[go: up one dir, main page]

WO2016058061A1 - Signatures de microarn pour troubles cardiaques - Google Patents

Signatures de microarn pour troubles cardiaques Download PDF

Info

Publication number
WO2016058061A1
WO2016058061A1 PCT/BE2015/000050 BE2015000050W WO2016058061A1 WO 2016058061 A1 WO2016058061 A1 WO 2016058061A1 BE 2015000050 W BE2015000050 W BE 2015000050W WO 2016058061 A1 WO2016058061 A1 WO 2016058061A1
Authority
WO
WIPO (PCT)
Prior art keywords
mir
patient
heart failure
biological sample
expression
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/BE2015/000050
Other languages
English (en)
Inventor
Stefan Janssens
Melissa SWINNEN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Katholieke Universiteit Leuven
Original Assignee
Katholieke Universiteit Leuven
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Katholieke Universiteit Leuven filed Critical Katholieke Universiteit Leuven
Publication of WO2016058061A1 publication Critical patent/WO2016058061A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/113—Antisense targeting other non-coding nucleic acids, e.g. antagomirs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • C12N2310/141—MicroRNAs, miRNAs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/10—Applications; Uses in screening processes
    • C12N2320/11—Applications; Uses in screening processes for the determination of target sites, i.e. of active nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • the invention relates to diagnosis, of cardiac disorders such as (chronic) heart failure, a physiological state in which cardiac output is insufficient to meet the needs of the body and lungs, in order to prevent and on-time treatment.
  • Changes in the expression of specific MicroRNAs are indicative of a transition from cardiac remodeling and cardiac hypertrophy to heart failure, a panel of microRNA biomarkers may be used to predict the likelihood of (chronic) heart failure.
  • novel methods of and compositions for inhibiting or silencing specific miRNAs for instance for antagomir-specific targeting of miR-764-3p or combined miR-764-3p and miR-130b) for treating those patients whose condition is progressing towards heart failure for instance such patients that have developed cardiac hypertrophy or are progressing towards cardiac hypertrophy.
  • Cardiovascular disease is the leading cause of morbidity and mortality worldwide and the incidence of chronic heart failure (HF) is increasing
  • HF chronic heart failure
  • state-of-the-art therapies emphasizes the need for a better understanding of the underlying key pathogenic events in the evolution of cardiovascular disease, in order to design tests for patients at risk of heart failure as well as next-generation treatments for heart failure.
  • Cardiac hypertrophy is an important cause of cardiovascular disease and can be described as an increase in cardiac mass.
  • an increase in load e.g. pressure overload induced by hypertension
  • the heart In response to an increase in load (e.g. pressure overload induced by hypertension), the heart must work harder under normal conditions. To cope with the elevated workload, the heart undergoes hypertrophic enlargement to normalize wall stress and permit normal cardiac function. This compensatory mechanism leads to an increase in size and mass. The increase in cardiac mass is largely due to an increase in ventricular weight. If the chronic increase in wall stress is not relieved, the hypertrophied heart can dilate, contractile function reduces and this could lead towards heart failure. (Levy, Larson et al. 1996, Kehat and Molkentin 2010) Heart failure is associated with significantly reduced physical and mental health, and there is no definitive cure.
  • the primary treatment options focus on improving symptoms and preventing progression of the disease. It is important for clinicians to identify the cases of cardiac hypertrophy which are progressing or which will progress towards heart failure.
  • Current methods for diagnosing heart failure are based on clinical history and examination, imaging, electrophysiology, blood tests, and angiography, but these methods lack the sensitivity to detect the transitional stages between cardiac hypertrophy and heart failure. Indeed, there remains a need in the art for tests that will indicate whether a patient with cardiac hypertrophy is likely to progress to HF and a need in the art for treatments that reverse, slow, or stop the development of heart failure.
  • miR microRNA
  • One aspect of the present disclosure relates to a method for identifying a patient at risk for developing heart failure, comprising (a) obtaining a biological sample from the patient; (b) measuring expression of miR-764-3p in the biological sample; and (c) comparing the expression of miR-764-3p in the biological sample with a reference sample; wherein an increase in expression of miR-764-3p in the biological sample as compared to the reference sample indicates that the patient is at risk for developing heart failure.
  • a further aspect of the present disclosure relates to a method for identifying a patient at risk for heart failure, comprising (a) obtaining a biological sample from the patient; (b) measuring expression of miR- 764-3p and miR-130b in the biological sample; and (c) comparing the expression of miR-764-3p and miR-130b in the biological sample with a reference sample, wherein an increase in expression of miR- 764-3p and miR-130b in the biological sample as compared to the reference sample indicates that the patient is at risk for developing heart failure.
  • Another aspect of the present disclosure relates to a method for identifying a patient at risk for developing heart failure, comprising (a) obtaining a biological sample from the patient; (b) measuring expression of miR-764-3p, miR-130b, miR-218, miR-9-3p, miR-322-3p, and miR-210 in the biological sample; and (c) comparing the expression of miR-764-3p, miR-130b, miR-218, miR-9-3p, miR-322-3p, and miR-210 in the biological sample with a reference sample, wherein an increase in expression of miR-764-3p and miR-130b and a decrease in expression of miR-218, miR-9-3p, miR-322-3p, and miR- 210 in the biological sample as compared to the reference sample indicates that the patient is at risk for developing heart failure.
  • the change in expression of miR As is a 2-fold change in expression.
  • the patient is identified as likely to develop heart failure.
  • the reference sample is biological sample from a healthy control subject.
  • the control subject may be age-matched, and may not have any heart disorders such as ventricular dysfunction (left ventricular (LV) dysfunction and/or right ventricular (RV) dysfunction).
  • the patient suffers from valvular heart disease, aortic stenosis (AS), aortic insufficiency, mitral stenosis or insufficiency, systemic or pulmonary hypertension, any form of dilated cardiomyopathy caused by ischemia, pressure- or volume overload.
  • the patient has cardiac hypertrophy, for example, left ventricular hypertrophy.
  • the patient has right ventricular hypertrophy.
  • the patient has at least one of dilation of one or more cardiac chambers, increased left and/or right ventricular diameter, and depressed systolic function.
  • the patient is a candidate for aortic or mitral valve replacement.
  • the patient has undergone aortic or mitral valve replacement.
  • the biological sample is a blood sample.
  • the biological sample is a tissue sample, such as a heart tissue sample.
  • the heart tissue sample may be a biopsy of heart tissue, such as a left ventricle biopsy. In some embodiments, the biopsy is a right ventricle biopsy.
  • the results of the methods described herein indicate that the patient has ventricular dysfunction and is at risk for developing heart failure.
  • the ventricular dysfunction is LV dysfunction.
  • the ventricular dysfunction is RV dysfunction.
  • the ventricular dysfunction is a combination of LV and RV dysfunction.
  • a particular embodiment of present invention concerns a pharmaceutical composition for use in a treatment of heart failure comprising an inhibitor molecule for inhibiting the activity of miR-764-3p or miR-764-3p and miR-130b-3p or a this pharmaceutical composition comprising a miR-764-3p inhibitor molecule or comprises molecules or a molecule that inhibit miR-764-3p and miR-130b-3p for use in a treatment of subgroup of cardiac disorder (CD) patients at risk for heart failure or this pharmaceutical composition comprising a miR-764-3p inhibitor molecule for use in a treatment of a patient at risk of heart failure by a miR-764-3p induced cardiac disorder.
  • CD subgroup of cardiac disorder
  • This pharmaceutical composition b be delivered to a patient for inhibiting the activity of miR-764-3p or miR-764-3p and miR-130b-3p into a cell.
  • inhibitor molecule that is a nucleic acid, in particular an oligonucleotide.
  • the nucleic acid molecule is perfectly complementary to miR-130b-3p or miR-764-3p and comprises a mispairing base at a cleavage site of Ago2 or a base modification for inhibiting cleavage by Ago2.
  • the nucleic acid molecule can be selected from the group consisting of antisense DNA-oligonucleotides, RNA-oligonucleotides, antisense DNA- RNA-oligonucleotides antisense 2'-0-methyl oligoribonucleotides, antisense oligonucleotides comprising a phosphorothiaote linkage, antisense oligonucleotides comprising a Locked Nucleic Acid base, morpholino antisense oligonucleotides, and antagomirs.
  • the cell inhibiting the activity of miR- 764-3p or miR-764-3p and miR-130b-3p is a mammalian cell, in particular a human cell, in particular a human heart cell.
  • a pharmaceutically for delivering the inhibitor to the patients can further comprise an acceptable diluent, carrier or adjuvant.
  • AS aortic stenosis
  • mitral stenosis or insufficiency aortic insufficiency
  • systemic or pulmonary hypertension any form of dilated cardiomyopathy caused by ischemia, pressure- or volume overload.
  • Treatment of heart failure by inhibiting by an inhibitor of miR-764-3p or of miR-764-3p and miR- 130b-3p or a pharmaceutical composition comprising this can be for cardiac hypertrophy, for instance a cardiac hypertrophy which is ventricular hypertrophy of the group consisting of left ventricular hypertrophy and right ventricular hypertrophy.
  • Treatment of heart failure by inhibiting by an inhibitor of miR-764-3p or of miR-764-3p and miR-130b-3p or a pharmaceutical composition comprising this can be for patients having dilation of one or more cardiac chambers, increased left and/or right ventricular diameter, and/or depressed systolic function, patient that are candidate for aortic or mitral valve replacement or patients that undergone aortic or mitral valve replacement.
  • FIG. 1 shows phenotypical characterization of different models of cardiac hypertrophy. Different timings of pressure overload have been studied: 2 wks , 4 wks and 10 wks after aortic banding (TAC) all compared to sham (SHAM). Additionally, angiotensin II (ANGII)-induced hypertrophy was studied for 4 wks (ANGII) compared with control treatment (SAL).
  • TAC aortic banding
  • SHAM sham
  • ANGII angiotensin II-induced hypertrophy was studied for 4 wks (ANGII) compared with control treatment (SAL).
  • Fig. 1A left ventricular end diastolic dimensions (LVEDD) were significantly increased after lOwks TAC compared with sham and compared with 2wks TAC, 4wks TAC and Angll-infusions.
  • Fig. IB left ventricular end systolic dimensions (LVESD) were significantly increased at lOwks TAC compared to sham treated animals.
  • Fig. 1C shows that
  • Fractional Shortening was significantly lower at lOwks TAC compared to sham treated animals.
  • FIG. 2 shows miR signatures in the hypertrophic heart.
  • Fig. 2A-2B miR arrays were performed on murine hearts at 2wks, 4 wks and lOwks after aortic banding (TAC) and at 4 weeks after angiotensin ll-infusions (ANGII), all compared to sham.
  • Fig. 2A shows up-regulated miRs.
  • miR-208b and miR-144 are two miRs commonly up-regulated in all 4 models whereas three miRs were uniquely up- regulated at lOwks TAC and not in the other models. Two of these three miRs have a common target gene that they can regulate.
  • Delta sarcoglycan is negatively regulated by miR-764-3p and miR-130b-3p.
  • Fig. 2B shows down-regulated miRs. Nine miRs were uniquely down-regulated at lOwks TAC and not in the other models. Four of these nine miRs have a common target gene that they can influence.
  • VAMP7 is negatively regulated by miR-218, miR-322-3p, miR-210 and miR-9-3p.
  • Figure 3 shows delta sarcoglycan (DSGC) in the dilated heart. In Fig.
  • a luciferase assay confirmed in vitro the negative regulation of miR-130b, miR-764-3p on DSGC while control miRs did not have an influence.
  • An empty plasmid (empty) and mutation in the miR-binding sites (DSGC MUT) were also applied.
  • quantitative RT-PC showed decreased DSGC expression after lOwks TAC.
  • Fig. 3C Immunohistological staining showed decreased DSGC levels after lOwks TAC.
  • Fig. 3D immunoblotting for DSGC showed decreased levels of DSGC at lOwks TAC compared to SHAM.
  • 3E and 3H shows immunohistological analysis for DSGC in lOwks SHAM and lOwks TAC.
  • Figs. 3F and 31 shows vascular Smooth muscle cell (VSMC) staining in lOwks SHAM and lOwks TAC.
  • Figs. 3G and 3J shows co-localization of DSGC and VSMC in lOwks SHAM and lOwks TAC.
  • Figure 4 shows phenotypical characterization with antagomiR treatment.
  • AntagomiR treatment to inhibit up-regulation of miR-764-3p or miR-130b-3p was administered and animals were studied for lOwks after aortic banding (TAC).
  • TAC aortic banding
  • PBS CTR
  • SCR scrambled miRs
  • Figs. 4A- 4C show echocardiographic analyses that have been performed in all animals.
  • LVIDd left ventricular end diastolic dimensions
  • LVIDs left ventricular end systolic dimensions
  • Fig. 4C Fractional Shortening (FS%) was significantly higher at lOwks TAC after antagomiR treatment compared to CTR or SCR administration.
  • Figs. 4D-4H show DSGC levels as determined by immunohistochemistry.
  • Fig. 4D DSGC was significantly increased after antagomiR treatment compared to CTR or SCR administration.
  • Figs. 4E-4H show immunohistological pictures representative for DSGC levels in TAC animals after CTR- (Fig. 4E), SCR-(Fig. 4F), antagomiR-764-3p- (Fig. 4G) and antagomiR-130b-3p-administration (Fig. 4H).
  • FIG. 5 shows VAMP7 in the dilated heart.
  • a luciferase assay confirmed in vitro the negative regulation of miR-218, miR-9-3p, miR-322-3p and miR-210 while control miRs did not have an effect. An empty plasmid was also tested.
  • RT-PCR Quantitative Real-Time PCR
  • Fig. 5C shows immunohistological analysis of VAMP7 in lOwks SHAM and lOwks TAC.
  • Fig. 5D shows immunoblotting for VAMP7 in lOwks SHAM and lOwks TAC.
  • Figs. 5E-5F show
  • FIG. 5G-5H shows downstream targets of VAMP7.
  • RT-PCR for MT1-MMP showed increased levels at lOwks TAC.
  • MMP2 and MMP9 zymographic activity was significantly increased at lOwks TAC compared to lOwks SHAM.
  • Figure 6 shows confirmation of murine miR signatures in chronic pressure overload in patients with severe aortic stenosis.
  • Figs. 6A-6B show up-regulated miR signatures.
  • Fig. 6A shows relative expression levels of miR-764-3p and miR-130b-3p in murine hearts at lOwks SHAM and lOwks TAC.
  • Fig. 6B shows relative expression levels of miR-764-3p and miR-130b in hearts of healthy individuals
  • Figs. 6C-6D show down-regulated miR signatures.
  • Fig. 6C shows relative expression levels of miR-218, miR-9, miR-322-3p and miR-210 in murine hearts at lOwks SHAM and lOwks TAC.
  • Fig. 6D shows relative expression levels of miR-218, miR-9, miR-322-3p and miR-210 in hearts of healthy individuals (CTR) and patients with severe aortic stenosis (AS).
  • Figure 7 shows an upregulation of both miR 764-3p and miR-130b in the plasma of aortic stenosis patients compared with healthy individuals.
  • the present disclosure relates to methods for identifying patients at risk for developing heart failure and methods for treating those patients whose condition is progressing towards heart failure or who have developed heart failure and it relates to a proper treatment of such patients.
  • the disclosure is based on observations of molecular changes occurring in hearts which are progressing towards heart failure. In particular, changes in the expression of micro As mark the transition towards heart failure.
  • Hypertrophy is generally defined as an increase in size of an organ or structure independent of natural growth that does not involve tumor formation. Hypertrophy of an organ or tissue is due either to an increase in the mass of the individual cells (true hypertrophy), or to an increase in the number of cells making up the tissue (hyperplasia), or both.
  • Cardiac hypertrophy is the enlargement of heart that is activated by both mechanical and hormonal stimuli and enables the heart to adapt to demands for increased cardiac output or to injury. This response is frequently associated with a variety of distinct pathological conditions, such as hypertension, aortic stenosis, myocardial infarction, cardiomyopathy, valvular regurgitation, cardiac shunt, congestive heart failure, etc.
  • mmu- miR-764-3p or MIMAT0003895 has seq.
  • ID1 AGGAGGCCAUAGUGGCAACUGU and hsa-mir-130b or MI0000748 has seq.
  • ID2 GGCCUGCCCGACACUCUUUCCCUGUUGCACUACUAUAGGCCG
  • non-myocytes are primarily fibroblast/mesenchymal cells, they also include endothelial and smooth muscle cells. Indeed, although myocytes make up most of the adult myocardial mass, they represent only about 30% of the total cell numbers present in heart. The enlargement of embryonic heart is largely dependent on an increase in myocyte number, which continues until shortly after birth, when cardiac myocytes lose their proliferative capacity. Further growth occurs through hypertrophy of the individual cells.
  • Hypertrophy of adult cardiac ventricular myocytes is a response to a variety of conditions which lead to chronic hemodynamic overload.
  • adult ventricular muscle cells can adapt to increased workloads through the activation of a hypertrophic process.
  • This response is characterized by an increase in myocyte cell size and contractile protein content of individual cardiac muscle cells, without concomitant cell division and activation of embryonic genes, including the gene for atrial natriuretic peptide (ANP)..
  • APP atrial natriuretic peptide
  • An increment in myocardial mass as a result of an increase in myocyte size that is associated with an accumulation of interstitial collagen within the extracellular matrix and around intramyocardial coronary arteries has been described in left ventricular hypertrophy secondary to pressure overload in humans. Cardiac hypertrophy due to chronic hemodynamic overload is the common end result of most cardiac disorders and a consistent feature of cardiac failure.
  • Cardiac Hypertrophy may involve medication or surgery.
  • the treatment of cardiac hypertrophy varies depending on the underlying cardiac disease.
  • Catecholamines, adrenocorticosteroids, angiotensin, prostaglandins, leukemia inhibitory factor (LIF), endothelin (including endothelin-1, -2, and -3 and big endothelin), cardiotrophin-1 (CT-1), cardiac hypertrophy factor (CHF) and blood pressure medication are among the factors identified as potential mediators of hypertrophy.
  • Cardiac remodeling involves molecular, cellular and interstitial modifications that manifest clinically as changes in size, shape and function of the heart.
  • Cardiac hypertrophy characterized by increased muscle mass in the heart, is a common type of cardiac remodeling that occurs when the heart sustains elevated workload.
  • pathological cardiac hypertrophy comprises cellular and molecular remodeling such as myocyte growth without significant proliferation, re- expression of fetal genes, alterations in the expression of structural proteins involved in excitation- contraction coupling, and changes in the energetic and metabolic state of the myocyte.
  • ECM extracellular matrix
  • Ventricular dysfunction can be either systolic, diastolic, or both combined.
  • Ventricular dysfuntion may be diagnosed by using a structured pathway of history, examination, and diagnostic testing with brain natriuretic peptide (BNP), electrocardiogram (ECG), ergospirometry, and echocardiography or other cardiac imaging modalities including cardiac magnetic resonance, multi-slice computer tomography or nuclear imaging.
  • BNP brain natriuretic peptide
  • ECG electrocardiogram
  • ergospirometry ergospirometry
  • echocardiography or other cardiac imaging modalities including cardiac magnetic resonance, multi-slice computer tomography or nuclear imaging.
  • cardiac output is insufficient to meet the needs of the body and lungs.
  • CHF congestive heart failure
  • CCF congestive cardiac failure
  • the heart attempts to compensate for decreased cardiac output by increasing its muscle mass, pumping blood faster, narrowing blood vessels, and diverting blood flow. Over time, these compensatory mechanisms stress the cardiovascular system and can lead to heart rhythm disorders, strokes or other blood vessel blockages, and failure of entire organ systems receiving inadequate blood supply.
  • cardiac remodeling and, in turn, heart failure may be caused by any condition which reduces the efficiency of the myocardium (heart muscle) through damage or overloading
  • many pathological conditions put the heart at risk for these conditions. Common causes include coronary heart disease, previous heart attack, high blood pressure, and cardiomyopathy. Other conditions which increases risk for heart failure include diabetes, obesity, lung disease, stress, sedentary lifestyle, smoking, hyperlipidemia and more.
  • Heart failure can further be described as chronic, congestive, acute, decompensated, systolic or diastolic.
  • the New York Heart Association (NYHA) classification describes the severity of the disease based on functional capacity of the patient; NYHA class can progress and/or regress based on treatment or lack of response to treatment.
  • in heart failure in heart failure, "increased severity" of cardiovascular disease refers to the worsening of disease as indicated by increased NYHA classification, to, for example, Class III or Class IV, and “reduced severity” of cardiovascular disease refers to an improvement of the disease as indicated by reduced NYHA classification, from, for example, class III or IV to class II or I.
  • Heart failure is diagnosed by examining clinical signs and symptoms, and by measuring indicators of decreased heart function, for example, by imaging the heart, measuring cardiac performance, coronary perfusion (angiography), intracoronary measurements, pressure-flow measurements, and evaluating myocardial structure through biopsy. Such tests can reveal cardiac remodeling and reduced cardiac output.
  • none of the tests or classifications can provide conclusive prognostic details of the likelihood that a patient's condition will deteriorate to develop heart failure. For example, there is currently no test to predict whether cardiac hypertrophy (for example, left ventricular hypertrophy) will progress to heart failure. Similarly, there is no test to determine whether a patient with LV dysfunction will develop heart failure. And there is currently no test to determine whether a patient who undergoes cardiac valve replacement will later suffer from heart failure.
  • one aspect of the present disclosure relates to a method for identifying a patient at risk for developing heart failure and/or for identifying a patient who will develop heart failure.
  • the method may also be used to identify LV dysfunction in a patient.
  • the method is used in patients who are already diagnosed with LV dysfunction, in order to determine whether these patients are at risk for developing heart failure.
  • the method comprises (a) obtaining a biological sample from the patient, (b) measuring expression one or more miRs in the biological sample, and (c) comparing the expression of the one or more miRs in the biological sample with a reference sample, wherein a change in the expression of the one or more miRs in the biological sample as compared to the reference sample indicates that a patient is at risk for developing heart failure.
  • the change in expression of the one or more miRs in the biological sample as compared with the reference sample may also indicate that a patient will develop heart failure.
  • the patient may have already been diagnosed with ventricular dysfunction, for example LV dysfunction.
  • the patient has already been diagnosed with LV dysfunction, RV dysfunction, or a combination of both.
  • the change in expression of one or more miRs in the biological sample as compared with the reference sample indicates that a patient has ventricular dysfunction, for example LV dysfunction.
  • the ventricular dysfunction is LV dysfunction, RV dysfunction, or both.
  • a patient may suffer from other conditions which are likely to lead to heart failure, so that the patient is at risk for heart failure.
  • the patient suffers from cardiac hypertrophy.
  • the cardiac hypertrophy may exist alone or may have further progressed to dilated cardiomyopathy.
  • the patient has aortic stenosis.
  • the patient may suffer from one or more of valvular heart disease, including aortic stenosis (AS), aortic insufficiency, mitral stenosis or insufficiency, tricuspid stenosis or insufficiency and systemic or pulmonary hypertension, and any form of dilated cardiomyopathy caused by ischemia, pressure- or volume overload.
  • the patient has not yet developed heart failure.
  • the patient has LV dysfunction.
  • LV dysfunction may be diastolic, systolic, or a combination of both. LV dysfunction may also be asymptomatic.
  • RNA refers to a molecule comprising at least one or more ribonucleotide residues.
  • a "ribonucleotide” is a nucleotide with a hydroxyl group at the 2' position of a beta-D- ribofuranose moiety.
  • the term RNA includes doublestranded RNA, singlestranded RNA, isolated RNA, such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly-produced RNA, as well as altered RNA that differs from naturally-occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Nucleotides of the RNA molecules can also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides.
  • a “mature microRNA” typically refers to a single-stranded RNA molecules of about 21-23 nucleotides in length, which regulates gene expression. miRNAs are encoded by genes from whose DNA they are transcribed, but miRNAs are not translated into protein; instead each primary transcript (pri-miRNA) is processed into a short stem-loop structure (precursor microRNA) before undergoing further processing into a functional mature miRNA. Mature miRNA molecules are partially complementary to one or more messenger RNA (mRNA) molecules, and their main function is to down-regulate gene expression. As used throughout, the term “microRNA” or “miRNA” includes both mature microRNA and precursor microRNA.
  • increase is meant an observable, detectable, or significant increase in a level as compared to a reference level or a level measured at an earlier or later time point in the same subject.
  • decrease is meant an observable, detectable, or significant decrease in a level as compared to a reference level or a level measured at an earlier or later time point in the same subject.
  • inhibitory RNA is meant a nucleic acid molecule that contains a sequence that is complementary to a target nucleic acid (e.g., a target microRNA).
  • target nucleic acid e.g., a target microRNA
  • inhibitory RNAs include interfering RNA, shRNA, siRNA, ribozymes, antagomiRs, and antisense oligonucleotides. Methods of making inhibitory RNAs are described herein. Additional methods of making inhibitory RNAs are known in the art.
  • an interfering RNA refers to any double stranded or single stranded RNA sequence, capable- either directly or indirectly (i.e., upon conversion)- of inhibiting or down regulating gene expression by mediating RNA interference.
  • Interfering RNA includes but is not limited to small interfering RNA ("siRNA”) and small hairpin RNA (“shRNA”).
  • siRNA small interfering RNA
  • shRNA small hairpin RNA
  • RNA interference refers to the selective degradation of a sequence-compatible messenger RNA transcript.
  • a shRNA small hairpin RNA refers to an RNA molecule comprising an antisense region, a loop portion and a sense region, wherein the sense region has complementary nucleotides that base pair with the antisense region to form a duplex stem.
  • the small hairpin RNA is converted into a small interfering RNA by a cleavage event mediated by the enzyme Dicer, which is a member of the RNase III family.
  • small interfering RNA or “siRNA” as used herein refers to any small RNA molecule capable of inhibiting or down regulating gene expression by mediating RNA interference in a sequence specific manner.
  • the small RNA can be, for example, about 18 to 21 nucleotides long.
  • an “antagomiR” refers to a small synthetic RNA having complementarity to a specific microRNA target, optionally with either mispairing at the cleavage site or one or more base modifications to inhibit cleavage.
  • risk of developing heart failure is meant the relative probability that a subject will develop a heart failure in the future as compared to a control subject or population (e.g., a healthy subject or population).
  • treating includes reducing risk of developing heart failure in a subject by delaying the onset of symptoms of cardiac disorders such as cardiac hypertrophy for instance ventricular hypertrophy (left ventricular hypertrophy and or right ventricular hypertrophy or of valvular heart disease, aortic stenosis (AS), aortic insufficiency, mitral stenosis or insufficiency, systemic or cardiac hypertrophy and preventing or decreasing the risk of developing heart failure or any form of dilated cardiomyopathy caused by ischemia, pressure- or volume overload that leads to heart failure. It also increasing the longevity of a subject having any one of these cardiac disorders.
  • cardiac hypertrophy for instance ventricular hypertrophy (left ventricular hypertrophy and or right ventricular hypertrophy or of valvular heart disease, aortic stenosis (AS), aortic insufficiency, mitral stenosis or insufficiency, systemic or cardiac hypertrophy and preventing or decreasing the risk of developing heart failure or any form
  • novel methods of and compositions for inhibiting or silencing specific miRNAs for instance for antagomiR-specific targeting of miR-764-3p or combined miR-764-3p and miR-130b) for treating those patients whose condition is progressing towards heart failure for instance such patients that have developed cardiac hypertrophy or are progressing towards cardiac hypertrophy.
  • one aspect of the present disclosure relates to an inhibitor of miR-130b-3p or an inhibitor of miR-764-3p (or a combination of inhibitors to both miR- 130-3p and miR-764-3p) for use in treating and/or preventing heart failure.
  • oligonucleotides which bind a miR may be used to inhibit miRs.
  • antagonistmiRs oligonucleotides which bind an mRNA sequence to which the miR binds
  • the inhibition of the activity of miR-130-3p and/or miR-764-3p can be achieved using a molecule chosen from the group consisting of antisense DNA-oligonucleotides, antisense RNA-oligonucleotides, antisense DNA-RNA-oligonucleotides, an antisense 2'-0-methyl oligoribonucleotides, antisense oligonucleotides containing phosphorothiaote linkages, antisense oligonucleotides containing Locked Nucleic Acid LNA (Registered trademark bases, morpholino antisense oligos, antagomiRs, and mixtures thereof.
  • inhibitor molecules are antisense oligonucleotides which are chemically modified to improve the thermal stability of the duplex between the antisense oligonucleotide and the miR, such as LNA (Registered trademark-antimiRs.
  • Other preferred chemical modified oligonucleotides include morpholinos, 2'-0-methyl, 2'-0-methoxyethyl oligonucleotides and cholesterol-conjugated 2'-0-methyl modified oligonucleotides (antagomiRs). Combinations of the above modifications can also occur in the inhibitor molecule.
  • LNA (Registered trademark is a modified RNA nucleotide.
  • the ribose moiety of an LNA nucleotide is modified with an extra bridge connecting the 2' oxygen and 4' carbon. This bridge locks the ribose in the 3'-endo conformation.
  • LNA nucleotides can be mixed with DNA or RNA bases in the oligonucleotide whenever desired.
  • Morpholino oligonucleotides comprise bases that are bound to morpholine rings instead of deoxyribose rings and linked through phosphorodiamidate groups instead of phosphates.
  • the inhibitor molecule is a nucleic acid, it is preferably an RNA.
  • the RNA may or may not comprise chemical modifications, e.g. a 2' O-methyl group, as described above and herein.
  • the inhibitory nucleic acids preferably have a length of 8 to 24 nucleotides, preferably of 15 to 22 nucleotides.
  • RNA molecules further comprising chemical modifications, namely: 3' cholesterol, 2' O-methyl, four consecutive phosphothiolate groups from the 3' side of the molecule, and two consecutive phosphothiolate groups from the 3' side of the molecule (according to Kriitzfeldt et al., Nature, 2005; 438: 685-689.).
  • a particular embodiment of present invention concerns a method for identifying a patient at risk for developing heart failure, comprising obtaining a biological sample from the patient, measuring expression of miR-764-3p in the biological sample; and comparing the expression of miR-764-3p in the biological sample with a reference sample; wherein an increase in expression of miR-764-3p in the biological sample as compared to the reference sample indicates that the patient is at risk for developing heart failure.
  • a method for identifying a patient at risk for developing heart failure comprising obtaining a biological sample from the patient, measuring expression of miR-130b in the biological sample; and comparing the expression of miR-130b in the biological sample with a reference sample; wherein an increase in expression of miR-130b in the biological sample as compared to the reference sample indicates that the patient is at risk for developing heart failure.
  • Patient at risk for developing heart failure can also been identified by a method, comprising obtaining a biological sample from the patient; measuring expression ot miR- 764-3p and miR-130b in the biological sample; and comparing the expression of miR-764-3p and miR- 130b in the biological sample with a reference sample, wherein an increase in expression of miR-764- 3p and miR-130b in the biological sample as compared to the reference sample indicates that the patient is at risk for developing heart failure.
  • Measuring expression of miR-764-3p and/or miR-130b herein may be combined with measuring expression of any one of miR-218, miR-9-3p, miR-322-3p, and miR-210 in the biological sample and comparing such expression of miR-764-3p, miR-130b, miR- 218, miR-9-3p, miR-322-3p, and miR-210 in the biological sample with a reference sample wherein an increase in expression of miR-764-3p and miR-130b and a decrease in expression of miR-218, miR-9- 3p, miR-322-3p, and miR-210 in the biological sample as compared to the reference sample indicates that the patient has is at risk for developing heart failure.
  • valvular heart disease aortic stenosis (AS), aortic insufficiency, mitral stenosis or insufficiency, systemic or cardiac hypertrophy and preventing or decreasing the risk of developing heart failure, any form of dilated cardiomyopathy caused by ischemia, pressure- or volume overload
  • ventricular hypertrophy such as left ventricular hypertrophy or right ventricular hypertrophy.
  • tissue sample for instance a blood sample or a heart tissue sample.
  • the invention pertains to a pharmaceutical composition
  • a pharmaceutical composition comprising an inhibitor of miR-764-3p and/or miR-130b, as described above and herein
  • a particular embodiment of present invention concerns method of inhibiting or silencing specific miRNAs (for instance for antagomiR-specific targeting of miR-764-3p or combined miR-764-3p and miR-130b) for treating those patients whose condition is progressing towards heart failure, for instance indicated by the here above mentioned method, for instance such patients that have developed cardiac hypertrophy or are progressing towards cardiac hypertrophy.
  • inhibitory nucleic acid comprising a sequence that is complementary to a contiguous sequence present in miR-764-3p and/or miR-130b, e.g., a contiguous sequence of at least 5 nucleotides, for use in treating cardiac hypertrophy in a subject and/or preventing heart failure.
  • the inhibitory nucleic acid van be any one of the following an antagomiR, an antisense oligonucleotide or a ribozyme and it can be an inhibitory RNA of the group consisting of interfering RNA, shRNA, siRNA, ribozymes, antagomiRs, and antisense oligonucleotides.
  • a particular embodiment is a method of treating can comprise administering to a subject in need at least one antagomiR comprising a sequence that is complementary to a contiguous sequence, e.g., a contiguous sequence of at least 5 nucleotides, present in any one of miR-764-3p and/or miR-130b.
  • Yet another embodiment is a pharmaceutical composition
  • inhibitory RNA of any one of miR-764-3p and/or miR-130b which inhibitory RNA is of the group consisting of interfering RNA, shRNA, siRNA, ribozyme, antagomiR and antisense oligonucleotide to treat, wherein the inhibitory RNA is complementary to a contiguous sequence present in miR-764-3p and/or miR-130b, e.g., a contiguous sequence of at least 5 nucleotides for use in such treatment to prevent or decrease cardiac hypertrophy to prevent heart failure and/or chronic heart failure.
  • the sequence or the complementary sequence thereof of said inhibitory RNA may be contained in vector replicable in the patient.
  • Heart failure is in the medical classification ( World Health Organization (WHO) under the ICD- 10 Version:2015) described as Heart failure (150), where under 150.0 Congestive heart failure, Congestive heart disease and Right ventricular failure (secondary to left heart failure), 150.1 Left ventricular failure, Cardiac asthma Left heart failure, oedema of lung or Pulmonary oedema with mention of heart disease nitric oxide synthase (NOS) or heart failure and 150.9 Heart failure, unspecified and Cardiac, heart or myocardial failure NOS.
  • WHO World Health Organization
  • Chronic heart failure is in the medical classification (World Health Organization (WHO) under the ICD-10 Version: 2015) described as Chronic rheumatic heart diseases Chronic rheumatic heart diseases (105-109), Chronic ischaemic heart disease (125.1 - 125.9), Chronic ischaemic heart disease, unspecified (125.9), Chagas disease (chronic) with heart involvement (B57.2) and other forms of chronic ischaemic heart disease (125.8).
  • WHO World Health Organization
  • Cardiomegaly is in the medical classification ( World Health Organization (WHO) under the ICD-10 Version:2015) described as code 151.7 (Cardiomegaly) due to cardiac ventricular dilatation, cardiac dilatation or cardiac hypertrophy (the heart's muscular left ventricle being abnormally thick).
  • WHO World Health Organization
  • the pharmaceutical composition comprises an inhibitor molecule (antagonist) of
  • the invention pertains to a pharmaceutical composition comprising an inhibitor of miR-764-3p and/or miR-130b, as described above and herein activity that is selected from the group consisting of antisense DNA- and/or RNA-oligonucleotides, antisense 2'-0-methyl oligoribonucleotides, antisense oligonucleotides containing phosphorothiaote linkages, antisense oligonucleotides containing Locked Nucleic Acid (LNA (Registered trademark) bases, morpholino antisense oligos, antagomiRs, and mixtures thereof. Particularly preferred is the presence of an antagomiR of
  • the invention pertains to a pharmaceutical composition comprising an inhibitor of mi ' R-764-3p and/or miR-130b, as described above and herein.
  • the antagonist of miRNA- 17 comprises a sequence that is complementary to the mature sequence of
  • the invention pertains to a pharmaceutical composition comprising an inhibitor of miR-764-3p, as described above and herein, or combinations thereof.
  • the invention also pertains to the use of an inhibitor molecule, such as a nucleic acid, that inhibits the activity of
  • an inhibitor molecule such as a nucleic acid
  • the invention pertains to a pharmaceutical composition comprising an inhibitor of miR-764-3p or miR-130b, as described above and herein in a cell for manufacturing a pharmaceutical composition for treating cardiac hypertrophy and preventing or decreasing the risk of developing heart failure.
  • the invention pertains to a method for treating a patient suffering from cardiac hypertrophy and preventing or decreasing the risk of developing heart failure, comprising introducing a nucleic acid molecule that inhibits the activity miR-764-3p or miR-130b into a cell.
  • the patient is a mammal, in particular a human.
  • the cell in which the inhibitor molecule inhibits the activity of miR-764-3p or miR-130b is preferably a mammalian cell, most preferably a human cell, in particular a vascular cell of the lung.
  • the cell can be part of an organism, organ or tissue, or it can be a single cell, e.g. in a tissue culture.
  • the cell can be treated ex vivo or in vivo.
  • the pharmaceutical composition of the invention may additionally comprise a pharmaceutically acceptable carrier, diluent, and/or adjuvant.
  • Liquid dosage forms for oral administration of the compounds of the invention include pharmaceutically acceptable emulsions, micro-emulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • the oral compositions can also include adjuvants such as wetting agents,
  • Suspensions in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • Powders can contain, in addition to a compound of this invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances.
  • compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.
  • the present invention therefore includes pharmaceutical formulations comprising the nucleic acids described herein, including pharmaceutically acceptable salts thereof, in pharmaceutically acceptable carriers for aerosol, oral and parenteral administration. Also, the present invention includes such compounds, or salts thereof, which have been lyophilized and which may be reconstituted to form pharmaceutically acceptable formulations for administration, as by intravenous, intramuscular, or subcutaneous injection. Administration may also be intradermal or transdermal.
  • a nucleic acid described herein, and pharmaceutically acceptable salts thereof may be administered orally or through inhalation as a solid, or may be administered intramuscularly or intravenously as a solution, suspension or emulsion. Alternatively, the compounds or salts may also be administered by inhalation, intravenously or intramuscularly as a liposomal suspension.
  • Pharmaceutical formulations are also provided which are suitable for administration as an aerosol, by inhalation. These formulations comprise a solution or suspension of the desired nucleic acid herein, or a salt thereof, or a plurality of solid particles of the compound or salt. The desired formulation may be placed in a small chamber and nebulized.
  • Nebulization may be accomplished by compressed air or by ultrasonic energy to form a plurality of liquid droplets or solid particles comprising the compounds or salts.
  • the liquid droplets or solid particles should have a particle size in the range of about 0.5 to about 5 micron m.
  • the solid particles can be obtained by processing the solid compound of any nucleic acid described herein, or a salt thereof, in any appropriate manner known in the art, such as by micronization. Most preferably, the size of the solid particles or droplets will be from about 1 micron m to about 2 micron m. In this respect, commercial nebulizers are available to achieve this purpose.
  • the formulation when the pharmaceutical formulation suitable for administration as an aerosol is in the form of a liquid, the formulation will comprise a water-soluble compound of any nucleic acid described herein, or a salt thereof, in a carrier that comprises water.
  • a surfactant may be present that lowers the surface tension of the formulation sufficiently to result in the formation of droplets within the desired size range when subjected to nebulization.
  • Peroral compositions also include liquid solutions, emulsions, suspensions, and the like.
  • the pharmaceutically acceptable carriers suitable for preparation of such compositions are well known in the art.
  • Typical components of carriers for syrups, elixirs, emulsions, and suspensions include ethanol, glycerol, propylene glycol, polyethylene glycol, liquid sucrose, sorbitol, and water.
  • typical suspending agents include methyl cellulose, sodium carboxymethyl cellulose, tragacanth, and sodium alginate;
  • typical wetting agents include lecithin and polysorbate 80; and typical preservatives include methyl paraben and sodium benzoate.
  • Peroral liquid compositions may also contain one or more components such as sweeteners, flavoring agents and colorants disclosed above.
  • compositions may also be coated by conventional methods, typically with pH or time-dependent coatings, such that the subject compound is released in the gastrointestinal tract in the vicinity of the desired topical application, or at various times to extend the desired action.
  • dosage forms typically include, but are not limited to, one or more of cellulose acetate phthalate, polyvinylacetate phthalate, hydroxypropyl methyl cellulose phthalate, ethyl cellulose, waxes, and shellac.
  • compositions useful for attaining systemic delivery of the nucleic acids include sublingual, buccal and nasal dosage forms.
  • Such compositions typically comprise one or more of soluble filler substances such as sucrose, sorbitol and mannitol; and binders such as acacia, microcrystalline cellulose, carboxymethyl cellulose and hydroxypropyl methyl cellulose. Glidants, lubricants, sweeteners, colorants, antioxidants and flavoring agents disclosed above may also be included.
  • Means of oral delivery have been described e.g. in Aourdi et al., Nature 458, 1180-1184, 2009.
  • compositions of this invention can also be administered topically to a subject, e.g., by the direct laying on or spreading of the composition on the epidermal or epithelial tissue of the subject, or transdermal ⁇ via a patch.
  • Such compositions include, for example, lotions, creams, solutions, gels and solids.
  • These topical compositions preferably comprise an effective amount, usually at least about 0.1 %, and preferably from about 1 % to about 5 %, of a nucleic acid of the invention.
  • Suitable carriers for topical administration preferably remain in place on the skin as a continuous film, and resist being removed by perspiration or immersion in water.
  • the carrier is organic in nature and capable of having dispersed or dissolved therein the therapeutic compound.
  • the carrier may include pharmaceutically acceptable emolients, emulsifiers, thickening agents, solvents and the like.
  • Appropriate dosage levels may be determined by any suitable method known to one skilled in the art. It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the condition to be treated. Dosage levels preferably lie in the range of 0.01 to 100 mg/kg body weight, more preferably in the range of 0.1 to 50 mg/kg body weight of the patient to be treated.
  • the respective active agents may be formulated together in a single dosage form. Alternatively, they may be formulated separately and packaged together, or they may be administered independently. In certain cases, a patient may be receiving one drug for the treatment of another indication; this invention then comprises administering the other drug.
  • Drugs which may be co-administered with a catecholamines, adrenocorticosteroid, angiotensin, prostaglandin, leukemia inhibitory factor (LIF), endothelin (including endothelin-1, -2, and -3 and big endothelin), cardiotrophin-1 (CT-1), cardiac hypertrophy factor (CHF) and blood pressure medication.
  • LIF leukemia inhibitory factor
  • CT-1 cardiotrophin-1
  • CHF cardiac hypertrophy factor
  • blood pressure medication a pharmaceutical composition of the invention
  • the respective drugs may be administered simultaneously, separately or sequentially.
  • the present invention does not refer to methods for treatment of the human or animal body by therapy or to diagnostic methods practiced on the human or animal body.
  • heart failure is characterized by myocyte lengthening, ECM remodeling, chamber dilation, and impaired systolic and/or diastolic function.
  • heart failure is characterized by regional degeneration of heart muscle fibers, inflammatory infiltration to the heart or vascular system, calcification of heart structures or vascular structures, areas of cell death in the heart, and/or occurrence of perivascular fibrosis.
  • Heart failure may be left-sided heart failure, right- sided heart failure, systolic heart failure, or diastolic heart failure.
  • the patient has a pre-existing condition such as a family history of cardiac disease, an existing heart condition, high blood pressure, and/or cardiomyopathy.
  • Other exemplary pre-existing conditions include but are not limited to diabetes, obesity, and lung disease, any of which may be combined with factors such as stress, a sedentary lifestyle, and/or environmental factors that contribute to cardiac pathologies.
  • the patient has cardiac hypertrophy.
  • the patient has left ventricular hypertrophy.
  • the patient may have right ventricular hypertrophy, or hypertrophy in both ventricles.
  • the patient has dilation of one or more of cardiac chambers, increased left and/or right ventricle diameter, and/or depressed systolic function.
  • the patient has cardiac hypertrophy and the methods described herein may be used to determine the patient's risk for heart failure in the future.
  • the methods may also be used to diagnose ventricular dysfunction, for example, LV dysfunction.
  • the methods may be used to diagnose LV dysfunction, RV dysfunction and/or a combination of both LV and RV dysfunction.
  • the patient has ventricular dysfunction such as LV dysfunction and the methods described herein may be used to determine the patient's risk for heart failure in the future.
  • the patient has RV dysfunction.
  • the patient may also have both LV and RV dysfunction.
  • the methods described herein indicate that a patient will develop heart future.
  • the patient is a candidate for aortic valve replacement or mitral valve replacement, or a procedure to restore function of a defective valve.
  • the patient's risk of developing heart failure may be determined before the intervention.
  • the biomarkers described herein may be used to determine if the patient will develop heart failure during or after the intervention.
  • the patient may have already undergone aortic valve replacement or mitral valve replacement, and the methods described herein may be used to determine the patient's risk for heart failure in the future. Similarly, the methods may be indicate that patient will develop heart failure.
  • the biological sample obtained from a patient is a blood sample.
  • the biological sample may be a plasma sample.
  • the biological sample is a tissue sample, for example, a sample of heart tissue.
  • the heart tissue may be obtained from a ventricle of the heart, for example, a left or right ventricular biopsy.
  • miRs are regulatory molecules of approximately 22 noncoding nucleotides. miRs regulate gene expression by hybridization to messenger RNAs (mRNAs) with the consequence of mRNA degradation or translational inhibition of targeted transcripts.(Ambros 2001, Thum, Galuppo et al. 2007) MiRs are involved in a variety of basic biological processes, including cell proliferation, apoptosis, and stress response.
  • miR expression profiles that reflect the condition of the heart. For example, in transverse aortic constriction (TAC) treatment model, when the initial CM cross-sectional area of the heart increases (2 weeks TAC), there is upregulation of 17 miRs and down regulation of 2 miRs. Over time (4 weeks TAC), 213 miRs are upregulated while none are down-regulated. Finally, at the stage of heart failure, as indicated by cardiac dilation, depressed systolic function, and/or increased wet/dry lung weight (10 weeks TAC), a panel of 5 miRs were upregulated and 9 miRs were down-regulated. Accordingly, a change of expression in one or more of the miRs in the following table may be indicative of heart failure:
  • the expression of one or more of miR 764-3p, miR-130b, and miR 2134 may be increased, while the expression of one or more of miR-218, miR-9-3p, miR-322-3p, miR-210, miR-208a, miR-135a, miR-19b, miR-29b, and miR-338 may be decreased.
  • both miR-130b-3p and miR-764-3p were increased in patients with severe aortic stenosis while miR-218, miR-9-3p, miR-322-3p and miR-210 were decreased. These patients are at great risk for developing heart failure; thus, the changes in expression of these biomarkers indicate a high risk of heart failure.
  • changes in expresison of these biomarkers indicate that the patient will develop heart failure.
  • changes in expression of the miRs indicate that the patient has ventricular dysfunction, such as LV dysfunction, RV dysfunction, or both.
  • the changes in expression of the miRs indicate that a patient with ventricular dysfunction (either LV dysfunction, RV dysfunction, or both) is at risk for heart failure and/or will develop heart failure.
  • the present data indicate that an increase in expression of one or more of miR 764-3p and miR-130b, and/or a decrease in expression of one or more of miR-218, miR-9-3p, miR-322-3p, and miR-210 are predictive for heart failure or risk for heart failure, as their expression is altered during progression from cardiac hypertrophy (such as ventricular hypertrophy) to ventricular dysfunction (LV dysfunction, RV dysfunction, and/or both) to heart failure.
  • cardiac hypertrophy such as ventricular hypertrophy
  • ventricular dysfunction LV dysfunction, RV dysfunction, and/or both
  • an aspect of the present disclosure relates to a method for identifying a patient at risk for developing heart failure, comprising (a) obtaining a biological sample from the patient, (b) measuring expression of miR-764-3p in the biological sample; and (c) comparing the expression of miR-764-3p in the biological sample with a reference sample; wherein an increase in expression of miR-764-3p in the biological sample as compared to the reference sample indicates that the patient is at risk for developing heart failure.
  • an increase in expression of miR-764-3p in the biological sample as compared to the reference sample indicates that the patient will develop heart failure.
  • a further aspect of the present disclosure relates to a method for identifying a patient at risk for heart failure, comprising (a) obtaining a biological sample from the patient; (b) measuring expression of miR- 764-3p and miR-130b in the biological sample; and (c) comparing the expression of miR-764-3p and miR-130b in the biological sample with a reference sample, wherein an increase in expression of miR- 764-3p and miR-130b in the biological sample as compared to the reference sample indicates that the patient is at risk for developing heart failure.
  • an increase in expression of miR- 764-3p and miR-130b in the biological sample as compared to the reference sample indicates that the patient will develop heart failure.
  • Another aspect of the present disclosure relates to a method for identifying a patient at risk for developing heart failure, comprising (a) obtaining a biological sample from the patient; (b) measuring expression of miR-218, miR-9-3p, miR-322-3p, and miR-210 in the biological sample; and (c) comparing the expression of miR-218, miR-9-3p, miR-322-3p, and miR-210 in the biological sample with a reference sample, wherein a decrease in expression of miR-218, miR-9-3p, miR-322-3p, and miR-210 in the biological sample as compared to the reference sample indicates that the patient is at risk for developing heart failure.
  • a decrease in expression of miR-218, miR-9-3p, miR- 322-3p, and miR-210 in the biological sample as compared to the reference sample indicates that the patient will develop heart failure.
  • Yet another aspect of the present disclosure relates to a method for identifying a patient at risk for developing heart failure, comprising (a) obtaining a biological sample from the patient; (b) measuring expression of miR-764-3p, miR-130b, miR-218, miR-9-3p, miR-322-3p, and miR-210 in the biological sample; and (c) comparing the expression of miR-764-3p, miR-130b, miR-218, miR-9-3p, miR-322-3p, and miR-210 in the biological sample with a reference sample, wherein an increase in expression of miR-764-3p and miR-130b and a decrease in expression of miR-218, miR-9-3p, miR-322-3p, and miR- 210 in the biological sample as compared to the reference sample indicates that the patient is at risk for developing heart failure.
  • an increase in expression of miR-764-3p and miR-130b and a decrease in expression of miR-218, miR-9-3p, miR-322-3p, and miR-210 in the biological sample as compared to the reference sample indicate that the patient will develop heart failure.
  • Still another aspect of the present disclosure relates to a method for diagnosing a patient's risk for developing heart failure, wherein risk for heart failure is characterized by an increase in expression of miR-130b and/or miR-764-3p, and/or a decrease in expression of mi ' R-218, miR-9-3p, miR-322-3p, and/or miR-210, comprising: (a) obtaining a biological sample from the patient; (b) applying a labeled probe specific to any one of miR-130b, miR-764-3p, miR-218, miR-9-3p, miR-322-3p, and miR-210 in order to form a probe-miRNA complex; (c) quantifying the probe-miRNA complex; (d) comparing the quantity of probe-miRNA complex to a reference measurement; and (e) diagnosing a patient at risk for heart failure where the probe-miRNA complex differs from the reference measurement.
  • the probe-miRNA complex differs from the reference measurement, the
  • the change in expression of miRNAs is at least a 2-fold change in expression as compared to the miRNA expression in a reference sample. For example, if a patient has a 2x increase in miR-764-3p as compared to a reference sample, this would indicate that the patient is likely to develop heart failure.
  • the increase in expression of miRs such as miR-130b and/or miR-764-3p is at least a 2x increase in expression as compared to the miRNA expression in a reference sample.
  • the increase is a 2x, 2.5x, 3x, 3.5x, 4x, 4.5x, 5x increase in expression.
  • the increase is a 5x-10x increase in expression.
  • the decrease in expression of miRs such as miR-218, miR-9-3p, miR-322-3p, and/or miR-210 is at least a 2x decrease in expression.
  • the decrease is a 2x, 2.5x, 3x, 3.5x, 4x, 4.5x, 5x decrease in expression.
  • the decrease is a 5x-10x decrease in expression.
  • the change in expression of miRs is at least 2x in patients with ventricular dysfunction, such as LV dysfunction, and increases over time as the ventricular dysfunction develops into heart failure.
  • the ventricular dysfunction is LV dysfunction, RV dysfunction, or both.
  • the reference sample is a biological sample from a healthy control subject.
  • the control subject may be age-matched and/or may not have any heart disorders such as cardiac hypertrophy (i.e., ventricular hypertrophy) or ventricular dysfunction (LV dysfunction, RV dysfunction, or both).
  • miRNA sequences may be found in publically-available databases, for example, miRBase (release 21 of June 2014), which includes human miRNA sequences (i.e., hsa-mir-764-3p) and mouse miRNA sequences, among others.
  • cardiac miR signatures can distinguish between ventricular hypertrophy (such as LV hypertrophy) and ventricular dysfunction (for example, LV dysfunction).
  • the cardiac miR signatures may also indicate that a patient is at risk for developing heart failure and/or will develop heart failure.
  • Serial analysis of cardiac miR profiles and comparison of different stress models allows identification of novel miRs that are associated with the transition of compensatory cardiac hypertrophy towards ventricular dysfunction.
  • One approach to preventing or slowing the transition towards ventricular dysfunction and/or lessening the severity of heart failure is to target the miRs associated with the process and/or to target the genes regulated by the miRs.
  • DGC dystrophin-glycoprotein complex
  • mice Because dystrophin and dystroglycan interact with actin and laminin, respectively, this molecular linkage directly connects the cytoskeleton and the ECM, where it is thought to provide support for the plasma membrane against forces generated during muscle contraction.
  • the absence of DSGC in mice resulted in regional degeneration of the muscle fibers accompanied by an inflammatory infiltrate and calcification was also frequently noted.
  • hearts lacking DSGC showed no histologic sign of cardiac degeneration, whereas after 12weeks of age, areas of cell death and inflammatory infiltration were seen together with frequent occurrence of perivascular fibrosis, 20 consistent with sudden death observed in these mice.
  • DSGC vascular smooth muscle cells
  • one aspect of the present disclosure relates to an inhibitor of miR-130b-3p or an inhibitor of miR-764-3p (or a combination of inhibitors to both miR-130-3p and miR-764-3p) for use in treating and/or preventing heart failure.
  • oligonucleotides which bind a miR may be used to inhibit miRs.
  • an inhibitor of miR-130b-3p and/or an inhibitor of miR-764-3p treats and/or prevents heart failure by reducing symptoms of cardiac dysfunction such as increased HW/BW ratios, increased CM size, cardiac dilation, and depression of systolic function.
  • the inhibitor may increase DSGC levels.
  • miRs are down-regulated in cardiac dysfunction.
  • 9 miRs were down-regulated after lOwks TAC. 4 of these down-regulated miRs (miR-218, miR-9-3p, miR-322-3p and miR-210) share a common potential target of VAMP7.
  • VAMP7 is responsible for the activation of MT1-MMP, 25 a protein which belongs to the family of matrix metalloproteinases (MMPs).
  • MMPs are a family of more than 20 zinc-dependent endopeptidases that collectively can degrade all ECM macromolecules.
  • MT1-MMP is a cell surfaced anchored MMP with a single transmembrane domain and an extracellular catalytic domain that also functions as a highly effective type I collagenase. 25 27 Increased levels of MT1-MMP have been associated with cardiomyopathic diseases. 28 Mice heterozygous for a null allele of MT1-MMP display functional advantages after myocardial infarction (Ml) because of attenuated ECM remodeling and better preserved cardiac function. Deschamps et al. also demonstrated increased MT1-MMP levels in a ischemia-reperfusion model in pigs.
  • MT1-MMP has been suggested to serve as a membrane receptor or activator of MMP-2 (Gelatinase A) 30 31 and indirectly as an activator of MMP-9. Increased MMP-2 and MMP-9 levels could be detected in patients with dilated cardiomyopathy.
  • MMP-2 and MMP-9 are expressed in different cell types including cardiac myoyctes and fibroblasts and they have been implicated both in clinical and experimental studies.
  • 33"35 Accordingly, one aspect of the present disclosure relates to an activator of miR-218, miR-9-3p, miR- 322-3p and/or miR-210 for use in preventing and/or treating heart failure.
  • miR mimics or AAV9-derived overexpression of single miRs may be used to increase expression of one or more of miR-218, miR-9-3p, miR-322-3p and/or miR-210.
  • Another aspect relates to an inhibitor of VAMP7 for use in preventing and/or treating heart failure.
  • Symptoms of cardiac dysfunction such as increased HW/BW ratios, increased CM size, cardiac dilation, and depression of systolic function may be treated or prevented with an activator of miR-218, miR-9-3p, miR-322-3p and/or miR-210 or an inhibitor of VAMP-7.
  • the activator may decrease VAMP7 levels.
  • An inhibitor of VAMP7 may decrease VAMP7 levels.
  • One aspect of the present disclosure relates to a method for diagnosing and treating a patient at risk for developing heart failure, comprising analyzing a patient sample for expression of at least one of miR-130b, miR-764-3p, miR-218, miR-9-3p, miR-322-3p, and miR-210, wherein the patient is diagnosed to be at risk for heart failure if the expression of miR-130b and/or miR-764-3p is increased, and/or the expression of miR-218, miR-9-3p, miR-322-3p, and/or miR-210 is decreased; and administering a treatment to the diagnosed patient.
  • a further aspect of the present disclosure relates to a method for treating a patient at risk for developing heart failure, comprising requesting a test providing results of an analysis of expression of at least one of miR-130b, miR-764-3p, miR-218, miR-9-3p, miR-322-3p, and miR-210 and administering a miR-specific or miR-targeted treatment to the patient if the patient shows increased expression of miR-130b and/or miR-764-3p and/or decreased expression of miR-218, miR-9-3p, miR- 322-3p, and/or miR-210.
  • CM cross-sectional area was significantly increased after 2wks, 4wks and lOwks TAC and after Angll-administration, compared to sham.
  • CM area no longer increased between 4wks and lOwks TAC despite greater HW/BW ratios at lOwks TAC, suggesting a switch towards cardiac dilation following sustained constriction.
  • CM density was significantly decreased in all models compared to sham (supplemental table 1).
  • Capillary density was slightly decreased as compared to sham animals but did not significantly differ between groups (supplemental table 1).
  • Further analysis of the hearts revealed increased cardiac fibrosis at lOwks TAC and after Angll-infusion. Echocardiographic analysis revealed progressively impaired cardiac function from 4wks TAC onward as indicated by increased cardiac dilation and depressed systolic function ( Figure 1 and supplemental table 1).
  • Angll-infusion caused less prominent reduction in systolic function then lOwks TAC, despite a similar degree of interstitial fibrosis. Marked cardiac failure was also indicated by increased wet/dry lung weight at lOwks TAC (supplemental table 1).
  • delta sarcoglycan (DSGC) was selected for validation as a central target for the up-regulated miRs 130b and 764-3p, whereas Vesicular Associated membrane protein-7 (VAMP7) was selected as a target for the down-regulated miRs-218, miR-9-3p, miR-322-3p and miR- 210 ( Figure 2).
  • DSGC is an identified target of the up-regulated miRs
  • miRs were selected that were uniquely up-regulated after lOwks TAC with a common potential target gene.
  • a dual luciferase reporter gene assay was performed to further examine DSGC as a target of miR-130b and miR-764-3p. Both miR-130b and miR-764-3p strongly repressed DSGC 3'-UTR-luciferase reporter expression. Specificity was confirmed using targeted mutations in the DSGC construct. Negative control miR without binding site in the 3'UTR had no effect on lucirerase expression. These results confirmed that DSGC is a target of both miR-130b and miR-764-3p (figure 3A).
  • DSGC expression was determined by quantitative RT-PCR (Figure 3B) and histological analysis (Figure 3C). At 2wks and 4wks TAC the expression of DSGC in the hearts was not different from these in shams whereas at lOwks TAC a significant decrease in DSGC was detected. Immunoblotting confirmed reduced DSGC levels at lOwks TAC ( Figure 3D). In addition, immunohistological analysis revealed reduced DSGC expression in cardiac muscle cells but also in vascular smooth muscle cells.
  • Figure 3E and 3H showed decreased DSGC levels in the hearts of mice after lOwks TAC.
  • Figure 3F and 31 revealed vascular smooth muscle cells in these hearts while Figure 3G and 3J clearly demonstrated co-localization of DSGC and VSMC, more present in sham animals compared to lOwks TAC hearts. It is known that DSGC protects against vascular spasm and this could explain the migration of miR-764-3p from the CMs toward the VSMC-positive blood vessels. Very low miR-764- 3p levels could be detected in all the other groups. In situ hybridization for miR-764-3p confirmed increased expression levels at lOwks (data not shown).
  • mice that underwent TAC were treated with antagomiR-764-3p or antagomiR-130b-3p to inhibit miR up- regulation. Both treatment groups were compared with a group injected with an antagomiR control (SCR) or with PBS treated animals (CTR). At lOwks TAC, antagomiR treatment diminished cardiac dilation and dysfunction, still present in the control-treated mice ( Figure 4A-C and Table 2). In addition, cardiac fibrosis and CM size were reduced after antagomiR treatment (Table 2 and supplemental Table 2).
  • VAMP7 is an identified target of the down-regulated miRs
  • VAMP7 mediates GLUT4 translocation but has this far an unidentified the role in the heart during cardiac hypertrophy.
  • a dual luciferase reporter gene assay was performed to investigate VAMP7 as a target of miRs-218, miR-9-3p, miR-322-3p and miR-210. These miRs strongly repressed VAMP7 3'- UTR-luciferase reporter expression. Negative control miR without binding site in the 3'UTR did not alter luciferase expression.
  • VAMP7 as a target of miRs-218, mi ' R-9-3p, miR- 322-3p and miR-210 (figure 5A).
  • RT-PCR for VAMP7 revealed similar expressions at 2wks TAC and 4wks TAC compared with the control animals but increased levels at lOwks after TAC (Figure 5B). Immunohistological analysis at lOwks after TAC showed increased VAMP7 levels as compared to sham, consistent with down-regulated levels of the miRs ( Figure 5C, 5E and 5F). In addition, immunoblotting revealed increased VAMP7 levels after lOwks TAC compared with sham ( Figure 5D). It has been shown that Membrane type 1-Matrix metalloproteinase (MTl-MMP)-dependent matrix degradation is regulated by VAMP7 15 . MT-1 MMP can directly degrade the extracellular matrix
  • MMP2 is a proteolytic enzyme involved in cell-matrix degradation causing cardiac dilation.
  • MMP9 can also be activated in a MTl-MMP/MMP2-dependent manner. 17
  • RT-PCR of MTl-MMP revealed similar mRNA levels at 2wks TAC and 4wks TAC but increased MTl-MMP levels at lOwks TAC ( Figure 5G). Increased MMP- 2- and MMP-9- activity could be detected by zymography in the hearts of lOwks TAC animals compared to lOwks sham ( Figure 5H).
  • a pressure overload model was obtained through transverse aortic constriction (TAC) as described previously. Briefly, after anesthetizing the mice a partial thoracotomy was performed and the transverse aortic arch was ligated between the innominate and left common carotid arteries with an overlying 27-gauge needle, which after removal of the needle left a reproducible discrete region of stenosis. Sham-operation included all procedures, except constriction of the aorta. Different study groups were designed and mice were studied after 2, 4 or 10 weeks of TAC.
  • angiotensin II (Angll)-induced cardiac hypertrophy was provoked by Angll (Bachem;1.5 mg/kg per day) administration for 4 weeks via osmotic minipumps (ALZET osmotic minipumps; model 2004). Sham-operation included all procedures, except Angll-administration. Echocardiographic measurements
  • the echocardiographic examination was performed by transthoracic echocardiography with a MS 400 transducer (Visualsonics inc.)in anesthetized mice (2% isloflurane, ecuphar, Oostkamp, Belgium) on a Vevo 2100 scanner (Visualsonics inc.) at baseline and at the end of the study period.
  • LVEDD end-diastole
  • LVESD septal wall muscle thickness
  • SW diast septal wall muscle thickness
  • PWdiast LV posterior wall in end diastole
  • FS fractional shortening
  • mice were anaesthetized, and hearts were taken out and prepared for further histological and molecular analysis.
  • Left (LV) and right (RV) ventricles, lungs, liver, kidney, pancreas were dissected, blotted dry and weighed.
  • Immunostainings on paraffin sections were performed using antibodies against laminin (Sigma,), CD31 (DAKO), DSGC (sigma), VAMP7 (Sigma). The quantity of collagen was determined as the percentage of Sirius Red staining area per total cardiac area.
  • Morphometric analysis were performed with a Zeiss Axiovert 200M (Zeiss) and analysed with Axiovision software (Zeiss).
  • RNA including the small RNA fraction, was extracted from myocardial biopsies using miRNeasy Qiagen kit according to the manufacturer's protocol (Qiagen). RNA recovery and sample purity were assessed with a NanoDrop spectrophotometer and RNA quality was checked by Bioanalyser (Agilent). miRNA expression profiles
  • nCounter assay (Nanostring Technologies) implicates the hybridization of fluorescently labeled, bar-coded probes to the miRNAs of interest, which are then scanned and counted to quantify miRNA expression in a multiplexed manner.
  • nCounter expression assays for mouse miRNA expression were performed on miRNAs extracted from LV tissue. All data were confirmed by performing quantitative Real Time-PCR (RT-PCR) using a real-time fluorescence detection method (Applied Biosystems). TaqMan ® MicroRNA assays were used and RT reactions were prepared according to the
  • miR signatures were determined comparing each study group with its SHAM group and calculating the fold change. A cutoff value of >2 fold up- or down regulation and a p-value ⁇ 0,01 was selected. Subsequently, these miR signatures were compared and common and distinct miR signatures were defined. These miR signatures were imported in Ingenuity pathway Analysis (IPA) to conduct further pathway analysis.
  • IPA Ingenuity pathway Analysis
  • the construct was transfected into HEK 293 cells with either specific mimic miRs (for miR 764-3p, miR-130b, miR-218, miR-9-3p, miR-322-3p or miR-210) or miR Negative control (miRIDIAN mimics 25nmol/L).
  • Relative luciferase expression was measured on a scintillation counter using a dual luciferase reporter system (Promega). Site-directed mutagenesis was performed by the deletion of the miR-763-3p and miR-130b seed sequences in the DSGC construct (Genecopoeia). Immunoblotting
  • Heart tissues were homogenized and extracted, and protein concentrations were determined. 30 ⁇ g of protein was subjected to non-reduced sodium dodecylsulfate polyacrylamide gel electrophoresis using 10% gels containing 0.1% gelatin (Invitrogen, Carlsbad, CA). (Alexander and Werb 1992, Kleiner and Stetler-Stevenson 1994) Gels were renatured by exchanging sodium dodecylsulfate with Triton X-100 (2.5%), followed by a 24 hours incubation at 37 C in developing buffer consisting of 50mM Tris-HCI, pH 7.5, supplemented with 7mM CaCI 2 , 0.2M NaCI and 0.02% Brij-35.
  • AntagomiR-764-3p or antagomiR-130b-3p was injected in mice that underwent TAC via repeated injections of 80mg/kg/day for 3 consecutive days. These injections were repeated every 3 weeks for the duration of the study period.
  • miR signatures in aortic stenosis (AS) patients, the most frequent human cause of chronic myocardial pressure overload.
  • AVR aortic valve replacement
  • miR signatures were analyzed in surgical LV biopsies from patients with symptomatic AS.
  • miRs as diagnostic or prognostic tool in AS patients.
  • miRNeasy serum/plasma kit Qiagen
  • RT-PCR was used according to the manufacturer's protocol as described above.
  • CM cardiomyocyte
  • LVEDD left ventricular end diastolic dimension
  • LVESD left ventricular end systolic dimension
  • FS fractional shortening
  • CM cardiomyocyte
  • sarcoglycanopathy Evolution of a concept of muscular dystrophy. Muscle Nerve. 1998;21:421-438

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Pathology (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

L'invention concerne le diagnostic de troubles cardiaques tels que l'insuffisance cardiaque chronique, un état pathophysiologique dans lequel le débit cardiaque est insuffisant pour satisfaire les besoins du corps et des poumons, et le ciblage de microARN spécifiques pour une intervention thérapeutique. Puisque les modification de l'expression de microARN spécifiques indiquent une transition entre une hypertrophie cardiaque et une insuffisance cardiaque, un ensemble de marqueurs biologiques de type microARN peut être utilisé pour prévoir la probabilité d'une insuffisance cardiaque chronique et pour servir de nouvelle cible pour une intervention thérapeutique.
PCT/BE2015/000050 2014-10-13 2015-10-13 Signatures de microarn pour troubles cardiaques Ceased WO2016058061A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GBGB1418043.4A GB201418043D0 (en) 2014-10-13 2014-10-13 MicroRNA signatures for cardiac disorders
GB1418043.4 2014-10-13

Publications (1)

Publication Number Publication Date
WO2016058061A1 true WO2016058061A1 (fr) 2016-04-21

Family

ID=52001297

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/BE2015/000050 Ceased WO2016058061A1 (fr) 2014-10-13 2015-10-13 Signatures de microarn pour troubles cardiaques

Country Status (2)

Country Link
GB (1) GB201418043D0 (fr)
WO (1) WO2016058061A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111154866A (zh) * 2020-03-05 2020-05-15 北京市心肺血管疾病研究所 用于预测高血压患者并发心肌肥厚的miRNA标记物及其应用
WO2020171889A1 (fr) * 2019-02-19 2020-08-27 University Of Rochester Blocage de l'accumulation des lipides ou de l'inflammation dans la maladie oculaire thyroïdienne
US20220213477A1 (en) * 2016-10-27 2022-07-07 The General Hospital Corporation Therapeutic Targeting of a microRNA to Treat Duchenne Muscular Dystrophy

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010019574A1 (fr) * 2008-08-11 2010-02-18 The Board Of Regents Of The University Of Texas System Micro-arn qui favorise une intégrité vasculaire et ses utilisations
WO2010126370A2 (fr) * 2009-04-29 2010-11-04 Academisch Medisch Centrum Bij De Universiteit Van Amsterdam Moyens et procédés pour contrebalancer, prévenir et/ou déterminer une insuffisance cardiaque, ou un risque d'insuffisance cardiaque
WO2010130351A1 (fr) * 2009-05-15 2010-11-18 Bayer Schering Pharma Ag Micro-arn comme biomarqueurs et cibles thérapeutiques pour l'insuffisance cardiaque
CN102416184A (zh) * 2011-12-08 2012-04-18 哈尔滨医科大学 microRNA-1的反义锁核苷酸序列在制备预防或治疗心肌梗死后心衰药物中的应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010019574A1 (fr) * 2008-08-11 2010-02-18 The Board Of Regents Of The University Of Texas System Micro-arn qui favorise une intégrité vasculaire et ses utilisations
WO2010126370A2 (fr) * 2009-04-29 2010-11-04 Academisch Medisch Centrum Bij De Universiteit Van Amsterdam Moyens et procédés pour contrebalancer, prévenir et/ou déterminer une insuffisance cardiaque, ou un risque d'insuffisance cardiaque
WO2010130351A1 (fr) * 2009-05-15 2010-11-18 Bayer Schering Pharma Ag Micro-arn comme biomarqueurs et cibles thérapeutiques pour l'insuffisance cardiaque
CN102416184A (zh) * 2011-12-08 2012-04-18 哈尔滨医科大学 microRNA-1的反义锁核苷酸序列在制备预防或治疗心肌梗死后心衰药物中的应用

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20220213477A1 (en) * 2016-10-27 2022-07-07 The General Hospital Corporation Therapeutic Targeting of a microRNA to Treat Duchenne Muscular Dystrophy
WO2020171889A1 (fr) * 2019-02-19 2020-08-27 University Of Rochester Blocage de l'accumulation des lipides ou de l'inflammation dans la maladie oculaire thyroïdienne
CN111154866A (zh) * 2020-03-05 2020-05-15 北京市心肺血管疾病研究所 用于预测高血压患者并发心肌肥厚的miRNA标记物及其应用

Also Published As

Publication number Publication date
GB201418043D0 (en) 2014-11-26

Similar Documents

Publication Publication Date Title
Martinez et al. MicroRNA-31 promotes adverse cardiac remodeling and dysfunction in ischemic heart disease
EP2882496B1 (fr) Traitement et diagnostic du mélanome
US8642751B2 (en) MicroRNA inhibitors comprising locked nucleotides
US8871731B2 (en) Micro-RNA for the regulation of cardiac apoptosis and contractile function
Wang et al. MicroRNA-132 attenuated cardiac fibrosis in myocardial infarction-induced heart failure rats
JP2016193888A (ja) ｍｉＲＮＡ−２９発現のアンタゴニスト、並びに大動脈瘤の予防及び治療におけるその使用
US10280422B2 (en) MiR-92 inhibitors and uses thereof
US11938129B2 (en) Methods and compositions for the treatment of vascular disease
Ball et al. Role and regulation of microRNAs in aldosterone-mediated cardiac injury and dysfunction in male rats
Lv et al. Metformin ameliorates cardiac conduction delay by regulating microRNA-1 in mice
WO2016058061A1 (fr) Signatures de microarn pour troubles cardiaques
Song et al. MicroRNA-19 upregulation attenuates cardiac fibrosis via targeting connective tissue growth factor
US20180250325A1 (en) Mir-19 modulators and uses thereof
CN108165632B (zh) Linc01426在肝细胞癌诊断和治疗中的应用
US8247389B2 (en) Treatment of scleroderma
Zhou et al. Fibroblast‐derived miR‐425‐5p alleviates cardiac remodelling in heart failure via inhibiting the TGF‐β1/Smad signalling
US20130089601A1 (en) Method for treating atherosclerosis
RU2811365C2 (ru) Лечение сердечной недостаточности у человека
US20250163420A1 (en) Promotion of Cardiomyocyte Proliferation and Regenerative Treatment of the Heart by Inhibition of microRNA-128
WO2024159113A1 (fr) Modulateurs de polyadénylation alternative et méthodes d'utilisation associées
García López et al. Sex-Specific Regulation of miR-29b in the Myocardium Under Pressure Overload is Associated with Differential Molecular, Structural and Functional Remodeling Patterns in Mice and Patients with Aortic Stenosis
JP2021038183A (ja) 心不全の予防又は治療薬及び心不全予防又は治療用医薬組成物
Bernardo et al. Silencing of miR-34a Attenuates Cardiac Dysfunction in a Setting of Moderate, but
Franch et al. miR-182 attenuates atrophy-related gene expression by targeting FoxO3 in skeletal 1 muscle 2 3 Matthew B. Hudson1, Jill A. Rahnert1, Bin Zheng1, Myra E. Woodworth-Hobbs2, Harold 4

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15801311

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 15801311

Country of ref document: EP

Kind code of ref document: A1