons, and baboons). Assays for inhibitors may be performed using animal models derived from any of the above species.

Treating the animal with the test compound can involve administering the compound to the animal in a suitable form. Administration can be by any route available for clinical purposes. Determining the efficacy of a compound in vivo can involve a variety of different criteria, including but not limited to, modification of hypertrophic signaling pathways and signs of hypertrophy. Measuring toxicity and dose response can also be performed in animals in a more meaningful way than in vitro and intracellular assays.

Vectors for cloning, Gene transfer and expression

In certain embodiments, an expression vector is employed to express miR-208 or an inhibitor thereof. Expression requires the provision of appropriate signals in the vector, including various regulatory elements, such as enhancers/promoters from both viral and mammalian sources that drive expression of the gene of interest in the host cell. Elements designed to optimize the stability and translatability of messenger RNAs in host cells are also defined. Also provided are conditions for using multiple dominant drug selection markers for establishing permanent, stable cell clones expressing the product, and elements linking expression of the drug selection marker to expression of the polypeptide.

A. Adjusting element

Throughout this application, the term "expression construct" is intended to include any type of genetic construct comprising a nucleic acid encoding a gene product, part or all of which nucleic acid encoding sequence is capable of being transcribed. The transcript may be translated into a protein, but it need not be translated into a protein. In certain embodiments, "expression" includes both transcription of a gene and translation of mRNA into a gene product. In other embodiments, "expression" includes only transcription of a nucleic acid encoding a gene of interest.

In certain embodiments, the nucleic acid encoding the gene product is under the transcriptional control of a promoter. "promoter" refers to a DNA sequence recognized by the synthetic machinery of a cell or introduced synthetic machinery and required to initiate specific transcription of a gene. The phrase "under transcriptional control" means that the promoter is in the correct position and orientation relative to the nucleic acid to control RNA polymerase initiation and gene expression.

The term "promoter" will be used herein to refer to a group of transcriptional control modules clustered around the initiation site of RNA polymerase II. The view of how promoters are organized is mostly derived from analysis of several viral promoters, including the promoters of HSV thymidine kinase (tk) and SV40 early transcription units. These studies, supported by subsequent work, showed that promoters were composed of discrete functional modules, each module consisting of approximately 7-20bp of DNA and containing one or more transcription activator recognition sites or repressor recognition sites.

At least one module in each promoter functions to determine the position of the RNA synthesis initiation site. The best known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter of the mammalian terminal deoxynucleotidyl transferase gene and the promoter of the SV40 late gene, a discrete element overlapping with the start site itself helps to determine the start position.

Other promoter elements regulate the frequency of transcription initiation. Typically, these elements are located in the region 30-110bp upstream of the start site, although many promoters have recently been shown to have functional elements downstream of the start site as well. The spacing between elements of a promoter is generally flexible such that promoter function is maintained when an element is inverted or moved relative to another element. In the tk promoter, the spacing between the promoter elements can be increased to 50bp apart, after which activity begins to decrease. Depending on the promoter, it appears that the individual elements may function cooperatively or independently to activate transcription.

In other embodiments, the human Cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, the Rous sarcoma virus long terminal repeat, the rat insulin promoter, and glyceraldehyde-3-phosphate dehydrogenase may be used to express high levels of a coding sequence of interest. The use of other viral or mammalian cell or bacteriophage promoters well known in the art to effect expression of a coding sequence of interest is also contemplated, provided that the level of expression is sufficient for a given purpose.

By using promoters with well-known properties, the expression level and expression pattern of the protein of interest after transfection or transformation can be optimized. In addition, by selecting promoters that are regulated in response to specific physiological signals, conditions can be provided for inducible expression of the gene product. Tables 1 and 2 list several regulatory elements that may be used to regulate the expression of a gene of interest in the context of the present invention. This table is not intended to be exhaustive of all the elements that may be involved in promoting gene expression, but is merely exemplary.

Enhancers are genetic elements that increase transcription by a promoter located at a distal position on the same DNA molecule. Enhancers are organized much like promoters. That is, they are composed of a number of individual elements, each of which binds to one or more transcribed proteins.

The basic distinction between enhancers and promoters is operational. The enhancer region as a whole must be capable of remotely stimulating transcription, while the promoter region or its constituent elements need not be. On the other hand, a promoter must have one or more elements that direct RNA synthesis to start at a specific site and in a specific orientation, while an enhancer lacks these properties. Promoters and enhancers are often overlapping and contiguous, and often appear to have very similar modular organization.

The following is a list of viral promoters, cellular promoters/enhancers and inducible promoters/enhancers that may be used in conjunction with the nucleic acid encoding the gene of interest in the expression construct (tables 1 and 2). In addition, any promoter/enhancer combination (based on the eukaryotic promoter database EPDB) can be used to drive gene expression. Eukaryotic cells can support cytoplasmic transcription of certain bacterial promoters if a suitable bacterial polymerase is provided (either as part of the delivery complex, or as another genetic expression construct).

Of particular interest are muscle-specific promoters, especially heart-specific promoters. These include the myosin light chain-2 promoter (Franz et al, 1994; Kelly et al, 1995), the alpha actin promoter (Moss et al, 1996), the troponin 1 promoter (Bhavsar et al, 1996); na (Na)⁴VCa²⁺Exchanger promoters (Barnes et al, 1997), dystrophin promoter (Kimura et al, 1997), α 7 integrin promoter (Ziober and Kramer, 1996), brain natriuretic peptide promoter (LaPointe et al, 1996) and α B-crystallin/small heat shock protein promoter (Gopal-Srivastava, 1995), α myosin heavy chain promoter (Yamauchi-Takihara et al, 1989) and ANF promoter (LaPointe et al, 1988).

If a cDNA insert is used, it will generally be desirable to include a polyadenylation signal to achieve proper polyadenylation of the gene transcript. The nature of the polyadenylation signal is not believed to be critical to the successful practice of the present invention, and any such sequence may be employed, such as the human growth hormone and SV40 polyadenylation signals. Elements of the expression cassette also encompass a terminator. These elements can be used to enhance the information level and minimize read-through from the cassette to other sequences.

B. Selection marker

In certain embodiments of the invention, the cell comprises a nucleic acid construct of the invention, and the cell may be identified in vitro or in vivo by introducing a marker into the expression construct. Such markers would confer an identifiable change to the cell, making it possible to readily identify the cell comprising the expression construct. In general, the introduction of drug selection markers aids in the selection of clones and transformants, for example, genes conferring resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selection markers. Alternatively, enzymes such as herpes simplex thymidine kinase (tk) or streptomycin acetyltransferase (CAT) may be employed. Immunological labels may also be employed. The selection marker employed is not considered to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding the gene product. Other examples of selection markers are well known to those skilled in the art.

C. Multigene constructs and IRES

In certain embodiments of the invention, an internal ribosome binding site (IRES) element is used to create multigene or polycistronic messages. IRES elements can bypass the ribosome scanning model of 5' methylated cap dependent translation and initiate translation at internal sites (Pelletier and Sonenberg, 1988). IRES elements from two members of the picornavirus (picornavirus) family (polio and encephalomyocarditis) have been described (Pelletier and Sonenberg, 1988), as well as IRES from mammalian information (Macejak and Sarnow, 1991). The IRES element can be linked to a heterologous open reading frame. Multiple open reading frames can be transcribed together, each separated by an IRES, creating a polycistronic message. By virtue of the IRES element, ribosomes can read each open reading frame, thereby achieving efficient translation. Multiple genes can be efficiently expressed by transcribing a single message with a single promoter/enhancer.

Any heterologous open reading frame can be linked to the IRES element. This includes secreted proteins, multi-subunit proteins encoded by separate genes, intracellular or membrane-bound proteins, and selectable marker genes. In this way, a cell can be engineered to have expression of several proteins simultaneously using a single construct and a single selectable marker.

D. Delivery of expression vectors

There are many ways in which an expression vector can be introduced into a cell. In certain embodiments of the invention, the expression construct comprises a virus or an engineered construct derived from a viral genome. The ability of certain viruses to undergo body-mediated endocytosis into cells, integration into the host cell genome, and stable and efficient expression of viral genes makes them attractive candidates for transferring foreign genes into mammalian cells (Ridgeway, 1988; Nicolas and Rubenstein, 1988; Baichwal and Sugden, 1986; Temin, 1986). The earliest viruses used as gene vectors were DNA viruses including papovaviruses (simian virus 40, bovine papilloma virus, and polyoma virus) (Ridgeway, 1988; Baichwal and Sugden, 1986) and adenoviruses (Ridgeway, 1988; Baichwal and Sugden, 1986). Their capacity for foreign DNA sequences is relatively low and the host spectrum is limited. In addition, their tumorigenic potential in permissive cells and cytopathic effects raise safety concerns. They can only accommodate up to 8kB of foreign genetic material, but can be readily introduced into a variety of cell lines and laboratory animals (Nicolas and Rubenstein, 1988; Temin, 1986).

One of the preferred methods for in vivo delivery involves the use of adenoviral expression vectors. "adenoviral expression vectors" are intended to include those constructs comprising sufficient (a) packaging to support the construct and (b) adenoviral sequences to express the antisense polynucleotide that has been cloned therein. In this context, expression does not require that the gene product be synthesized.

Expression vectors include genetically engineered forms of adenovirus. Knowledge of the genetic organization of adenovirus, a 36kB linear double stranded DNA virus, allows one to replace large segments of adenovirus DNA with up to 7kB of foreign sequences (Grunhaus and Horwitz, 1992). In contrast to retroviruses, infection of host cells by adenovirus does not result in chromosomal integration, since adenoviral DNA can replicate episomally and without potential genotoxicity. Also, adenoviruses are structurally stable and no genomic rearrangements are detected after extensive amplification. Adenoviruses are capable of infecting almost all epithelial cells, regardless of their cell cycle phase. To date, adenovirus infection appears to be associated with only mild diseases, such as acute respiratory disease in humans.

Adenoviruses are particularly suitable for use as gene transfer vectors because of their medium-sized genome, ease of manipulation, high titer, broad target cell range, and high infectivity. Both ends of the viral genome contain 100-200bp inverted repeats (ITRs), which are cis-elements essential for viral DNA replication and packaging. The early (E) and late (L) regions of the genome contain distinct transcriptional units, separated by the onset of viral DNA replication. The E1 region (E1A and E1B) encodes proteins and several cellular genes responsible for regulating transcription of the viral genome. Expression of the E2 region (E2A and E2B) results in the synthesis of proteins for viral DNA replication. These proteins are involved in DNA replication, late gene expression and host cell shut-off (shanan, 1990). The products of late genes, including most viral capsid proteins, are expressed only after significant processing of single-processing transcripts produced by the Major Late Promoter (MLP). MLPs (at 16.8m.u.) are particularly efficient late in infection and all mRNAs produced by this promoter have the 5' -ternary leader (TPL) sequence, making them the preferred mRNA for translation.

In one existing system, recombinant adenovirus is generated from homologous recombination between a shuttle vector and a proviral vector. Due to possible recombination between the two proviral vectors, it is possible to generate wild-type adenovirus from this process. Therefore, it is crucial to isolate a single clone of the virus from a single plaque and to examine its genomic structure.

The generation and propagation of existing replication-defective adenovirus vectors relies on a unique helper cell line, designated 293, which is obtained by transforming human embryonic kidney cells with Ad5DNA fragments and constitutively expresses the E1 protein (Graham et al, 1977). Since the E3 region is not essential for the adenoviral genome (Jones and Shenk, 1978), existing adenoviral vectors carry foreign DNA in either or both regions of E1 and D3 by means of 293 cells (Graham and Prevec, 1991). In nature, adenoviruses can package approximately 105% of the wild-type genome (Ghosh-Choudhury et al, 1987), providing additional capacity for approximately 2kb of DNA. In combination with the alternative about 5.5kb DNA in the E1 and E3 regions, the maximum capacity of the existing adenoviral vectors is below 7.5kb, or about 15% of the total length of the vector. More than 80% of the adenovirus genome remains in the vector backbone and is a source of vector-transmitted cytotoxicity. Also, the replication defect of the E1 deleted virus was incomplete.

Helper cell lines may be derived from human cells, such as human embryonic kidney cells, muscle cells, hematopoietic cells, or other human embryonic mesenchymal or epithelial cells. Alternatively, the helper cells may be derived from cells of other mammalian species permissive for human adenovirus. Such cells include, for example, Vero cells or other monkey embryonic mesenchymal or epithelial cells. As mentioned above, the preferred helper cell line is 293.

Racher et al (1995) disclose improved methods for culturing 293 cells and propagating adenovirus. In one mode, the cell individual was seeded into a 1 liter siliconized spinner flask (Techne, Cambridge, UK) containing 100-200ml of medium to culture native cell aggregates. After agitation at 40rpm, cell viability was assessed using trypan blue. In another mode, Fibra-Cel microcarriers (Bibby Sterlin, Stone, UK) (5g/l) were used as follows. The cell inoculum resuspended in 5ml of medium was added to the carrier (50ml) in a 250ml conical flask and left for 1-4 hours with occasional stirring. The medium was then replaced with 50ml of fresh medium and shaking was started. For virus production, cells were grown to approximately 80% confluence, after which the medium was changed (to 25% of the total volume) and adenovirus was added at an MOI of 0.05. The culture was allowed to stand overnight, after which the volume was increased to 100% and shaken for a further 72 hours.

In addition to the requirement that the adenoviral vector is replication-defective or at least conditionally defective, it is believed that the nature of the adenoviral vector is not critical to the successful practice of the invention. The adenovirus may be of any of 42 different known serotypes or subgroups A-F. In order to obtain a conditionally defective adenovirus vector for use in the present invention, adenovirus type 5 of subgroup C is a preferred starting material. This is because adenovirus type 5 is a human adenovirus, much biochemical and genetic information on which has been known, and it is mostly used in the construction methods that have traditionally employed adenovirus as a vector.

As mentioned above, typical vectors according to the invention are replication-defective and do not have the adenoviral E1 region. Thus, it may be most convenient to introduce a polynucleotide encoding a gene of interest at a position where the coding sequence for E1 has been removed. However, the location of the construct insertion within the adenoviral sequence is not critical to the invention. The polynucleotide encoding the gene of interest may also be inserted into an E3 replacement vector in place of the deleted E3 region, as described by Karlsson et al (1986), or into the E4 region, where the E4 deficiency is complemented by a helper cell line or helper virus.

Adenoviruses are easy to culture and manipulate, and exhibit a wide host range in vitro and in vivo. This group of viruses can be obtained at high titers, e.g. 10⁹-10¹²Individual plaque forming units per ml and they are highly infectious. The life cycle of an adenovirus does not require integration into the host cell genome. The foreign genes delivered by the adenoviral vector are episomal and therefore less genotoxic to the host cell. No side effects were reported in studies of vaccination with wild-type adenovirus (Couch et al, 1963; Top et al, 1971), demonstrating their safety and therapeutic potential as gene transfer vectors in vivo.

Adenoviral vectors have been used for 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 have suggested that recombinant adenoviruses may be used for gene therapy (Stratford-Perricaudet and Perricaudet, 1991; Stratford-Perricaudet et al, 1990; Rich et al, 1993). Studies using recombinant adenovirus to different tissues include tracheal instillation (Rosenfeld et al, 1991; Rosenfeld et al, 1992), intramuscular injection (Ragout et al, 1993), peripheral intravenous injection (Herz and Gerard, 1993), and stereotactic (stereotactic) intracerebral vaccination (Le GaI La Salle et al, 1993).

Retroviruses are a group of single-stranded RNA viruses characterized by the ability to convert their RNA into double-stranded DNA by reverse transcription in infected cells (Coffin, 1990). The resulting DNA is then stably integrated into the cellular chromosome as a provirus and directs the synthesis of viral proteins. Integration results in the maintenance of viral gene sequences in the recipient cell and its progeny. The retroviral genome contains three genes, gag, pol, and env, which encode capsid proteins, polymerase, and envelope components, respectively. One sequence present upstream of the gap gene comprises a signal to package the genome into a virion. Two Long Terminal Repeat (LTR) sequences are present at the 5 'and 3' ends of the viral genome. They contain strong promoter and enhancer sequences and are also required for integration into the host cell genome (Coffin, 1990).

To construct retroviral vectors, certain viral sequences are replaced (in the place of) by inserting nucleic acid encoding a gene of interest into the viral genome to produce a replication-defective virus. To generate virions, packaging cell lines were constructed that contained the gag, pol, and env genes but no LTR and packaging components (Mann et al, 1983). If a recombinant plasmid comprising cDNA as well as retroviral LTRs and packaging sequences is introduced into this cell line (e.g.by calcium phosphate precipitation), the packaging sequences allow the RNA transcripts of the recombinant plasmid to be packaged into viral particles which are then secreted into the culture medium (Nicolas and Rubenstein, 1988; Temin, 1986; Mann et al, 1983). The culture broth containing the recombinant retrovirus is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors are capable of infecting a wide variety of cell types. However, integration and stable expression requires division of the host cell (Passkind et al, 1975).

Based on chemical modification of retroviruses by chemical addition of lactose residues to the viral envelope, a new approach aimed at specific targeting of retroviral vectors has recently been developed. This modification can allow specific infection of hepatocytes via the sialoglycoprotein receptor.

A different approach to targeting recombinant retroviruses has been devised in which biotinylated antibodies to retroviral envelope proteins and specific cellular receptors are used. The antibody was coupled via biotin using streptavidin (Roux et al, 1989). They demonstrated in vitro infection of a variety of human cells bearing those surface antigens with a avid virus using antibodies against major histocompatibility complex class I and class II antigens (Roux et al, 1989).

Certain conditions impose limitations on the use of retroviral vectors in all aspects of the invention. For example, retroviral vectors typically integrate into a random site in the genome of a cell. This can lead to insertional mutagenesis by disruption of host genes or by insertion of viral regulatory sequences that interfere with the function of flanking genes (Varmus et al, 1981). Another concern with the use of defective retroviral vectors is the potential for wild-type replication-competent virus to be present in the packaging cell. The reason for this phenomenon may be a recombination event where the complete sequence from the recombinant virus is inserted into the genome of the host cell upstream of the gag, pol, env sequences. However, there are now new packaging cell lines which may greatly reduce the possibility of recombination (Markowitz et al, 1988; Hersdorffer et al, 1990).

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 herpes virus may be used. They provide several attractive features for various mammalian cells (Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al, 1988; Horwich et al, 1990).

With the knowledge of defective hepatitis B viruses, there is a new understanding of the structure-function associations of different viral sequences. In vitro studies have shown that viruses retain the ability to aid in (virus) -dependent packaging (helper-dependent packaging) and reverse transcription even when up to 80% of the genome is deleted (Horwich et al, 1990). This suggests that most of the genome can be replaced with foreign genetic material. Hepatic and persistent (integration) are particularly attractive features for liver-directed gene transfer. Chang et al introduced the streptomycin acetyltransferase (CAT) gene into the genome of duck hepatitis B virus in place of the polymerase coding sequence, surface coding sequence, and pre-surface coding sequence. It was co-transfected with wild-type virus into avian liver cancer cell lines. Primary duckling hepatocytes were infected using culture medium containing high titer recombinant virus. Stable CAT gene expression was detected at least 24 days after transfection (Chang et al, 1991).

To achieve expression of the sense or antisense gene construct, the expression construct must be delivered into the cell. This delivery can be accomplished in vitro, as in laboratory procedures for transforming cells, or in vivo or ex vivo, as in the treatment of certain disease states. One of the delivery mechanisms is viral infection, in which the viral construct is encapsulated in a viral particle.

The invention also encompasses several non-viral methods for transferring expression constructs into mammalian cells. 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), liposomes loaded with DNA (Nicolau and Sene, 1982; Fraley et al, 1979) and Lipofectamine-DNA complexes, cell sonication (Fechheimer et al, 1987), gene bombardment with high velocity microparticles (Yang et al, 1990), and receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu, 1988). Some of these techniques can be successfully adapted for in vivo or ex vivo use.

Once the expression construct is delivered into the cell, the nucleic acid encoding the gene of interest can be localized 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 occur at a natural location and with a natural orientation by homologous recombination (gene replacement), or it may be a random, non-specifically targeted integration (gene augmentation). In other embodiments, the nucleic acid may be stably maintained in the cell as a separate, episomal (episomal) DNA segment. Such nucleic acid segments, or "episomes," encode sequences sufficient to permit maintenance and replication independent of, or in synchronization with, the host cell cycle. How the expression construct is delivered to the cell and where in the cell the nucleic acid is maintained depends on the type of expression construct employed.

In yet another embodiment of the invention, the expression construct may consist of naked recombinant DNA or plasmid only. The transfer of the construct may be carried out by any of the methods described above for permeabilizing cell membranes by physical or chemical means. This is particularly applicable to in vitro transfer, but it may also be applied for in vivo use. Dubensky et al (1984) successfully injected polyomaviral DNA as calcium phosphate precipitates into the liver and spleen of adult and neonatal mice, showing positive viral replication and acute infection. Benveninsty and Neshif (1986) also demonstrated that direct intraperitoneal injection of calcium phosphate-precipitated plasmids resulted in expression of the transfected gene. DNA encoding a gene of interest may also be transferred in vivo and the gene product expressed in a similar manner.

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

Selected organs, including liver, kidney, 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 tissue or cells to clear any tissue between the gun and the target organ, i.e., ex vivo treatment. Also, DNA encoding a particular gene may be delivered by this method and still be encompassed by the present invention.

In yet another embodiment of the invention, the expression construct may be encapsulated in a liposome. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by an aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid component undergoes self-rearrangement, followed by formation of a compact structure and trapping of water and dissolved solutes between lipid bilayers (Ghosh and Bachhawat, 1991). The invention also encompasses Lipofectamine-DNA complexes.

Liposome-mediated nucleic acid delivery and in vitro foreign DNA expression have been very successful. Wong et al (1980) demonstrated the feasibility of liposome-mediated delivery and foreign DNA expression in cultured chick embryos, HeLa and hepatoma cells. Nicolau et al (1987) achieved successful liposome-mediated gene transfer in rats following intravenous injection.

In certain embodiments of the invention, the liposomes may be complexed with Hemagglutinating Virus (HVJ). This has been shown to promote fusion with cell membranes and to facilitate entry of liposome-encapsulated DNA into cells (Kaneda et al, 1989). In other embodiments, liposomes can be complexed or conjugated to nuclear non-histone chromosomal proteins (HMG-I) (Kato et al, 1991). In other embodiments, liposomes can be complexed or combined with both HVJ and HMG-I. Since such expression constructs have been successfully used for the transfer and expression of nucleic acids in vitro and in vivo, they are suitable for use in the present invention. If a bacterial promoter is used in the DNA construct, it may also be desirable to include a suitable bacterial polymerase within the liposome.

Other expression constructs that can be used to deliver nucleic acids encoding a particular gene into a cell are receptor-mediated delivery vehicles (recipient-mediated delivery vehicles). They take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis in almost all eukaryotic cells. Delivery can be highly specific due to the cell type specific distribution of the various receptors (Wu and Wu, 1993).

Receptor-mediated gene targeting agents 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 well understood ligands are the non-salivary blood mucoid (ASOR) (Wu and Wu, 1987) and transferrin (Wagner et al, 1990). Recently, a synthetic glycoprotein mimetic (neoglyoprotein) that 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) observed increased uptake of insulin genes by hepatocytes by incorporating lacosyl-ceramide, a galactose-terminated asialoglycoside, into liposomes. It can thus be seen that specific delivery of a nucleic acid encoding a particular gene into a cell type, with or without liposomes, is also possible via any of a variety of receptor-ligand systems. For example, Epidermal Growth Factor (EGF) may be used as a receptor to mediate delivery of nucleic acids into cells exhibiting upregulation of EGF receptors. Mannose can be used to target mannose receptors on hepatocytes. Also, antibodies against CD5(CLL), CD22 (lymphoma), CD25 (T-cell leukemia), and MAA (melanoma) can be similarly used as targeting moieties.

In one particular example, the oligonucleotide 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 disclosure of WO0071096 (expressly incorporated herein by reference) describes different formulations that can be effectively used in gene therapy, such as DOTAP: cholesterol or cholesterol derivative formulations. Other publications also discuss different lipid or liposome formulations (including nanoparticles) and methods of administration; these include, but are not limited to, U.S. patent publications 20030203865, 20020150626, 20030032615, and 20040048787, which are expressly incorporated herein by reference for their disclosure of formulations and other related aspects of administering and delivering nucleic acids. Methods 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 herein by reference.

In certain embodiments, gene transfer can be more easily performed under ex vivo conditions. Ex vivo gene therapy refers to the isolation of cells from animals, the delivery of nucleic acids into cells in vitro, and the return of modified cells into animals. This may involve surgical removal of tissue/organs or primary cultures of cells and tissues from the animal.

Method for producing transgenic mice

A particular embodiment of the invention provides a transgenic animal lacking one or both of the two functional miR-208 alleles. Also, transgenic animals expressing miR-208 under the control of inducible, tissue-selective, or constitutive promoters, recombinant cell lines derived from such animals, and transgenic embryos can be used to determine the precise role that miR-208 plays in the development and differentiation of cardiomyocytes and in the development of pathologic cardiac hypertrophy and heart failure. In addition, these transgenic animals may provide insight into cardiac development. The use of constitutively expressed miR-208-encoding nucleic acids provides a model for over-or down-regulated expression. Transgenic animals in which one or both alleles of miR-208 are "knocked out" are also contemplated.

In one general aspect, transgenic animals are generated by integrating a given transgene into the genome in a manner that allows for expression of the transgene. Methods for generating transgenic animals are generally described in Wagner and Hoppe, U.S. Pat. No. 4,873,191 (incorporated herein by reference) and Brinster et al, 1985 (incorporated herein by reference).

Typically, genes flanked by genomic sequences are transferred into fertilized eggs by microinjection. The microinjected eggs are implanted into host females and the offspring are screened for expression of the transgene. Transgenic animals can be produced from fertilized eggs of many 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, using standard techniques, DNA clones for microinjection can be cleaved with enzymes suitable for excision of bacterial plasmid sequences and the DNA fragments electrophoresed on 1% agarose in TBE buffer. The DNA bands were visualized by staining with ethidium bromide and the bands containing the expressed sequences were excised. The excised band was then placed in a dialysis bag containing 0.3M sodium acetate pH 7.0. The DNA was electroeluted into dialysis bags, extracted with a 1: 1 phenol: chloroform solution, and precipitated by two volumes of ethanol. The DNA was redissolved in 1ml of low salt buffer (0.2M NaCl, 20mM Tris pH7.4, and 1mM EDTA) and applied at Elutip-D^TMAnd (5) purifying on a column. The column was first pretreated (prime) with 3ml of high salt buffer (1M NaCl, 20mM Tris pH7.4, and 1mM EDTA) followed by washing with 5ml of low salt buffer. The DNA solution was allowed to flow through the column three times to allow the DNA to bind to the column matrix. After washing once with 3ml of low salt buffer, the DNA was eluted with 0.4ml of high salt buffer and precipitated with two volumes of ethanol. The DNA concentration was measured by absorbance at 260nm in a UV spectrophotometer. For microinjection, the DNA concentration was adjusted to 3. mu.g/ml in 5mM Tris pH7.4 and 0.1mM EDTA. Other methods for purifying DNA for microinjection are described in Palmiter et al, 1982; and Sambrook et al, 2001.

In an exemplary microinjection protocol, six-week-old female mice were induced to superovulate by injection of 5 IU (0.1cc, ip) of pregnant mare serum gonadotropin (PMSG; Sigma) followed 48 hours later by 5 IU (0.1cc, ip) of human chorionic gonadotropin (hCG; Sigma). Female and male mice were placed together immediately after hCG injection. 21 hours after hCG injection, by CO₂Mated females were sacrificed by choking or cervical dislocation and embryos recovered from excised oviducts and placed in Dulbecco's phosphate buffered saline containing 0.5% bovine serum albumin (BSA; Sigma). Surrounding cumulus cells were removed with hyaluronidase (1 mg/ml). The prokaryotic embryos were then washed (pronuclear embryo) and placed in Earle's Balanced Salt Solution (EBSS) containing 0.5% BSA in 5% CO₂Humidified atmosphere of 95% air 37.5 ℃ incubator until injection. Embryos can be implanted at the two-cell stage.

Adult female mice with random cycles (randomly cycling) were paired with vasectomized males. C57BL/6 or Swiss mice or other equivalent strains can be used for this purpose. Recipient females were mated simultaneously with donor females. At the time of embryo transfer, recipient females were anesthetized with intraperitoneal injection of 0.015ml of 2.5% avertin (avertin) per gram of body weight. The fallopian tubes are exposed through a single incision in the back. An incision is then made through the body wall just above the fallopian tube. The ovarian cysts were torn open with Watchmakersforceps fine forceps (watchmakersforceps). Embryos to be transferred were placed in DPBS (Dulbecco phosphate buffered saline) in a pipette tip (approximately 10-12 embryos). A pipette tip was inserted into the oviduct funnel (infundibulum) and the embryos were transferred. After transfer, the incision was closed with two sutures.

IX. definition

As used herein, the term "heart failure" broadly refers to any condition that reduces the ability of the heart to pump blood. As a result, congestion and edema are formed in the tissue. Most commonly, heart failure is caused by a decrease in myocardial contractility due to decreased coronary blood flow; however, many other factors can lead to heart failure, including damage to heart valves, vitamin deficiencies, and primary heart muscle disease. Although the precise physiological mechanisms of heart failure are not fully understood, it is generally believed that heart failure involves a disturbance in several cardiac autonomic properties, including sympathetic, parasympathetic, and baroreceptor responses. The phrase "manifestations of heart failure" is used broadly to encompass all outcomes associated with heart failure, such as shortness of breath, foveal edema, enlarged liver tenderness, jugular vein filling, pulmonary rales, and the like, including findings from experiments associated with heart failure.

The terms "treatment" and "treating" or grammatical equivalents encompass the amelioration and/or reversal of heart failure symptoms (i.e., the ability of the heart to pump blood). The "improvement in physiological function" of the heart can be assessed using any of the metrics described herein (e.g., measuring ejection fraction, fractional shortening, left ventricular size, heart rate, etc.), as well as any effect on animal survival. In using the animal model, the response of the treated transgenic animal and the untreated transgenic animal is compared using any of the assays described herein (in addition, treated and untreated non-transgenic animals can be included as controls). Compounds that cause an improvement in any parameter associated with heart failure in the screening methods of the invention may thus be identified as therapeutic compounds.

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, by the frequent involvement of the right ventricle.

The term "compound" refers to any chemical entity, drug, pharmaceutical, etc., that can be used to treat or prevent a disease or disorder of bodily function. The compounds include known and potential therapeutic compounds. The screening methods of the present invention can be used to screen to determine a compound as therapeutic. "known therapeutic compound" refers to a therapeutic compound that has been shown to be effective in such treatment (e.g., by animal testing or prior experiments administered to humans). In other words, known therapeutic compounds are not limited to compounds that are effective in the treatment of heart failure.

As used herein, the term "agonist" refers to a molecule or compound that mimics the action of a "native" or "natural" compound. Agonists may be homologous to these native compounds in terms of conformation, charge or other characteristics. Thus, agonists are recognized by receptors expressed on the cell surface. This recognition can result in physiological and/or biochemical changes within the cell, such that the cell responds to the presence of the agonist in the same manner as if the native compound was present. Agonists may include proteins, nucleic acids, carbohydrates, or any other molecule that reacts with a molecule, receptor, and/or pathway of interest.

As used herein, the term "cardiac hypertrophy" refers to the process by which adult cardiomyocytes respond to stress by hypertrophic growth. Such growth is characterized by an increase in cell size without cell division, assembly of additional sarcomere within the cell to maximize force generation, and activation of fetal cardiac gene programs. Cardiac hypertrophy is often associated with an increased risk of morbidity and mortality, and as such, studies aimed at understanding the molecular mechanisms of cardiac hypertrophy will have a significant impact on human health.

As used herein, the terms "antagonist" and "inhibitor" refer to a molecule, compound, or nucleic acid that inhibits the action of a cellular factor that may be involved in cardiac hypertrophy. Antagonists may be homologous or different in conformation, charge, or other characteristics from these native compounds. Thus, an antagonist may be recognized by the same receptor as that recognized by the agonist, or by a different receptor. Antagonists may have allosteric effects that prevent the action of agonists. Alternatively, an antagonist may prevent the function of an agonist. In contrast to agonists, antagonistic compounds do not cause pathological and/or biochemical changes within the cell such that the cell responds to the presence of the antagonist in the same manner as if a cytokine were present. Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates, or any other molecules that bind to or interact with a receptor, molecule, and/or pathway of interest.

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

The term "beta-adrenergic receptor antagonist" refers to a chemical compound or entity capable of partially or completely blocking beta (beta) -type adrenergic receptors (i.e., receptors of the adrenergic system that respond to catecholamines, particularly norepinephrine). Some beta-adrenergic receptor antagonists exhibit activity at one receptor subtype (generally beta)₁) A certain degree of specificity; such antagonists are referred to as "beta₁Specific adrenergic receptor antagonists "and" beta₂A specific adrenergic receptor antagonist. The term "beta-adrenergic receptor antagonist" refers to a chemical compound that is both a selective and a non-selective antagonist. Examples of beta-adrenergic receptor antagonists include, but are not limited to, acebutolol, atenolol, butoxyamine, carteolol, esmolol, labetalol (labeolol), metoprolol, nadolol, penbutolol, propranolol, and timolol. The methods of the invention encompass the use of derivatives of known beta-adrenergic receptor antagonists. In fact, the methods of the present invention encompass any compound that functionally behaves as a beta-adrenergic receptor antagonist.

The term "angiotensin converting enzyme inhibitor" or "ACE inhibitor" refers to a chemical compound or entity capable of partially or completely inhibiting the enzymes involved in the chymosin-angiotensin system that convert relatively inactive angiotensin I to active angiotensin. In addition, ACE inhibitors simultaneously inhibit the degradation of bradykinin, which has the potential to significantly enhance the antihypertensive effect of ACE inhibitors. Examples of ACE inhibitors include, but are not limited to, benazepril (benazepril), captopril, enalapril (enalopril), fosinopril, lisinopril, quinapril (quinapril), and ramipril. The methods of the present invention encompass the use of derivatives of known ACE inhibitors. Indeed, the methods of the present invention encompass any compound that functionally behaves as an ACE inhibitor.

As used herein, the term "genotype" refers to the actual genetic makeup of an organism, while "phenotype" refers to the physical trait exhibited by an individual. In addition, a "phenotype" is the result of genome-selective expression (i.e., it is the cell's historical expression and its response to the extracellular environment). In fact, the human genome is estimated to contain 30,000-35,000 genes. In each cell type, only a small fraction (i.e., 10-15%) of these genes are expressed.

X example

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 inventor 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.

Example 1 materials and methods

Northern blot analysis. Cardiac tissue samples were obtained from Gilead Colorado (Westminster, CO) from anonymous human subjects diagnosed as heart failure or heart failure. Total RNA was isolated from mouse, rat and human heart tissue samples using Trizol reagent (Gibco/BRL). Northern blotting was performed to detect microRNAs as described previously (l). U6 probe was used as a loading control (U6 forward: 5-GTGCTCGCTTCGGCAGC-3(SEQ ID NO: 18), U6 reverse: 5-AAAATATGGAACGCTTCACGAATTTGCG-3(SEQ ID NO: 19)). To detect alpha MHC expression, Northern blots containing 10 μ g RNA from cardiac tissue from both adult wild-type and miR-208 mutant animals were probed with alpha MHC cDNA fragments covering the 5' UTR region and first exon.

And (7) PTU processing. Thyroid hormone deficiency was induced by feeding the animals iodine-free food supplemented with 0.15% PTU (purchased from Harlan Teklad Co. (TD 97061) (Madison, WI)) for the indicated duration.

Microarray and real-time PCR analysis. Total RNA was isolated from heart tissue using trizol (invitrogen). Microarray analysis was performed using a mouse genome 4302.0 array (Affymetrix). RT-PCR was performed on RNA samples using random hexamer primers (Invitrogen), and then quantitative real-time PCR using Taqman probes purchased from ABI was used to analyze the expression of one subset of genes.

Generation of miR-208 mutant mice. To generate the miR-208 targeting vector, a 0.4kb fragment (5' arm) extending upstream of the miR-208 coding region was digested with SacII and NotI and ligated into the pGKneoF2L2dta targeting plasmid, upstream of the loxP site and the neomycin cassette flanking the Frt. The 3.3kb fragment (3' arm) was digested with SalI and HindIII and ligated into the vector between the neomycin cassette and the Dta negative selection cassette. Target ES cells carrying the disrupted allele are identified by Southern blot analysis using 5 'and 3' probes. Three miR-208 target ES clones were identified and used for blastocyst injections. The chimeric mice thus obtained were bred with C57BL/6 to obtain germline transmission of mutant alleles (germlinetransmision). The PCR primers can be obtained on request.

Western blotting. Myosin was extracted from heart tissue as described (Morkin, 2000). MHC isoforms were separated by SDSPAGE and Western blots were performed with mouse monoclonal α MHC (BA-G5) (ATCC, Rockville, MD) and mouse monoclonal anti-myosin (slow, skeletal M8421) (Sigma, MO), the latter being highly specific for β MHC. To detect all striated myosins, a pan-specific antibody (mouse monoclonal 3-48; Accurate Chemical & Scientific Corporation, NY) was used. THRAP1 was detected by immunoprecipitation from 400 μ g cardiac protein lysate. After pre-clarification of the samples at 4 ℃ for 1 hour, the supernatant was incubated overnight at 4 ℃ with 1. mu.l of rabbit polyclonal anti-THRAP 1 (presented by R.Roeder of the university of LokeFiller) and 15. mu.l of protein A beads. The beads were washed three times with lysis buffer and boiled in SDS sample buffer. Immunoprecipitated THRAP1 protein was resolved by SDS-PAGE and analyzed using 1: 3000 diluted rabbit polyclonal anti-THRAP 1 and 1: 5000 diluted horseradish peroxidase conjugated anti-rabbit IgG, detected by Luminol reagent (Santa Cruz).

Histological analysis and RNA in situ hybridization. Tissues for histological analysis were incubated in Krebs-Henselheit (Krebs-Henselheit) solution, fixed in 4% paraformaldehyde, sectioned, and processed into hematoxylin and eosin (H) by standard techniques (Krenz and Robbins, 2004)&E) And Masson's trichrome stain or in situ hybridization. Generated using Maxissript kit (Amersham)³⁵S-labeled RNA probe. The signal was pseudo-colored red using Adobe Photoshop.

Transathorax echocardiography. Cardiac function and heart size (dimensions) were assessed by two-dimensional echocardiography in conscious mice using the Vingmed System (GE Vingmed Ultrasound, hospital, Norway) and an 11.5MHz linear array transducer. The anterior and posterior wall thicknesses at end diastole and end systole were measured using M-mode tracking. The largest anteroposterior diameter in either diastole (LVIDd) or Systole (LVIDs) was measured as the Left Ventricular (LV) internal diameter (LVID). Data were analyzed by a single observer blinded to mouse genotype. The LV shortening score (fractional shorteningFS) is calculated according to the following formula: FS (%) [ (LVIDd-LVIDs)/LVIDd ] × 100.

Generation of transgenic mice. The mouse genomic fragment flanking the miRNA of interest was subcloned into a heart-specific expression plasmid (Kiriazis and Kranias, 2000) containing α -MHC and human GH poly (a) + signal. DNA was isolated from mouse tail biopsies and PCR analysis was performed using primers specific for human GH poly (a) + signal.

Treatment and transfection assays. A305 bp genomic fragment encompassing the miR-208 coding region was amplified by PCR and ligated into pCMV 6. A1 kb fragment encompassing the entire murine THRAP 1-UTR was amplified by PCR and ligated into an HA-tagged pCMV6 expression construct and a firefly luciferase (f-luc) reporter construct (pMIR-REPORT)^TMAmbion). Mutations of the UCGUCUUAmiR-208 seed binding sequence were constructed by PCR-based mutagenesis.

Example 2 results

miR-208 is a central regulator of cardiac contractile function. Intron micrornas are transcribed as part of the host gene transcript, spliced out, and processed into mature mirnas. miR-208 is an intronic miRNA located within the 27 th intron of the alpha-MHC gene. FIG. 1 like α -MHC, miR-208 is expressed only in the heart. Figure 2 postnatal thyroid hormone regulates the expression of ventricular myosin isozymes by stimulating alpha-MHC synthesis and inhibiting beta-MHC expression. To examine whether blocking thyroid hormone signaling also affects miRNA-208 expression, the inventors used a cardiac rat sample, which was exposed to Propylthiouracil (PTU) for a predetermined period of time. PTU blocks thyroid hormone biosynthesis by inhibiting the "organization" of iodine (i.e., iodine incorporation into T3 and T4) and thereby suppresses α -MHC expression and increases β -MHC. Northern blot analysis indicated that there is an excellent correlation between the level of α -MHC expression and the level of pre-miRNA, the so-called "stem-loop", whereas mature miRNA still exists for several weeks thereafter. FIGS. 3A-C and FIGS. 4A-C.

To investigate the role of miR-208, the inventors created miR-208-deleted mice. FIG. 5. although this did not interfere with α -MHC transcription or translation, microarray analysis of heart tissue from 2-month old wild-type and miR-208KO mice showed that abolishing miR-208 can result in strong induction of rapid skeletal muscle genes. Fig. 6A-B and fig. 7.

To examine the effect of miR-208 elimination during cardiac stress, the inventors received wild-type and miR-208KO animals with a transverse aortic belt constriction (TAB). TAB is a strong cause of cardiac hypertrophy and its associated hypertrophic gene expression. While wild-type animals showed a dramatic increase in β -MHC expression, KO animals did not show this induction. Fig. 8.

TABLE 3 KO vs WT 3 weeks after TAB

Gene	Fold change after TAB compared to wild type
		Cardiac troponin I, fast bone	194.0X Up-Regulation
Cardiac troponin T3, fast bone	194.0X Up-Regulation
		MLC, fast bone	3.7X Up-Regulation
Alpha skeletal actin	2.8X Up-Regulation
		βMHC	29.8S Down-Regulation

Taken together, these data indicate that expression of the α -MHC gene further induces expression of a miRNA that down-regulates expression of the rapid skeletal muscle gene program. miR-208 is embedded in the α -MHC gene, which is regulated by developmental, physiological, and developmental signals. alpha-MHC is the major determinant of rapid contractility. miR-208 suppresses the fast skeletal muscle gene in the heart, and its deletion can result in a significant increase in the expression of the fast skeletal muscle gene (fig. 13). miR-208 is also required for β -MHC upregulation in the heart. Since micrornas act as repressors, we propose the hypothesis that miR-208 can repress the repressor of β -MHC expression, as illustrated in figure 9. During cardiac stress, this miRNA is responsible for the induction of β -MHC at the RNA and protein levels, whereas in the absence of miR-208, this induction is completely absent and α -MHC remains the only myosin heavy chain isoform. Analysis of α -MHC expression in samples of failing and non-failing human hearts showed that α -MHC expression was reduced in the failing heart compared to the non-failing heart (fig. 10). These data demonstrate that miR-208 is a central regulator in cardiac contractile function and appears to be involved in maladaptive myosin switching during cardiac disease.

Using the miRanda software (available from Computational Biology Center of memory Sloan-Kettering Cancer Center) and the PicTar algorithm for identifying miRNA targets (Krek et al, 2005), it was determined that thyroid hormone receptor-related protein 1(THRAP1) is a predicted target for miR-208. FIG. 12 shows an alignment of miR-208 with THRAP 13' UTR sequences from human, chimpanzee, mouse, rat, dog, chicken, puffer, and zebrafish.

miR-208 regulates pathological cardiac remodeling. Homozygous miR-208-deleted mice were viable and did not exhibit significant abnormalities in heart size, shape, or structure until 20 weeks. To further investigate the potential function of miR-208, the inventors compared the response of wild-type and miR-208 mutant mice to Thoracic Aortic Banding (TAB), which induces cardiac hypertrophy by elevating cardiac afterload and is accompanied by α MHC down-regulation and β MHC up-regulation (Hill et al, 2000). Alpha MHC mRNA expression decreased after TAB as expected (fig. 14A), but miR-208 was still abundantly expressed 21 days after TAB (fig. 14B), consistent with its relatively long half-life.

In response to TAB, wild type mice showed a significant increase in cardiac mass with hypertrophic growth of cardiomyocytes and ventricular fibrosis (fig. 15A). In contrast, miR-208 mutant animals showed little cardiomyocyte hypertrophy or fibrosis in response to TAB (fig. 15A). The echocardiography proves that the miR-208^-/-Animals displayed blunted (blunted) hypertrophic responses and reduced contractility (fig. 14C). Most notably, mutant animals are unable to upregulate β MHC. Instead, alpha MHC protein expression is elevated in response to TAB in miR-208 mutant hearts, which likely reflects a complementary mechanism for maintaining MHC expression in the absence of beta MHC upregulation. Other stress-responsive genes, such as those encoding natriuretic peptides ANF and BNP, were strongly induced in miR-208 mutant animals (fig. 15B-C). For cells from wild type and miR-208^-/-Microarray analysis of the hearts of animals demonstrated that miR-208 deletion resulted in highly specific blockade of β MHC expression (tables 4-5).

TABLE 4 Pair from wild type and miR-208^-/-Microarray analysis of animal heart tissue. Shows the presence of miR208 in each category^-/-The first 20 genes differentially expressed compared to wild type animals.

Table 5: for the field from 3 weeks after TABBiotype and miR-208^-/-Microarray analysis of animal heart tissue. Shows miR208 3 weeks post-TAB surgery in each category^-/-The first 20 genes differentially expressed compared to wild type animals.

miR-208^-/-Mice were also resistant to fibrosis and cardiomyocyte hypertrophy in response to transgene expression of activated calcineurin, a particularly potent stimulator of cardiac hypertrophy and heart failure (fig. 15D). Similarly, miR-208 of β MHC mRNA and protein at 6 weeks of age^-/-(ii) a There was no upregulation in the heart of CnA-Tg mice; whereas ANF and BNP were strongly induced (fig. 15E-F). It can be seen that miR-208 is essential for β MHC upregulation and cellular remodeling, but not for expression of other cardiac stress markers.

To test that miR-208 is a sufficient condition for upregulation of β MHC expression, the inventors created transgenic mice that overexpress miR-208 under the control of an α MHC promoter. The α MHC-miR-208 transgenic mice were viable and expressed miR-208 at a level approximately 3-fold over wild-type heart (FIG. 14D). Hearts from transgenic lines exhibiting mean transgene overexpression did not show signs of overt pathological remodeling at 2 months of age, but, notably, exhibited significant upregulation of β MHC expression (fig. 15G and 14E). This activity of miR-208 is specific, as transgene overexpression of miR-214 (which is induced during cardiac hypertrophy) has no effect on β MHC expression. Considering that endogenous levels of miR-208 in the heart of adult mice are insufficient to upregulate β MHC expression, a 3-fold increase in miR-208 expression in these transgenic mice could lead to the discovery that β MHC expression is upregulated, suggesting that such micrornas have a mutated (sharp) threshold for control of β MHC expression.

miR-208 regulates T3-dependent repression of β MHC. T3 signaling induces alpha MHC transcription via a positive T3 Response Element (TRE), while a negative TRE in the promoter of the beta MHC gene mediates transcriptional repression (Ojamaa et al, 2000). To test whether miR-208 is required for T3-dependent regulation of β MHC, mutant and wild-type littermates were fed PTU-containing chow for 2 weeks to block T3 signaling. Northern blot analysis confirmed that miR-208 is abundant 2 weeks after PTU treatment (FIG. 16A). As expected, PTU induced a decrease in heart rate and contractility and an increase in relaxation, with no dramatic difference between wild type and mutant animals (fig. 16B). However, while wild-type animals showed a decrease in α MHC and an increase in β MHC in response to PTU as expected, miR-208^-/-Animals still showed resistance to β MHC upregulation, although trace amounts of β MHC expression could be detected (fig. 17A-B). PTU up-regulates miR-208^-/-ANF and BNP in animals, confirming the specific role of miR-208 in β MHC expression (figure 16C). Since PTU induces an isotype switch from α MHC to β MHC by merely interfering with thyroid hormone receptor (TR) signaling, these findings suggest that miR-208 potentiates β MHC expression through a mechanism involving TR.

miR-208 targets TR-related protein 1. Among the relatively few miR-208 predictive targets, mRNA encoding thyroid hormone receptor-related protein 1 (thap 1), also known as TRAP240, was evaluated by the PicTar target prediction program (Krek et al, 2005) as the strongest predictive target. THRAP1 is a component of the TR-associated TRAP complex, which regulates TR activity by recruiting RNA polymerase II and a universal initiator (Ito and Roeder, 2001). The putative miR-208 binding site in the 3 '-UTR of thap 1 mRNA showed high complementarity to the 5' arm of miR-208, which is the most critical determinant for miRNA targeting and evolutionary conservation (fig. 18A). Based on the incomplete complementarity of miR-208 with THRAP 13' -UTR sequence, miR-208 is expected to inhibit the translation of THRAP 1.

To examine whether the putative miR-208 target sequence in the thap 13 '-UTR could mediate translational repression, the inventors inserted the full-length 3' -UTR of thap 1 transcript into a luciferase expression plasmid and transfected into COS1 cells. An increase in the amount of CMV-driven miR-208 resulted in a dose-dependent decrease in luciferase activity, whereas a comparable amount of miR-126 as a control had no effect (fig. 18B). CMV-miR-208 also dose-dependently abolished translation of the HA-tagged Malonyl CoA Decarboxylase (MCD) expression cassette linked to thap 13' UTR binding sequence, but the mutant miR-208 target sequence was not (fig. 18C). In addition, from miR-208, as compared to wild-type littermates^-/-Expression of thap 1 protein was elevated in heart protein lysates of mice (fig. 18D), while thap 1 mRNA in hearts of both genotypes was comparable (fig. 19), consistent with the conclusion that miR-208 functions as a negative regulator of translation in vivo. Given that recent studies have shown that stress enhances miRNA suppression by promoting miRNA binding to Argonaute (Leung et al, 2006), we believe that the negative impact of miR-208 on thap 1 protein expression may still be greater in the case of stress.

miR-208 is required for expression of miR-499. To further explore the mechanism of action of miR-208 in the heart, the inventors determined microrna expression patterns in the heart from wild-type and miR-208-deleted mice by microarray analysis. In several micrornas, up-and down-regulated in mutant hearts, the inventors found that miR-499 is highly abundant in normal hearts, but expression in miR-208 mutants did not exceed background levels. These findings were confirmed by Northern blotting (FIG. 21).

Analysis of the genomic localization of the miR-499 gene shows that it is contained within the 20 th intron of the Myh7b gene, Myh7b is a homologue of the α -Mhc gene (Myh7b) (FIG. 22). The Myh7b gene was conserved in vertebrates and was only expressed in heart and slow skeletal muscle (soleus) (fig. 23). In addition, miR-499 is down-regulated during cardiac hypertrophy (FIG. 24).

MEF2 regulates miR-499 expression in heart and skeletal muscle. Within the 5' flanking region of the Myh7 gene, the inventors identified a potential MEF2 consensus sequence that is conserved across species. This sequence binds MEF2 tightly in a gel mobility shift assay, and mutation of this sequence abrogates expression of the lacZ reporter in transgenic mice. MEF2 site is juxtaposed with a conserved E-box sequence (CANNTG) which serves as a binding site for a MyoD family member of bHLH proteins that drive skeletal muscle gene expression with MEF 2. In fact, MyoD binds the E-box from the promoter together with the ubiquitous bHLH protein E12. Mutation of this sequence prevented expression of the lacZ transgene in skeletal muscle, but did not affect expression in the heart.

The target ID. In summary, the data reported herein indicate that Myh7b gene expression regulated by MEF2 further induces the expression of a slow muscle and heart specific miRNA that down-regulates the expression of the fast skeletal muscle gene program. These data provide evidence that miRNA 499 is a central regulator in skeletal muscle fiber types.

miR-208 is highly homologous to miR-499 and it is noteworthy that both microRNAs are encoded by introns of the Mhc gene, suggesting that they share a common regulatory mechanism. Since mirnas negatively affect gene expression in a sequence-specific manner, the high degree of homology predisposes miR-208 and miR-499 to function similarly due to the overlap of target genes. The inventors have identified transcriptional regulators of Mhc expression that appear to serve as targets for miR-499. The inventors have also shown that miR-499 expression is under the control of miR-208 in the heart, such that miR-208 knockdown can abolish miR-499 expression.

Since the inventors previous data demonstrated that miR-208 gene disruption can lead to strong induction of specific rapid skeletal muscle genes in the heart, it is likely that miR-499 has a similar function in skeletal muscle and can act as a dominant regulator of fiber type. Consistent with this hypothesis, promoter analysis of this transcript indicated that expression of miR-499 and its host transcript was regulated by the myogenic transcription factor MEF2, a central regulator of skeletal muscle fiber type and slow fiber gene expression. The inventors have demonstrated that MEF2 activity can promote muscle endurance and prevent muscle fatigue after prolonged exercise. As such, they suggested that these effects of MEF2 were at least partially dependent on direct activation of miR-499 expression (fig. 25).

Taken together, these data indicate that Myh7b gene expression regulated by MEF2 further induces the expression of a slow muscle and heart specific miRNA, which can down-regulate the expression of the fast skeletal muscle gene program. These data provide evidence that miRNA 499 is a central regulator in skeletal muscle fiber types. The remarkable fact that miR-208 and-499 are highly homologous and that both microRNAs are encoded by introns of the Mhc gene suggests that they share a common regulatory mechanism. Since mirnas negatively affect gene expression in a sequence-specific manner, the high degree of homology predisposes miR-208 and miR-499 to function equivalently due to the overlap of target genes. The inventors have identified transcriptional regulators of Mhc expression that appear to serve as targets for miR-499, and they have also shown that miR-499 expression is controlled in the heart by miR-208, such that miR-208 knockdown can abolish miR-499 expression.

Modulation of cardiac hypertrophy and heart failure by stress-responsive mirnas. Based on their role in the regulation of cellular phenotypes, we hypothesized that mirnas might play a role in regulating cardiac responses to cardiac stress, which is known to lead to transcriptional and translational changes in gene expression. To investigate the potential role of mirnas in cardiac hypertrophy, the inventors performed a parallel (side-by-side) miRNA microarray analysis in 2 established mouse models of cardiac hypertrophy. This assay used a microarray representing 186 different mirnas (Babak et al, 2004). Mice were subjected to a Thoracic Aortic Banding (TAB) (treatment which induces hypertrophy by elevating cardiac afterload) (Hill et al, 2000) and such mice were compared to sham operated mice. In a second model, transgenic mice expressing activated calcineurin (CnA) in the heart, which results in a well-known severely hypertrophic form (Molkentin et al, 1998), were compared to wild-type littermates (fig. 26A). RNA isolated from hearts of mice subjected to TAB showed increased expression of 27 mirnas compared to the sham-operated control, and CnA Tg mice showed increased expression of 33 mirnas compared to the non-transgenic littermate control, 21 of which were upregulated in both models. Similarly, TAB and CnA induced hypertrophy were accompanied by decreased expression of 15 and 14 mirnas, respectively, with 7 mirnas co-downregulated (fig. 26B). Northern analysis (our unpublished data) and previous microarray analysis (Barad et al, 2004; Sempere et al, 2004; Shingara et al, 2005; Liu et al, 2004) of these miRNAs indicated that they were expressed in a wide range of tissues. Based on their relative expression levels, conservation of human, rat and mouse sequences, and expression levels during hypertrophy, the inventors focused on 11 upregulated mirnas and 5 downregulated mirnas (fig. 26C).

Northern blot analysis of cardiac RNA from WT and can Tg mice confirmed elevated miR-21, -23, -24, -125b, -195, -199a and-214 expression, while decreased miR-29C, -93, -150 and-181 b expression (FIG. 26C and FIG. 27). Collectively, these data suggest that different mirnas are regulated during cardiac hypertrophy, suggesting that they may function as modulators of this process.

The miR-29 family acts as a downstream target for miR-208 regulation. The inventors performed miRNA microarrays on hearts from wild-type and miR-208-depleted mice to graphically identify downstream mirnas that might mediate miR-208 action (fig. 28). They found that multiple members of the miR-29 family were up-regulated in miR-208-deficient mice (FIG. 29). Target prediction suggests that miR-29 family members target mRNA encoding various collagenases and other extracellular matrix components (fig. 30). Thus, upregulation of miR-29 family members in miR-208-deficient mice is likely responsible for the blockade of fibrosis found in these animals (fig. 31).

And (6) summarizing. The findings that miR-29 is down-regulated in diseased heart and targets coding for collagenases and extracellular matrix proteins suggest that strategies to enhance expression of miR-29 or enhance its binding to target mRNA are likely to produce beneficial effects on the heart in the context of pathological cardiac remodeling and fibrosis. In addition, an increase in miR-29 expression or function has the potential to prevent fibrosis associated with many diseases in tissues such as liver, lung, kidney, etc. In addition, the fact that miR-28 represses miR-29 expression and miR-208 upregulates miR-29 expression indicates that miR-29 is a downstream mediator of the action of miR-208 on the heart.

Example 3 discussion

These results demonstrate that miR-208, encoded by introns of the alpha MHC gene, can regulate stress-dependent cardiomyocyte growth and gene expression. In the absence of miR-208, expression of β MHC is severely blunted in adult hearts in response to stress overload, calcium dependent phosphatase activation, or hypothyroidism, etc., suggesting that these stimuli share a common miR-208 sensitive component for the inducible pathway of β MHC transcription (FIG. 9). In contrast, beta MHC is expressed in nascent miR-208^-/-No changes in the heart of the mice demonstrated that miR-208 is specifically involved in a stress-dependent β MHC expression regulatory mechanism.

One clue of miR-208 action mechanism comes from miR-208^-/-The similarity of the heart to hyperthyroid hearts, both of which exhibit blockade of β MHC expression, upregulation of stress responsive genes (Wei et al, 2005; Pantos et al, 2006), and protection against pathological hypertrophy and fibrosis (Yao and Eghbali, 1992; Chen et al, 2000). miR-208^-/-Upregulation of fast skeletal muscle genes in the heart is also analogous to the induction of fast skeletal muscle fibers in hyperthyroidism states (Vadaszova et al, 2004). T3 signaling suppresses beta MHC expression in postnatal heart, while PTU, which causes hypothyroidism, induces beta MHC (Morkin, 2000; Schuyler and Yarbrough, 1990). PTU can not be in miR-208^-/-Induction of beta MHC expression in the heart further suggests that miR-208 is involved in the T3 signaling pathway.

These results suggest that miR-208 functions at least in part by suppressing the expression of the TR co-regulator THRAP1, and that THRAP1 can exert both positive and negative effects on transcription (Pavri et al, 2005; Park et al),2005). TR acts through a negative TRE to suppress β MHC expression in adult hearts (Morkin, 2000). Therefore, the increase of THRAP1 expression in the absence of miR-208 is expected to enhance the inhibitory activity of TR on beta MHC expression, and the miR-208^-/-Blockade of β MHC expression in the heart is in concert. In contrast, regulation of α and β MHC expression during development is independent of T3 signaling (Morkin, 2000) and is not affected by miR-208. Notably, other TR target genes, such as Phospholamban (PLB) and sarcoplasmic (endoplasmic reticulum calcium ATPase (SERCA)2a and glucose transporter (GLUT)4, are at miR-208^-/-Normal expression in mice (fig. 20). It has been suggested that the β MHC gene may respond to specific TR isoforms (Kinugawa et al, 2001; Mansen et al, 2001; Kinugawa et al, 2001). It is possible that thap 1 acts on a particular TR isoform or selectively on a subset of TR-dependent genes via interaction with promoter-specific factors. Since mirnas generally exert their effects through a variety of downstream targets, it is also possible that other targets contribute to the effects of miR-208 on cardiac growth and gene expression.

A relatively slight increase in β MHC composition, as occurs during cardiac hypertrophy and heart failure, can decrease myofibrillar atpase activity and contractile function (Abraham et al, 2002). As such, therapeutic manipulation of miR-208 expression or interaction with its mRNA target may be able to enhance cardiac function by suppressing β MHC expression. Based on the prominent effect of miR-208 on cardiac stress response and the modulation of numerous mirnas in diseased hearts (van Rooij et al, 2006), the inventors expect that mirnas would prove to be key regulators of function and response to disease in adult hearts and possibly other organs.

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, in the steps and in the sequence of steps of the method 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 (9)

1. Use of an inhibitor of expression or activity of miR-208 for the manufacture of a medicament for treating or preventing pathologic cardiac hypertrophy, myocardial infarction, or heart failure in a subject in need thereof, wherein said inhibitor of expression or activity of miR-208 is encoded by a sequence that differs from SEQ ID NO: 5, and a sequence complementary to the sequence of 5.
2. 2. The use of claim 1, wherein said inhibitor of expression or activity of miR-208 is an antagomir or a 2' -O-methyl antisense oligonucleotide.
3. 3. The use of claim 1, wherein the medicament is formulated for intravenous administration or direct injection into cardiac tissue.
4. 4. The use of claim 1, wherein the medicament is formulated for oral, transdermal, sustained release, controlled release, delayed release, suppository, or sublingual administration.
5. 5. The use of claim 1, wherein the medicament further comprises a second cardiac hypertrophy medicament.
6. 6. The use of claim 5, wherein the second cardiac hypertrophy drug is a beta blocker, a inotropic, a diuretic, an ACE-I, AII antagonist, BNP, Ca⁺⁺-a blocker, an endothelin receptor antagonist, or an HDAC inhibitor.
7. 7. The use of claim 1, wherein the medicament ameliorates one or more symptoms of pathologic cardiac hypertrophy or heart failure.
8. 8. The use of claim 1, wherein the medicament delays the transition from cardiac hypertrophy to heart failure.
9. 9. The use of claim 1, wherein the subject has one or more risk factors selected from the group consisting of: long-term uncontrolled hypertension, uncorrected valvular disease, chronic angina, recent myocardial infarction, congenital predisposition to heart disease, or cardiogenic hypertrophy.