WO2010130351A1

WO2010130351A1 - Micrornas as biomarkers and therapeutic targets for heart failure

Info

Publication number: WO2010130351A1
Application number: PCT/EP2010/002702
Authority: WO
Inventors: Georgia Xouri; Stefan Golz; Ulf Brüggemeier; Peter Ellinghaus
Original assignee: Bayer Schering Pharma Ag
Priority date: 2009-05-15
Filing date: 2010-05-04
Publication date: 2010-11-18

Abstract

The invention provides micro RNAs hsa-miR-152, hsa-miR-155 and hsa-miR-497 which are associated with cardiovascular diseases. The invention also features compounds which bind to and/or activate or inhibit the expression of hsa-miR-152, hsa-miR-155 and hsa-miR-497 as well as pharmaceutical compositions comprising such compounds. The invention also provided the use of hsa-miR-152, hsa-miR-155 and hsa-miR-497 regulated genes as biomarkers or targets for cardiovascular diseases. The invention also provides hsa-miR-152, hsa-miR-155 and hsa-miR-497 as a biomarker and therapeutic target for diseases as cardiovascular diseases

Description

microRNAs as biomarkers and therapeutic targets for heart failure

Technical field of the invention

The present invention is in the field of molecular biology, more particularly, the present invention relates to nucleic acid sequences of microRNAs as biomarkers and therapeutic targets of cardiovascular diseases in mammals.

Background of the invention Introduction

MicroRNAs (miRNAs) are a class of small naturally occurring non-coding RNAs (18-24 nucleotides) that regulate gene expression. Many microRNAs are well conserved across species and they are present in a broad range of species: plants, nematodes, fruit flies and humans. MicroRNAs have partially or perfect complementary sequence to one or more messenger RNA molecules (mRNAs) and their main function is to negatively regulate the expression of genes. In particular, microRNAs bind to the 3' untranslated regions of mRNAs (3-UTR) thus leading to down regulation of mRNAs in a variety of ways such as mRNA cleavage, translational repression and deadenylation.

History of microRNAs

The first microRNA, lin-4, was discovered in the worm Caenorhabditis elegans in 1993, where it was found that can negatively regulate the LIN- 14 protein level, thus playing a role in developmental timing in worm. These first data suggested that lin-4 regulates Hn- 14 translation via an antisense RNA-RNA interaction, a mechanism that at that time considered novel but however specific in worms. It was not until 2000 that a second microRNA, let-7, was discovered also in worms and was shown to be evolutionary conserved in a variety of organisms such as vertebrate, ascidian, hemichordate, mollusc, annelid and arthropod, hi 2001, the term 'microRNA' was introduced for the short non coding RNAs (1) and since then the role of microRNAs as central regulators of the gene expression have been slowly started to reveal. Nowadays, more than 600 hundreds microRNAs have been identified in humans. Biogenesis of microRNAs

The genes encoding microRNAs are much longer than the processed mature microRNAs, which are about 18-23 nucleotide (nt) in length. These genes can reside in intergenic regions, either in an exon or in an nitron of non-coding transcripts, or can be found in introns of protein-coding genes. In addition, other microRNAs are clustered in the genome with an expression pattern that implies transcription as polycistronic primary transcripts. Moreover, several microRNAs are expressed in a tissue-, disease- or development-specific manner.

In a first step, microRNAs are transcribed by RNA polymerase II as large RNA precursors called primary miRNAs (pri-miRNAs) (2). These pri-miRNAs have length that varies from several hundred to several thousand nucleotides and can encode for one or for more microRNAs. hi a next step, these pri-miRNAs undergo nuclear cleavage by the microprocessor complex in which Drosha, an RNA II endonuclease, and the double-stranded RNA binding protein DGCR8 (or Pascha) produce a 60-70 nucleotide long intermediate precursor microRNA (pre-miRNA) that has a stem-loop-like structure. The latter precursor is transported to the cytoplasm by exportin-5 in a ran-guanine triphosphatase-dependent manner, where is further processed by Dicer, a RNase ITI endonuclease, to generate a mature microRNA duplex, which has 18-23 nucleotide length (3). From this duplex mature microRNA, only one strand is incorporated into the RNA-induced silencing complex (RISC)₃ while the other one is presumably degraded.

MicroRNAs and their mechanism of function The RNA-induced silencing complex (RISC) is a ribonucleoprotein complex responsible for the miRNA-mediated negative regulation of gene expression. The RISC complex consists of the Argonaute proteins family members and of some accessory factors. Regulation of gene expression by RISC is mediated through interaction of the microRNAs with the Argonaute protein, which in turn guides the RISC complex to the target mRNAs and most favorably to the 3'- untranslated region. A microRNA can either inhibit translation or induce degradation of its target mRNAs and this depends primarily on the overall degree of complementary between the sequence of the microRNA and mRNAs. What is important in this interaction is the 7-8 nucleotides at the 5' - end of the microRNA, which called 'seed region', and must be exactly complementary to the target mRNAs. The rest of the microRNA sequence can be partially complementary to the sequence of the target mRNAs. However, the more complementary the sequence between microRNA and the target mRNA is, the more likely the mRNA will be degraded. When there is no perfect complementation between microRNA and the target mRNAs, then this leads to translation inhibition of mRNAs (4). Therefore, microRNAs can act as global gene expression regulators, and that is why the last few years, have been attracted, much _ attention. Given this universal role of microRNAs, it is of no surprise that more and more studies have started to implicate microRNAs in a variety of biological processes. However, until now a specific function has been assigned to relative few microRNAs.

Prediction of microRNA regulated genes.

As microRNAs can regulate some thousands of mRNAs, it is equally important to try to identify the targets of these mRNAs as this will give us a better insight into the role of microRNAs and into the mechanism of action in the different biological processes. To this end, several computational methods have been developed in order to predict the microRNA regulated genes such as the miRanda program. These predictions are mainly based on the perfect or near perfect complementation between the 'seed region' of microRNA and the mRNAs of the different genes. Moreover, another determinant is an additional base pairing at the 3' end of the microRNA as well as the content of the AU nucleotide which is in close proximity to the target site of the mRNA, thus making the mRNA more accessible to the RISC complex. These computational approaches in several cases have successfully predicted the target mRNAs of specific microRNAs. However as this is only a prediction, experimental data should be provided in order to verify these predictions.

miRNAs in Cardiovascular Development

Heart development and pathology are complex processes as they are linked to multiple genetic pathways. Until recently, most of the studies have focused on the expression of cardiac genes and how this may change during the heart development and heart diseases. However, very recent studies have suggested that microRNAs are also involved in heart development and their expression can be altered in heart pathology. The requirement of microRNAs during heart development was mainly shown in a paper in 2007 as tissue-specific deletion of Dicer, an endonuclease important for processing the pre-miRNAs into the mature microRNAs, caused lethality of embryos due to defects in cardiogenesis. However, such kind of approach has limitations as Dicer deletion inhibits the overall process of all microRNAs, thus leaving questions such as: what is the precise function of specific microRNAs and which are the microRNAs important for the cardiac development. To this end, several researchers have started to do profiling of microRNAs expression in both in vitro and in vivo models for cardiac hypertrophy in order to give a better insight into which microRNAs are important for heart development and pathology (5, 6, 7). One of the first studies is from van Rooij et al (5) where it was shown that there is a relative small number of microRNAs change in two models of pathological cardiac hypertrophy, in thoracic aortic-banded hearts (TAB) and in calcineurin over- expression transgenic mice. Since then, it has been become apparent that the expression profile of miRNAs is disease dependent. In particular, pathological processes (e.g. cardiovascular diseases) are associated with a specific expression pattern of miRNAs. This expression pattern could be useful for human cardiovascular diseases as a prognostic and diagnostic marker.

The most well studied microRNAs in the regulation of cardiac hypertrophy are: miR-1, miR-21, miR-133, miR-195 and miR-208.

MicroRNA 1 (miR-1): It is abundantly expressed in cardiac muscles. MicroRNA 1 (miR-1) has been proposed to be required for cardiac development. MiR-I has been found to be downregulated in three murine models (TAC mice, transgenic mice with selective cardiac overexpression of a constitutively active mutant of the Akt kinase) as well as in the cardiac tissue of patients with dilated cardiomyopathy (DCM) or aortic stenosis. However, different studies suggest that miR-1 is overexpressed in the disease status rather than downregulated. Particularly, miR-1 has been found to overexpressed in heart tissues from patients with coronary artery disease that are at high risk to develop arrhythmias and in left ventricular tissues of patients with end- stage heart failure. In agreement with the latter in vivo data, overexpression of miR-1 in infracted rat hearts exacerbated, whereas elimination of miR-1 by antisense inhibitors relieved arrhythmias. Moreover, miR-1 is upregulated in the rat myoblast cell line H9C2 cells upon oxidative stress.

MicroRNA-21 (miR-21): MicroRNA-21 is a miRNA that has been implicated in tumor celLgrowth andin apoptosis. However,_recently this mjcroRNA has also been implicated in heart failure. In particular, miR-21 has been shown to be upregulated in agonist-induced cardiac hypertrophy in vitro and in pressure-overload-induced hypertrophy in vivo. To this end, inhibition of miR-21 by antisense oligonucleotide (LNA-antisense) was shown to induce hypertrophy in vitro (rat cardiomyocytes), while ectopic overexpression of miR-21 reduced hypertrophy as indicated by reduced expression of hypetrophic marker genes.

MicroRNA-133 (miR-133): MicroRNA-133 is also one of the most abundant microRNAs in cardiac muscles and has also been proposed to be required for cardiac development. MicroRNA-133 has been found to be downregulated in the left ventricle in three independent animal models (TAC mice, transgenic mice with selective cardiac overexpression of a constitutively active mutant of the Akt kinase) and as well as in heart tissues from patients with cardiac hypertrophy. Likewise, in vitro overexpression of miR-133 inhibited cardiac hypertrophy, while the opposite effect, induced hypertrophy, was observed when miR-133 were inhibited. In vivo inhibition of miR-133 by an antisense oligonucleotide (antimiR) caused sustained cardiac hypertrophy.

MicroRNA-195 (miR-195): MicroRNA-195 was found to be overexpressed in human heart failing hearts and it has been demonstrated that overexpression of only microRNA-195 is sufficient to induce hypertrophic growth both in vitro (primary cardiomyocytes) and in vivo (transgenic mice) (5). MicroRNA-208 (miR-208): MicroRNA 208 (miR-208) is encoded by an intron for the oMHC gene. MiR-208 is shown to required for cardiomyocyte hypertrophy and fibrosis as loss of miR-208 protects mice against cardiac hypertrophy.

Detection and expression profiling of microRNAs

A variety of experimental approaches and different techniques have been used the last decade in order to identify new microRNAs, as well as to study their expression pattern in the different biological processes. One of the challenges for cloning and detecting microRNAs is their small size as well as the fact that for more biological relevant data detection of only the mature microRNA form is usually required. The cloning and identification of new microRNAs have been successfully done from size fractioned RNA samples using small RNA cloning approaches. Other approaches is as putative microRNAs homologues to microRNAs that already have been described in other species or using computational approaches alone or in combination with microarray analysis and sequence-directed cloning. One of the first techniques used for detection and profiling of microRNAs was Northern Blotting, where hybridization is done with a complementary ³²P, digoxigenin-labeled oligo or modified Locked-nucleic-acid (LNA) oligonucleotides after gel separation. Other techniques that have been developed to specifically detect microRNAs are a modified invader assay (a synthetic oligonucleotide, the probe, which is in an appropriate overlap-flap structure is enzymatically cleavage by a structure-specific 5* nuclease) and in situ hybridization (using fluorescent-labeled complementary probes containing chemically modified nucleotides e.g. LNAs). Another widely used technique for detection and profiling of microRNAs is the use of oligonucleotide micro-array based detection platforms either with DNA capture probes or using modified Locked-nucleic-acid (LNA) oligonucleotides in which the ribose moiety is modified with an extra bridge that connects the 2'-0 and 4'-C atoms.

hi addition, quantitative real-time PCR (reverse transcriptase/ polymerase chain reaction using Taqman or SYBR green technology) has been used for detection and profiling of precursor or mature microRNAs. This technique is very successful as is sensitive, require low amounts of starting material and through newly developed approaches for RT primer design the detection of individual mature microRNAs is possible. Interestingly, quite recently a Taqman microRNA arrays have been developed that provide the sensitivity of the qRT-PCR, while at the same time enables the simultaneously detection of different microRNAs in one sample.

The methods described here are the more used or known ones, for a more comprehensive list of the methods used for microRNA-profiling can be found in Wark et al^2008 (8)

Description of the invention

Until now, the profiling of microRNAs in the field of heart failure has been done mainly using oligonucleotide microRNA micro-arrays based on DNA capture probes or using modified oligonucleotides. Using a combination of Taqman microRNA array and real time PCR technologies, we have surprisingly identified three microRNAs that are highly regulated in heart tissues from human heart failure patients. In particular, microRNA- 152 (miR-152 /hsa-miR-152 in humans), microRNA- 155 (miR-155 /hsa-miR-155 in humans) and microRNA-497 (miR-497 /hsa-miR-497 in humans) are shown here to be up-regulated in heart tissues from human heart failure patients in comparison to the heart tissues from non-failing patients. Interestingly, microRNA- 155 (miR-155 /hsa-miR-155 in humans) is shown here in addition to be regulated in heart tissues from human heart failure patients before (pre-, heart failure patients ) and after (post-, 'partially recovered' patients) the implantation of a left ventricular assist device (LVAD) that supports the heart function until heart transplantation.

Therefore, microRNAs miR-152, miR-155 and miR-497 could be used as biomarkers for heart failure as well as therapeutic targets for heart failure either alone and/or in combination with the other microRNAs and/ or in combination with proteins in the signaling pathway of these microRNAs.

MicroRNAs

The identified microRNAs are: hsa-miR-152, hsa-miR-155 and hsa-miR-497

The stem-loop and mature nucleotide sequences of hsa-miR-152 are accessible in the miRbase database by the accession number MI0000462 and MMAT0_000438_ respectively. The sequences are given in SEQ ID NO:1 and 2 (figure 1 and figure 2).

The stem-loop and mature nucleotide sequences of hsa-miR-155 are accessible in the miRbase database by the accession number MI0000681 and MIMAT0000646 respectively. The sequences are given in SEQ ID NO:3 and 4 (figure 3 and figure 4).

The stem-loop and mature nucleotide sequences of hsa-miR-497 are accessible in the miRbase database by the accession number MI0003138 and respectively MIMAT0002820. The sequences are given in SEQ ID NO:5 and 6 (figure 5 and figure 6).

Summary of the invention

The invention relates to the identification and use of microRNAs hsa-miR-152, hsa- miR-155 and hsa-miR-497 as biomarkers and therapeutic targets in cardiovascular diseases. The invention also relates to the use of microRNAs hsa-miR-152, hsa-miR- 155 and hsa-miR-497 as biomarkers and therapeutic targets for heart failure. The invention also relates to novel disease associations of microRNAs hsa-miR-152, hsa- miR-155 and hsa-miR-497 polynucleotides. The invention also relates to genes regulated by microRNAs hsa-miR-152, hsa-miR-155 and hsa-miR-497 in cardiovascular diseases. The invention further comprises methods of diagnosing cardiovascular diseases in mammals. Brief Description of the Drawings

Fig. 1 shows the stem-loop nucleotide sequence of hsa-miR-152 (SEQ ID NO:1). Fig. 2 shows the mature nucleotide sequence of hsa-miR-152 (SEQ ID NO:2). Fig. 3 shows the stem-loop nucleotide sequence of hsa-miR-155 (SEQ ID NO:3). Fig. 4 shows the mature nucleotide sequence of hsa-miR-155 (SEQ ID NO:4).

Fig. 5 shows the stem-loop nucleotide sequence of hsa-miR-497 (SEQ ID NO:5). Fig. 6 shows the mature nucleotide sequence of hsa-miR-497 (SEQ ID NO:6). Fig. 7 shows the results of the Taqman microarray expression analysis of hsa-miR- 152 in.human hearts. X axis : disease status; Y axis. : relative expression X2_^Λ(ΔΔCt)); NF : non- failure; HF: heart failure. The expresion of hsa-miR-152 is disease state- regulated in human heart (p value≤O.01, SEM: standard error, nNF=5 and nHF=8,). Fig. 8 shows the results of the Taqman microarray expression analysis of hsa-miR- 155 in human hearts. X axis : disease status; Y axis : relative expression (2^Λ(ΔΔCt)); NF : non- failure; HF: heart failure. The expresion of hsa-miR-155 is disease state- regulated in human heart (p value < 0.001, SEM: standard error, nNF=5 and nHF=8).

Fig. 9 shows the results of Taqman microarray expression analysis of hsa-miR-497 in human hearts. X axis : disease status; Y axis : relative expression (2^^ΔΔCt^); NF : non-failure; HF: heart failure. The expresion of hsa-miR-497 is disease state- regulated in human heart (p value < 0.002, SEM: standard error, nNF=5 and nHF=8). Fig. 10 shows the results of the real-time expression analysis of hsa-miR-152 in human hearts. X axis : disease status; Y axis : relative expression (2^Λ(ΔΛCt)); NF : non- failure; HF: heart failure. The expresion of hsa-miR-152 is disease state- regulated in human heart (p value < 0.03, SEM: standard error, nNF=5 and nHF=5). Fig. 11 shows the results of the real-time expression analysis of hsa-miR-155 in human hearts. X axis : disease status; Y axis : relative expression (2^^MCt^); NF : non- failure; HF: heart failure. The expresion of hsa-miR-155 is disease state- regulated in human heart (p value < 0.03, SEM: standard error, nNF=5 and nHF=5). Fig. 12 shows the results of the real-time expression analysis of hsa-miR-497 in human hearts. X axis : disease status; Y axis : relative expression (2^^MCt)); NF : non- failure; HF: heart failure. The expresion of hsa-miR-497 is disease state- regulated in human heart(p value < 0.07, SEM: standard error, nNF=5 and nHF=5). Fig. 13 shows the results of Taqman microarray expression analysis of hsa-miR- 155 in heart samples from the same individual before (pre-LVAD) and after (post-LVAD) the addition of left ventricular assist device. X axis : disease status; Y axis : relative expression (2^Λ(ΔΔCt)); PRE : pre-LVAD (heart failure patients); POST: post-LVAD ('partially recovered' patients). The expresion of hsa-miR-155 is disease state- regulated in human heart (p value < 0.0006, SEM: standard error, n=4). Fig. 14 shows the results of the real-time expression analysis of hsa-miR-155 in heart samples from the same individual before (pre-LVAD) and after (post-LVAD) the addition of left ventricular assist device_^ X axis : disease status; Y axis :_relative expression (2^A(ΔΔCt)); PRE : pre-LVAD(heart failure patients); POST: post-LVAD

('partially recovered' patients). The expresion of hsa-miR-155 is disease state- regulated in human heart (p value < 0.02, SEM: standard error, n=4). Fig. 15 shows the results of the real-time expression analysis of angiotensin π type 1 receptor gene (AGTRl), a predicted gene target for the microRNA-155, in heart samples from the same individual before (pre-LVAD) and after (post-LVAD) the addition of left ventricular assist device,. X axis : disease status; Y axis : absolute expression ; PRE : pre-LVAD (heart failure patients); POST: post-LVAD ('partially recovered' patients) (p value < 0.003, SEM: standard error, n=4). The expresion of AGTRl is disease state- regulated in human heart and anti-corellates with the expression of the microRNA- 155.

Detailed description of the invention Definition of terms

"Animal" as used herein may be defined to include human, domestic (e.g., cats, dogs, etc.), agricultural (e.g., cows, horses, sheep, etc.) or test species (e.g., mouse, rat, rabbit, etc.).

"Biomarkers" are measurable and quantifiable biological parameters (e.g. specific enzyme concentration, specific hormone concentration, specific gene phenotype distribution in a population, presence of biological substances) which serve as indices for health - and physiology related assessments, such as disease risk, psychiatric disorders, environmental exposure and its effects, disease diagnosis, metabolic processes, substance abuse, pregnancy, cell line development, epidemiologic studies, etc. Parameter that can be used to identify a toxic effect in an individual organism and can be used in extrapolation between species. Indicator signalling an event or condition in a biological system or sample and giving a measure of exposure, effect, or susceptibility.

Biological markers can reflect a variety of disease characteristics, including the level of exposure to an environmental or genetic trigger, an element of the disease process itself, _an intermediate stage between exposure and disease onset, _or an independent factor associated with the disease state but not causative of pathogenesis. Depending on the specific characteristic, biomarkers can be used to identify the risk of developing an illness (antecedent biomarkers), aid in identifying disease (diagnostic biomarkers), or predict future disease course, including response to therapy (prognostic biomarkers).

"Probes" may be derived from naturally occurring or recombinant single- or double- stranded nucleic acids or may be chemically synthesized. They are useful in detecting the presence of identical or similar sequences. Such probes may be labeled with reporter molecules using nick translation, Klenow fill-in reaction, PCR or other methods well known in the art. Nucleic acid probes may be used in southern, northern or in situ hybridizations to determine whether DNA or RNA encoding a certain protein is present in a cell type, tissue, or organ.

An "oligonucleotide" is a stretch of nucleotide residues which has a sufficient number of based to be used as an oligomer, amplimer or probe in a polymerase chain reaction (PCR). Oligonucleotides are prepared from genomic or cDNA sequence and are used to amplify, reveal or confirm the presence of a similar DNA or RNA in a particular cell or tissue.

"Chimeric" molecules may be constructed by introducing all or part of the nucleotide sequence of this invention into a vector containing additional nucleic acid sequence which might be expected to change any one or several of the following microRNAs hsa-miR-152, hsa-miR-155 and hsa-miR-497 characteristics: cellular location, distribution, ligand-binding affinities, signaling, etc.

"Relative or absolute expression" is a quantitative or qualitative measurement for comparison. It is based on a statistically appropriate number of normal samples and is created to use as a basis of comparison when performing diagnostic assays, running clinical trials, or following patient treatment profiles.

The nucleotide sequences encoding microRNAs hsa-miR-152, hsa-miR-155 and hsa-miR-497 (or their complement) have numerous applications in techniques known to those skilled in the art of molecular biology. These techniques include use as hybridization probes, use in the construction of oligomers for PCR and use in generation of antisense DNA or RNA, their chemical analogs and the like. Uses of nucleotides encoding microRNAs hsa-miR-152, hsa-miR-155 and hsa-miR-497 disclosed herein are exemplary of known techniques and are not intended to limit their use in any technique known to a person of ordinary skill in the art.

Furthermore, the nucleotide sequences disclosed herein may be used in molecular biology techniques that have not yet been developed, provided the new techniques rely on properties of nucleotide sequences that are currently known, e.g specific base pair interactions, etc.

Nucleotide sequences encoding microRNAs hsa-miR-152, hsa-miR-155 and hsa- miR-497 may be joined to a variety of other nucleotide sequences by means of well established recombinant DNA techniques. Useful nucleotide sequences for joining to microRNAs hsa-miR-152, hsa-miR-155 and hsa-miR-497 polynucleotides include an assortment of cloning vectors such as plasmids, cosmids, lambda phage derivatives, phagemids, and the like. Vectors of interest include expression vectors, replication vectors, probe generation vectors, sequencing vectors, vectors that contain additional nucleic acid sequences in order to generate "chimeric" molecules etc. In general, vectors of interest may contain an origin of replication functional in at least one organism, convenient restriction endonuclease sensitive sites, and selectable markers for one or more host cell systems. Another aspect of the subject invention is to provide for microRNAs hsa-miR-152, hsa-miR-155 and hsa-miR-497-specific hybridization probes capable of hybridizing with naturally occurring nucleotide sequences encoding microRNAs hsa-miR-152, hsa-miR-155 and hsa-miR-497. Such probes may also be used for the detection of similar microRNAs encoding sequences. The hybridization probes of the subject invention may be derived from the nucleotide sequences presented as SEQ ID NO: 1-

6 or from genomic sequences including promoter, enhancers or introns of the native gene. Hybridization probes may be labelled by a variety of reporter molecules using techniques well known in the art.

Nucleic acid molecules that will hybridize to microRNAs hsa-miR-152, hsa-miR-155 and hsa-miR-497 polynucleotides under stringent conditions can be identified functionally. Without limitation, examples of the uses for hybridization probes include: histochemical uses such as identifying tissues that express microRNAs hsa- miR-152, hsa-miR-155 and hsa-miR-497; measuring mRNA levels, for instance to identify a sample's tissue type or to identify cells that express abnormal levels of microRNAs hsa-miR-152, hsa-miR-155 and hsa-miR-497.

PCR provides additional uses for oligonucleotides based upon the nucleotide sequences which encode microRNAs hsa-miR-152, hsa-miR-155 and hsa-miR-497. Such probes used in PCR may be of recombinant origin, chemically synthesized, or a mixture of both. Oligomers may comprise discrete nucleotide sequences employed under optimized conditions for identification of microRNAs hsa-miR-152, hsa-miR- 155 and hsa-miR-497 in specific tissues or diagnostic use. The same two oligomers, a nested set of oligomers, or even a degenerate pool of oligomers may be employed under less stringent conditions for identification of closely related DNAs or RNAs.

Rules for designing polymerase chain reaction (PCR) primers are now established. Degenerate primers, i.e., preparations of primers that are heterogeneous at given sequence locations, can be designed to amplify nucleic acid sequences that are highly homologous to, but not identical with microRNAs hsa-miR-152, hsa-miR-155 and hsa-miR-497. Strategies are now available that allow for only one of the primers to be required to specifically hybridize with a known sequence. For example, appropriate nucleic acid primers can be ligated to the nucleic acid sought to be amplified to provide the hybridization partner for one of the primers, hi this way, only one of the primers need be based on the sequence of the nucleic acid sought to be amplified.

PCR methods for amplifying nucleic acid will utilize at least two primers. One of these primers will be capable of hybridizing to a first strand of the nucleic acid to be amplified and of priming enzyme-driven nucleic acid synthesis in a first direction.

The other will be capable of hybridizing the reciprocal sequence of the first strand (if the sequence to be amplified is single stranded, this sequence will initially be hypothetical, but will be synthesized in the first amplification cycle) and of priming nucleic acid synthesis from that strand in the direction opposite the first direction and towards the site of hybridization for the first primer. Conditions for conducting such amplifications, particularly under preferred stringent hybridization conditions, are well known.

Other means of producing specific hybridization probes for microRNAs hsa-miR- 152, hsa-miR-155 and hsa-miR-497 include the cloning of nucleic acid sequences encoding microRNAs hsa-miR-152, hsa-miR-155 and hsa-miR-497 into vectors for the production of mRNA probes. Such vectors are known in the art, are commercially available and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA polymerase as T7 or SP6 RNA polymerase and the appropriate reporter molecules.

It is possible to produce a DNA sequence, or portions thereof, entirely by synthetic chemistry. After synthesis, the nucleic acid sequence can be inserted into any of the many available DNA vectors and their respective host cells using techniques which are well known in the art. Moreover, synthetic chemistry may be used to introduce mutations into the nucleotide sequence. Alternately, a portion of sequence in which a mutation is desired can be synthesized and recombined with longer portion of an existing genomic or recombinant sequence.

Patients Exhibiting Symptoms of Disease A number of diseases are associated with changes in the relative expression of a certain nucleotide or microRNA. For patients having symptoms of a disease, the real-time PCR method can be used to determine if the patient has copy number alterations which are known to be linked with diseases that are associated with the symptoms the patient has.

Therapeutic Indications and Methods

Cardiovascular Disorders

Cardiovascular disorders refer to the class of diseases that affect the heart and/or blood vessels. These disorders include but are not limited to disorders of the heart and the vascular system like congestive heart failure, myocardial infarction, ischemic diseases of the heart, all kinds of atrial and ventricular arrhythmias, hypertensive vascular diseases, peripheral vascular diseases, and atherosclerosis. A more detailed definition of some disorders are listed below.

Heart failure is defined as a pathophysiological state in which an abnormality of cardiac function is responsible for the failure of the heart to pump blood at a rate commensurate with the requirement of the metabolizing tissue. It includes all forms of pumping failures such as high output and low output, acute and chronic, right sided or left sided, systolic or diastolic, independent of the underlying cause. Dilated cardiomyopathy (DCM), also known as congestive cardiomyopathy, primarily affects the myocardium as a portion of the myocardium is dilated. Left and/or right ventricular systolic pump function of the heart is impaired, leading to progressive cardiac enlargement and hypertrophy (remodeling process). Applications

The present invention provides microRNAs hsa-miR-152, hsa-miR-155 and hsa- miR-497 for prophylactic, therapeutic and diagnostic methods for cardiovascular diseases.

The regulatory method of the invention involves contacting a cell with an agent that modulates the expression of microRNAs hsa-miR-152, hsa-miR-155, and hsa-miR- 497. An agent that modulates expression can be an agent as described herein, such as a nucleic acid or a- protein, a-peptide, a-peptidomimetic, or any-small molecule. In one embodiment, the agent mimics the biological activities of microRNAs hsa-miR-

152, hsa-miR-155 and hsa-miR-497. Examples of such agents include the active microRNAs hsa-miR-152, hsa-miR-155 and hsa-miR-497 and nucleic acid molecules encoding a portion of microRNAs hsa-miR-152, hsa-miR-155 and hsa-miR-497. In another embodiment, the agent inhibits the biological activities of microRNAs hsa- irήR-152, hsa-miR-155 and hsa-miR-497. Example of such inhibitory agents include antisense nucleic acid molecules. These regulatory methods can be performed in vitro (e.g. by culturing the cell with the agent) or, alternatively, in vivo (e.g by administering the agent to a subject). As such, the present invention provides methods of treating an individual afflicted with a disease or disorder characterized by unwanted expression of microRNAs hsa-miR-152, hsa-miR-155 and hsa-miR-497 or a protein in the microRNAs hsa-miR-152, hsa-miR-155 and hsa-miR-497 signaling pathway. In one embodiment, the method involves administering an agent like any agent identified or being identifiable by a screening assay as described herein, or combination of such agents that modulate say upregulate or downregulate the expression of microRNAs hsa-miR-152, hsa-miR-155 and hsa-miR-497 or of any protein in the microRNAs hsa-miR-152, hsa-miR-155 and hsa-miR-497 signaling pathway. In another embodiment, the method involves administering a regulator of microRNAs hsa-miR-152, hsa-miR-155 and hsa-miR-497 as therapy to compensate for reduced or undesirably low expression of microRNAs hsa-miR-152, hsa-miR-155 and hsa-miR-497 or a protein in the microRNAs hsa-miR-152, hsa-miR-155 and hsa- miR-497 signaling pathway. Overexpression of microRNAs hsa-miR-152, hsa-miR-155 and hsa-miR-497 is desirable in situations in which expression is abnormally low and in which increased expression is likely to have a beneficial effect. Restoring the levels of the altered miRNA expression can be done for example by using a vector that overexpress a specific miRNA sequence, by transient transfection of double-stranded miRNA (eg microRNA mimics), by addition of endogenous pri-miRNA (or pre-miRNA) that can be used as scaffolds for the induction of RNA interference machinery or by artificial miRNAs. These examples should not be construed as limiting.

Conversely, inhibition of expression of microRNAs hsa-miR-152, hsa-miR-155 and hsa-miR-497 is desirable in situations in which the expression of microRNAs hsa- miR-152, hsa-miR-155 and hsa-miR-497 is abnormally high and in which decreasing its expression is likely to have a beneficial effect. Methods for inhibiting microRNAs expression could be utilized strategies where directly inhibit the mature microRNA sequence by using modified anti-miRNA oligonucleotides (AMOs - also designated as "antagomirs). Different types of modified AMOs complementary to mature miRNAs have been shown to successfully inhibit specific endogenous miRNAs in vitro and in vivo. Such examples are the modified 2-OH residues of the ribose by T- O-methyl (2'-0Me), 2'-O-methoxyethyl (2'-MOE) and locked nucleic acid (LNA). These molecules can be further modified by addition of cholesteryl at the 3' end of the nucleic acid, to improve the pharmacokinetic properties and enhanced cellular uptake Another strategy for inhibiting microRNAs expression is to down-regulate components of the miRNA biogenesis pathway, thereby reducing mature miRNA levels. This could be achieved by inhibiting components of the miRNA pathway such as Drosha, Dicer, or others. These methods are served as an example and the inhibition of microRNAs expression is not limited only to these.

The present invention provides for the use of microRNAs hsa-miR-152, hsa-miR-155 and hsa-miR-497 or fragments of microRNAs hsa-miR-152, hsa-miR-155 and hsa- miR-497 as a biomarker for cardiovascular diseases. Another object of the invention is a method of screening for therapeutic agents useful in the treatment of cardiovascular diseases in a mammal comprising the steps of (i) determining the activity of a microRNA hsa-miR-152, hsa-miR-155 or hsa-miR-497 at a certain concentration of a test compound or in the absence of said test compound, (ii) determining the activity of said microRNA at a different concentration of said test compound. E.g., compounds that lead to a difference in the activity of the microRNA hsa-miR-152, hsa-miR-155 or hsa-miR-497 in (i) and (ii) are identified potential therapeutic agents for such a disease.

Another object of the invention is a method of screening for therapeutic agents useful in the treatment of a disease comprised in a group of diseases consisting of cardiovascular diseases in a mammal comprising the steps of (i) determining the activity of a microRNA hsa-miR-152, hsa-miR-155 or hsa-miR-497 at a certain concentration of a test compound, (ii) determining the activity of a microRNA hsa- miR-152, hsa-miR-155 or hsa-miR-497 at the presence of a compound known to be a regulator of a microRNA hsa-miR-152, hsa-miR-155 or hsa-miR-497. E.g., compounds that show similar effects on the activity of the microRNA hsa-miR-152, hsa-miR-155 or hsa-miR-497 in (i) as compared to compounds used in (ii) are identified potential therapeutic agents for such a disease.

Other objects of the invention are methods of the above, wherein the step of contacting is in or at the surface of a cell.

Other objects of the invention are methods of the above, wherein the cell is in vitro.

Other objects of the invention are methods of the above, wherein the step of contacting is in a cell- free system.

Other objects of the invention are methods of the above, wherein the microRNA is coupled to a detectable label.

Other objects of the invention are methods of the above, wherein the compound is coupled to a detectable label. Other objects of the invention are methods of the above, wherein the test compound displaces a ligand which is first bound to the microRNA.

Other objects of the invention are methods of the above, wherein the microRNA is attached to a solid support.

Other objects of the invention are methods of the above, wherein the compound is attached to a solid support.

Another object of the invention is a method of screening for therapeutic agents useful in the treatment of cardiovascular disease in a mammal comprising the steps of (i) contacting a test compound with a microRNA hsa-miR-152, hsa-miR-155 or hsa- miR-497 polynucleotide, (ii) detect binding of said test compound to said microRNA hsa-miR-152, hsa-miR-155 or hsa-miR-497 polynucleotide. Compounds that, e.g., bind to the microRNA hsa-miR-152, hsa-miR-155 or hsa-miR-497 polynucleotide are potential therapeutic agents for the treatment of a cardiovascular disease.

Another object of the invention is a method of the above, wherein the contacting step is in or at the surface of a cell.

Another object of the invention is a method of the above, wherein the contacting step is in a cell-free system.

Another object of the invention is a method of the above, wherein the polynucleotide is coupled to a detectable label.

Another object of the invention is a method of the above, wherein the test compound is coupled to a detectable label.

The invention provides methods (also referred to herein as "screening assays") for identifying compounds which can be used for the treatment of diseases related to microRNA hsa-miR-152, hsa-miR-155 or hsa-miR-497. The methods entail the identification of candidate or test compounds or agents (e.g., AMOs, LNAs, small molecules or other molecules) which bind to microRNA hsa-miR-152, hsa-miR-155 or hsa-miR-497 and/or have a stimulatory or inhibitory effect on the biological activity of microRNA hsa-miR-152, hsa-miR-155 or hsa-miR-497 or its expression and then determining which of these compounds have an effect on symptoms or diseases related to microRNA hsa-miR-152, hsa-miR-155 or hsa-miR-497 in an in vivo assay. In a preferred embodiment the compound is an inhibitor of the the biological activity of microRNA hsa-miR-152, hsa-miR-155 or hsa-miR-497. In another preferred embodiment the compound is an activator of the the biological activity of microRNA hsa-miR-152, hsa-miR-155 or hsa-miR-497.

Pharmaceutical Compositions

This invention further pertains to novel agents identified by the above-described screening assays and uses thereof for treatments as described herein.

The nucleic acid molecules (also referred to herein as "active compounds") of the invention can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the nucleic acid molecule and a pharmaceutically acceptable carrier. As used herein the language "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art.

Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

The invention includes pharmaceutical compositions comprising a regulator of microRNAs hsa-miR-152, hsa-miR-155 and hsa-miR-497 expression (and/or a regulator of the microRNAs hsa-miR-152, hsa-miR-155 and hsa-miR-497 signaling pathway) as well as methods for preparing such compositions by combining one or more such regulators and a pharmaceutically acceptable carrier. Also within the invention are pharmaceutical compositions comprising a regulator identified using the screening assays of the invention packaged with instructions for use. For regulators that reduce the microRNAs hsa-miR-152, hsa-miR-155 and hsa-miR-497 expression, the instructions would specify use of the pharmaceutical composition for treatment of cardiovascular diseases.

An inhibitor of the microRNAs hsa-miR-152, hsa-miR-155 and hsa-miR-497 may be produced using methods which are generally known in the art. In particular, to screen libraries of pharmaceutical agents to identify those which specifically bind the microRNAs hsa-miR-152, hsa-miR-155and hsa-miR-497. In another embodiment of the invention, the polynucleotides encoding microRNAs hsa-miR-152, hsa-miR-155 and hsa-miR-497, or any fragment or complement thereof, may be used for therapeutic purposes. In one aspect, the complement of the polynucleotide encoding microRNAs hsa-miR-152, hsa-miR-155 and hsa-miR-497 may be used in situations in which it would be desirable to block the transcription of the mRNA. In particular, cells may be transformed with sequences complementary to polynucleotides encoding microRNAs hsa-miR-152, hsa-miR-155 and hsa-miR-497. Thus, complementary molecules or fragments may be used to modulate microRNAs hsa- miR-152, hsa-miR-155 and hsa-miR-497 expression, or to achieve regulation of gene function. Such technology is now well known in the art, and sense or antisense oligonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding microRNAs hsa-miR-152, hsa-miR- 155 and hsa-miR-497.

Expression vectors derived from retroviruses, adenoviruses, or herpes or vaccinia viruses, or from various bacterial plasmids, may be used for delivery of nucleotide sequences to the targeted organ, tissue, or cell population. Methods which are well known to those skilled in the art can be used to construct vectors which will express nucleic acid sequence complementary to the polynucleotides of the gene encoding microRNAs hsa-miR-152, hsa-miR-155 and hsa-miR-497.

Any of the therapeutic methods described above may be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans. An additional embodiment of the invention relates to the administration of a pharmaceutical composition containing microRNAs hsa-miR-152, hsa-miR-155 and hsa-miR-497 in conjunction with a pharmaceutically acceptable carrier, for any of the therapeutic effects discussed above. Such pharmaceutical compositions may consist of mimetics or inhibitors of microRNAs hsa-miR-152, hsa-miR-155 and hsa-miR-

497. The compositions may be administered alone or in combination with at least one other agent, such as a stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier including, but not limited to, saline, buffered saline, dextrose, and water. The compositions may be administered to a patient alone, or in combination with other agents, drugs or hormones.

A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EM™ (BASF, Parsippany, NJ.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a pharmaceutically acceptable polyol like glycerol, propylene glycol, liquid polyetheylene glycol, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like, hi many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin. Sterile injectable solutions can be prepared by incorporating the active compound (e.g., DNA/RNA) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above, hi the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a pressurized container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. 4,522,811.

It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used-herein refers to physically discrete units, suited as jmitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration. For pharmaceutical compositions which include an antagonist of microRNAs hsa-miR- 152, hsa-miR- 155 and hsa-miR-

497, a compound which reduces expression of microRNAs hsa-miR-152, hsa-miR- 155 and hsa-miR-497, or a compound which reduces expression or activity of a protein in the microRNAs hsa-miR-152, hsa-miR-155 and hsa-miR-497 signaling pathway or any combination thereof, the instructions for administration will specify use of the composition for cardiovascular diseases. For pharmaceutical compositions which include an agonist of microRNAs hsa-miR-152, hsa-miR-155 and hsa-miR- 497, a compound which increases expression of microRNAs hsa-miR-152, hsa-miR- 155 and hsa-miR-497, or a compound which increases expression or activity of a protein in the microRNAs hsa-miR-152, hsa-miR-155 and hsa-miR-497 signaling pathway or any combination thereof, the instructions for administration will specify use of the composition for cardiovascular diseases. Diagnostics

One embodiment of the invention describes microRNAs hsa-miR-152, hsa-miR-155 and hsa-miR-497 as a biomarker for diagnostic use.

Use of microRNAs hsa-miR-152, hsa-miR-155 and hsa-miR-497 as a biomarker in diagnostics is based by the comparison of microRNAs hsa-miR-152, hsa-miR-155 and hsa-miR-497 level in a biological sample from a diseased mammal with the microRNAs hsa-miR-152, hsa-miR-155 and hsa-miR-497 level in a control sample from a healthy or normal mammal. When the microRNAs hsa-miR-152, hsa^njR- 155 and hsa-miR-497 level in the diseased mammal differs from the microRNAs hsa- miR-152, hsa-miR-155 and hsa-miR-497 level in a normal or healthy mammal then the diseased mammal is diagnosed with a disease associated with an altered microRNAs hsa-miR-152, hsa-miR-155 and hsa-miR-497 level. Furthermore, comparing microRNAs hsa-miR-152, hsa-miR-155 and hsa-miR-497 levels of a biological sample from a diseased mammal with microRNAs hsa-miR-152, hsa-miR-

155 and hsa-miR-497 levels of control samples from mammals with a microRNAs hsa-miR-152, hsa-miR-155 and hsa-miR-497-associated disease already diagnosed with different stages or severity of said disease, allows the diagnose of a microRNAs hsa-miR-152, hsa-miR-155 and hsa-miR-497-associated disease of said first diseased mammal and specifying the severity of the microRNAs hsa-miR-152, hsa-miR-155 and hsa-miR-497-associated disease. The biological sample is taken from the analogue tissue or body fluid (eg blood, urine) than the control sample.

Normal or standard values for microRNAs hsa-miR-152, hsa-miR-155 and hsa-miR- 497 expression are established by using control samples from healthy or diseased mammalian subjects. A control sample can be obtained by collecting body fluids, tissue or cell extracts taken from normal mammalian subjects, preferably human, achieving statistical relevant numbers. To obtain the normal or standard microRNAs hsa-miR-152, hsa-miR-155 and hsa-miR-497 level of the control samples, the samples were subjected to suitable detection methods to detect microRNAs hsa-miR-

152, hsa-miR-155 and hsa-miR-497 polynucleotide. The determination of microRNAs hsa-miR-152, hsa-miR-155 and hsa-miR-497 level in a mammal subjected to diagnosis is performed analogously by collecting a biological sample from said mammal. Quantities of microRNAs hsa-miR-152, hsa-miR-155 and hsa- miR-497 levels in biological samples from a mammal subjected to diagnosis are compared with the standard or normal values measured from a control sample. Deviation between standard value (determined from control sample) and subject value (determined from biological sample) establishes the parameters for diagnosing disease. Absolute quantification of microRNAs hsa-miR-152, hsa-miR-155 and hsa- miR-497 levels measured from biological or control samples may be achieved by comparing those values with values obtained from an experiment in which a known amount of a substantially purified polynucleotide is used.

In another embodiment of the invention, the polynucleotides encoding microRNAs hsa-miR-152, hsa-miR-155 and hsa-miR-497 may be used for diagnostic purposes. The polynucleotides which may be used include oligonucleotide sequences, complementary RNA and DNA molecules, and PNAs. The polynucleotides may be used to detect and quantified gene expression in control and biological samples in which expression of the biomarker microRNAs hsa-miR-152, hsa-miR-155 and hsa- miR-497 may be correlated with disease. The diagnostic assay may be used to distinguish between absence, presence, and excess expression of microRNAs hsa- miR-152, hsa-miR-155 and hsa-miR-497, and to monitor regulation of microRNAs hsa-miR-152, hsa-miR-155 and hsa-miR-497 levels during therapeutic intervention.

Polynucleotide sequences encoding microRNAs hsa-miR-152, hsa-miR-155 and hsa- miR-497 may be used for the diagnosis of cardiovascular diseases associated with expression of microRNAs hsa-miR-152, hsa-miR-155 and hsa-miR-497. The polynucleotide sequences encoding microRNAs hsa-miR-152, hsa-miR-155 and hsa- miR-497 may be used in Northern or other membrane-based technologies; in PCR technologies; and in microarrays utilizing a biological sample from diseased mammals to detect altered microRNAs hsa-miR-152, hsa-miR-155 and hsa-miR-497 expression. Such qualitative or quantitative methods are well known in the art. In a particular aspect, the nucleotide sequences encoding microRNAs hsa-miR-152, hsa-miR-155 and hsa-miR-497 may be useful in assays that detect the presence of associated disorders, particularly those mentioned above. The nucleotide sequences encoding microRNAs hsa-miR-152, hsa-miR-155 and hsa-miR-497 may be labeled by standard methods and added to a biological sample from diseased mammals under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantified and compared with a standard value. If the amount of signal in the patient sample is altered from that-of a comparable control sample,_the nucleotide sequences have hybridized_with nucleotide sequences in the sample, and the presence of altered levels of nucleotide sequences encoding microRNAs hsa-miR-152, hsa-miR-155 and hsa-miR-497 in the sample indicates the presence of the associated disorder. Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or in monitoring the treatment of an individual patient.

Biomarker

Use of microRNAs hsa-miR-152, hsa-miR-155 and hsa-miR-497 as biomarkers.

One of ordinary skill in the art knows several methods and devices for the detection and analysis of the markers of the instant invention.

The analysis of a plurality of markers may be carried out separately or simultaneously with one test sample. Several markers may be combined into one test for efficient processing of a multiple of samples. In addition, one skilled in the art would recognize the value of testing multiple samples (for example, at successive time points) from the same individual. Such testing of serial samples will allow the identification of changes in marker levels over time. Increases or decreases in marker levels, as well as the absence of change in marker levels, would provide useful information about the disease status that includes, but is not limited to identifying the approximate time from onset of the event, the presence and amount of salvagable tissue, the appropriateness of drug therapies, the effectiveness of various therapies, identification of the severity of the event, identification of the disease severity, and identification of the patient's outcome, including risk of future events.

An assay consisting of a combination of the markers referenced in the instant invention may be constructed to provide relevant information related to differential diagnosis. Such a panel may be constucted using 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more or individual markers. The analysis of a single marker or subsets of markers comprising a larger panel of markers could be carried out methods described within the instant invention to optimize clinical _.sensitivity or specificity in various clinical settings.

The analysis of markers could be carried out in a variety of physical formats as well. For example, the use of microtiter plates or automation could be used to facilitate the processing of large numbers of test samples. Alternatively, single sample formats could be developed to facilitate immediate treatment and diagnosis in a timely fashion, for example, in ambulatory transport or emergency room settings. Particularly useful physical formats comprise surfaces having a plurality of discrete, addressable locations for the detection of a plurality of different analytes.

Cardiac markers serve an important role in the early detection and monitoring of cardiovascular disease. Markers of disease are typically substances found in a bodily sample that can be easily measured. The measured amount can correlate to underlying disease pathophysiology, presence or absence of a current or imminent cardiac event, probability of a cardiac event in the future. In patients receiving treatment for their condition the measured amount will also correlate with responsiveness to therapy. Markers can include elevated levels of blood pressure, cholesterol, blood sugar, homcysteine and C-reactive protein (CRP). However, current markers, even in combination with other measurements or risk factors, do not adequately identify patients at risk, accurately detect events (i.e., heart attacks), or correlate with therapy. For example, half of patients do not have elevated serum cholesterol or other traditional risk factors. Use of markers in diagnosis of cardiac conditions is described in a number of publications (9-11).

Cardiovascular biomarker BNP (as an example for cardiovascular biomarkers)

B-type natriuretic peptide (BNP), also called brain- type natriuretic peptide is a 32 amino acid, 4 kDa peptide that is involved in the natriuresis system to regulate blood pressure and fluid balance. The precursor to BNIMs synthesized as a 108-amino acid molecule, referred to as "pre pro BNP," that is proteolytically processed into a 76- amino acid N-terminal peptide (amino acids 1-76), referred to as "NT pro BNP" and the 32-amino acid mature hormone, referred to as BNP or BNP 32 (amino acids 77- 108). It has been suggested that each of these species-NT pro- BNP, BNP-32, and the pre pro BNP- can circulate in human plasma. The 2 forms, pre pro BNP and NT pro BNP, and peptides which are derived from BNP, pre pro BNP and NT pro BNP and which are present in the blood as a result of proteolyses of BNP, NT pro BNP and pre pro BNP, are collectively described as markers related to or associated with BNP. Proteolytic degradation of BNP and of peptides related to BNP have also been described in the literature and these proteolytic fragments are also encompassed it the term "BNP related peptides". BNP and BNP-related peptides are predominantly found in the secretory granules of the cardiac ventricles, and are released from the heart in response to both ventricular volume expansion and pressure overload. Elevations of BNP are associated with raised atrial and pulmonary wedge pressures, reduced ventricular systolic and diastolic function, left ventricular hypertrophy, and myocardial infarction (12). Furthermore, there are numerous reports of elevated BNP concentration associated with congestive heart failure and renal failure. While BNP and BNP-related peptides are likely not specific for ACS₅ they may be sensitive markers of ACS because they may indicate not only cellular damage due to ischemia, but also a perturbation of the natriuretic system associated with ACS. The term "BNP" as used herein refers to the mature 32-amino acid BNP molecule itself. As the skilled artisan will recognize, however, other markers related to BNP may also serve as diagnostic or prognostic indicators in patients with ACS. For example, BNP is synthesized as a 108-amino acid pre pro-BNP molecule that is proteolytically processed into a 76-amino acid "NT pro BNP" and the 32- amino acid BNP molecule. Because of its relationship to BNP, the concentration of NT pro-BNP molecule can also provide diagnostic or prognostic information in patients. The phrase "marker related to BNP or BNP related peptide" refers to any polypeptide that originates from the pre pro-BNP molecule, other than the 32-amino acid BNP molecule itself. Thus, a marker related to or associated with BNP includes the NT pro-BNP molecule, the pro domain, a fragment of BNP that is smaller than the entire 32-amino acid sequence, a fragment of pre pro-BNP other than BNP, and a fragment of the pro domain.

Biomarker classes

The microRNAs hsa-miR-152, hsa-miR-155 and hsa-miR-497 could be used alone or as combination as a biomarkers for cardiovascular diseases in different classes :

Disease Biomarker: a biomarker that relates to a clinical outcome or measure of disease.

Efficacy Biomarker: a biomarker that reflects beneficial effect of a given treatment.

Staging Biomarker: a biomarker that distinguishes between different stages of a chronic disorder. Surrogate Biomarker: a biomarker that is regarded as a valid substitute for a clinical outcomes measure.

Toxicity Biomarker: a biomarker that reports a toxicological effect of a drug on an in vitro or in vivo system.

Mechanism Biomarker: a biomarker that reports a downstream effect of a drug. Target Biomarker: a biomarker that reports interaction of the drug with its target. One embodiment of the invention is a method of use of microRNAs hsa-miR-152, hsa-miR-155 and hsa-miR-497 as a biomarker for a disease comprising :

(a) obtaining a biological sample from a mammal,

(b) measuring the level of microRNAs hsa-miR-152, hsa-miR-155 or hsa- miR-497 in the biological sample,

(c) obtaining a control sample from a mammal,

(d)-measuring-the level of microRNAs-hsa-miR-152, hsa-miR-l-55-or hsa- miR-497, respectively, in the control sample,

(e) comparing the level of microRNAs hsa-miR-152, hsa-miR-155 or hsa-miR-497 in the biological sample with the level of microRNAs hsa-miR-152, hsa-miR-155 or hsa-miR-497, respectively, in a control sample, and

(f) diagnosing a disease based upon the microRNAs hsa-miR-152, hsa- miR-155 or hsa-miR-497 level of the biological sample in comparison to the control sample.

One embodiment of the invention is a method of use of microRNAs hsa-miR-152, hsa-miR-155 or hsa-miR-497 as a biomarker for a disease comprising :

(a) measuring the level of microRNAs hsa-miR-152, hsa-miR-155 or hsa- miR-497 in a biological sample taken from a mammal,

(b) measuring the level of microRNAs hsa-miR-152, hsa-miR-155 or hsa- miR-497, respectively, in a control sample taken from a mammal,

(c) comparing the level of microRNAs hsa-miR-152, hsa-miR-155 or hsa-miR-497 in the biological sample with the level of microRNAs hsa-miR-152, hsa-miR-155 or hsa-miR-497, respectively, in a control sample, and (d) diagnosing a disease based upon the microRNAs hsa-miR-152, hsa- miR-155 or hsa-miR-497 level of the biological sample in comparison to the control sample.

Use of microRNAs hsa-miR-152, hsa-miR-155 or hsa-miR-497 as a biomarker in disease diagnostics is based by the comparison of microRNAs hsa-miR-152, hsa- miR-155 or hsa-miR-497 level in a biological sample from a diseased mammal with the microRNAs hsa-miR-152, hsa-miR-155 or hsa-miR-497 microRNAs hsa-miR- 152, hsa-miR-155 or hsa-miR-497 level in a control sample from a healthy or normal mammal. Does the microRNAs hsa-miR-152, hsa-miR-155 or hsa-miR-497 level in the diseased mammal differs from the microRNAs hsa-miR-152, hsa-miR-155 or hsa-miR-497 level in a normal or healthy mammal then the diseased mammal is diagnosed with a disease associated with an altered microRNAs hsa-miR-152, hsa- miR-155 or hsa-miR-497 level. Furthermore, comparing microRNAs hsa-miR-152, hsa-miR-155 or hsa-miR-497 levels of a biological sample from a diseased mammal with microRNAs hsa-miR-152, hsa-miR-155 or hsa-miR-497 levels of control samples from mammals with a microRNAs hsa-miR-152, hsa-miR-155 or hsa-miR- 497-associated disease already diagnosed with different stages or severity of said disease, allows the diagnose of a microRNAs hsa-miR-152, hsa-miR-155 or hsa- miR-497-associated disease of said first diseased mammal and specifying the severity of the microRNAs hsa-miR-152, hsa-miR-155 or hsa-miR-497-associated disease. The biological sample is taken from the analogue tissue or body fluid than the control sample.

The biological sample in step (a) of the methods is in a preferred embodiment a biological sample comprised in a group of samples consisting of a blood sample, a plasma sample, a serum sample, a tissue sample, a oral mucosa sample, a saliva sample, an interstitial fluid sample or an urine sample. The blood sample is for example a whole blood sample, a fractionated blood sample, a platelet sample, a neutrophil sample, a leukocyte sample, a white blood cell sample, a monocyte sample, a red blood cell sample, a granulocyte sample, and a erythrocyte sample. A tissue sample is for example a sample collected from muscle, adipose, heart or skin. A preferred sample is a heart tissue sample.

hi a preferred embodiment microRNAs hsa-miR-152, hsa-miR-155 or hsa-miR-497 are used as a biomarker diagnosing a disease which is associated with altered levels of microRNAs hsa-miR-152, hsa-miR-155 or hsa-miR-497. Another preferred embodiment microRNAs hsa-miR-152, hsa-miR-155 or hsa-miR-497 are used as a biomarker for identifying an individual risk for developing a disease, or for predicting-an-adverse-outeome-in-a-patient-diagnosed-with-a-disease.-In-a-preferred embodiment the disease is a cardiovascular disease, in a more preferred embodiment the disease is heart failure.

Use of microRNAs hsa-miR-152, hsa-miR-155 or hsa-miR-497 as a disease biomarker in diagnostics is based by the comparison of microRNAs hsa-miR-152, hsa-miR-155 or hsa-miR-497 level in a biological sample from a diseased mammal with the microRNAs hsa-miR-152, hsa-miR-155 or hsa-miR-497, respectively, level in a control sample from a healthy or normal mammal or a group of healthy or normal mammals. When the microRNAs hsa-miR-152, hsa-miR-155 or hsa-miR-497 level in the diseased mammal differs from the microRNAs hsa-miR-152, hsa-miR- 155 or hsa-miR-497 level, respectively, in a normal or healthy mammal, then the diseased mammal is diagnosed with a disease associated with altered microRNAs hsa-miR-152, hsa-miR-155 or hsa-miR-497 level.

Furthermore, using microRNAs hsa-miR-152, hsa-miR-155 or hsa-miR-497 as a staging biomarker, the microRNAs hsa-miR-152, hsa-miR-155 or hsa-miR-497 levels of a diseased mammal are compared with microRNAs hsa-miR-152, hsa-miR- 155 or hsa-miR-497 levels, respectively, of a mammal with a microRNAs hsa-miR- 152, hsa-miR-155 or hsa-miR-497-associated disease already diagnosed with different stages or severity of said disease, allows the diagnose of said first diseased mammal specifying the severity of the microRNAs hsa-miR-152, hsa-miR-155 or hsa-miR-497-associated disease, respectively. A control sample can be a sample taken from a mammal. A control sample can be a previously taken sample from a mammal, as a microRNA hsa-miR-152, hsa-miR- 155 or hsa-miR-497 level in a control sample can be a predetermined level of microRNA hsa-miR-152, hsa-miR-155 or hsa-miR-497 measured in a previously taken sample. The level of microRNA hsa-miR-152, hsa-miR-155 or hsa-miR-497 in a control sample or in a biological sample can be determined for example as a relative value or as an absolute value. A previously measured microRNAs hsa-miR- 152, hsa-miR-155 and hsa-miR-497 level from a control sample can be for example stored in a database, in an internet publication, in an electronically accessible form, in a publication. Comparing the level of microRNA hsa-miR-152, hsa-miR-155 or hsa- miR-497 of a biological sample to a control sample may be comparing relative values or absolute quantified values.

Another embodiment is a method of use of microRNA hsa-miR-152, hsa-miR-155 or hsa-miR-497 as a biomarker for guiding a therapy of a disease comprising:

(a) obtaining a baseline level of microRNA hsa-miR- 152, hsa-miR- 155 or hsa-miR-497 in biological sample from a diseased mammal,

(b) administering to the diseased mammal a treatment for the disease,

(c) obtaining one or more subsequent biological samples from the diseased mammal

(d) measuring the level of microRNA hsa-miR-152, hsa-miR-155 or hsa- miR-497, respectively, in the one or more subsequent biological samples,

(e) comparing the level of microRNA hsa-miR-152, hsa-miR-155 or hsa- miR-497, respectively, in the one or more subsequent biological samples with the baseline sample, and

(f) determining whether increased dosages, additional or alternative treatments are necessary based on microRNA hsa-miR-152, hsa-miR- 155 or hsa-miR-497 levels obtained from one or more subsequent biological samples compared to the baseline microRNA hsa-miR-152, hsa-miR-155 or hsa-miR-497 level, respectively.

(a) obtaining a baseline level of microRNA hsa-miR-152, hsa-miR-155 or hsa-miR-497 in biological sample taken from a diseased mammal,

(b) obtaining one or more subsequent biological samples taken from the diseased mammal with subsequent treatment for the disease,

(c) measuring the level of microRNA hsa-miR-152, hsa-miR-155 or hsa- miR-497, respectively, in the one or more subsequent biological samples,

(d) comparing the level of microRNA hsa-miR-152, hsa-miR-155 or hsa- miR-497, respectively, in the one or more subsequent biological samples with the baseline sample, and

(e) determining whether increased dosages, additional or alternative treatments are necessary based on microRNA hsa-miR-152, hsa-miR- 155 or hsa-miR-497 levels obtained from one or more subsequent biological samples compared to the baseline microRNA hsa-miR-152, hsa-miR-155 or hsa-miR-497 level, respectively.

In a preferred embodiment microRNA hsa-miR-152, hsa-miR-155 or hsa-miR-497 is used as a biomarker for guiding a therapy in a disease which is associated with altered microRNA hsa-miR-152, hsa-miR-155 or hsa-miR-497 levels.

Use of microRNA hsa-miR-152, hsa-miR-155 or hsa-miR-497 as a disease, efficacy or surrogate endpoint biomarker in diagnostics is based by the comparison of microRNA hsa-miR-152, hsa-miR-155 or hsa-miR-497 level, respectively, in a biological sample from a diseased mammal before treatment (the baseline sample level) with the microRNA hsa-miR-152, hsa-miR-155 or hsa-miR-497 level, respectively, in subsequent samples from said mammal receiving a treatment for the disease. Does the microRNA hsa-miR-152, hsa-miR-155 or hsa-miR-497 level in the baseline sample differs from the microRNA hsa-miR-152, hsa-miR-155 or hsa- miR-497 level in the subsequent samples then the therapy can be considered as successful. Does the microRNA hsa-miR-152, hsa-miR-155 or hsa-miR-497 level in the baseline sample does not differ or differs only slightly from the microRNA hsa- miR-152, hsa-miR-155 and hsa-miR-497 level in the subsequent samples then the therapy- -can_be_considered .as. _not_ successful.. Jf_ Jhe_ therapy is considered not successful increased dosages of the same therapy, repeat of the same therapy or an alternative treatment which is different from the first therapy can be considered.

The biological sample in step (a) of the methods is in a preferred embodiment a biological sample comprised in a group of samples consisting of a blood sample, a plasma sample, a serum sample, a tissue sample, a oral mucosa sample, a saliva sample, an interstitial fluid sample or an urine sample. The blood sample is for example a whole blood sample, a fractionated blood sample, a platelet sample, a neutrophil sample, a leukocyte sample, a white blood cell sample, a monocyte sample, a red blood cell sample, a granulocyte sample, and a erythrocyte sample. A tissue sample is for example a sample collected from muscle, adipose, heart, skin or a biopsy.

In a preferred embodiment the level of microRNA hsa-miR-152, hsa-miR-155 or hsa- miR-497 is determined by determining the level of microRNAs hsa-miR-152, hsa- miR-155 and hsa-miR-497 polynucleotide.

In a preferred embodiment of the invention the mammal is a human.

In a preferred embodiment of the invention the level of microRNA hsa-miR-152, hsa-miR-155 or hsa-miR-497 of the biological sample is elevated compared to the control sample.

Another embodiment of the present invention prefers the use of microRNA hsa-miR- 152, hsa-miR-155 or hsa-miR-497 in combination with the use of one or more biomarkers, more preferably with biomarkers used in diagnosing microRNA hsa- miR-152, hsa-miR-155 or hsa-miR-497-associated diseases.

In a preferred embodiment of the invention the use of microRNA hsa-miR-152, hsa- miR-155 or hsa-miR-497 is combined with the use of one or more biomarkers which are comprised in a group of biomarkers consisting of eg ANP, BNP, microRNA 21

(hsa-miR-21), microRNA 1 (hsa-miR-1), microRNA 133 (hsa-miR-133) or other microRNAs known to be regulated in cardiovascular diseases or genes that are regulated by the microRNA hsa-miR-152, hsa-miR-155 or hsa-miR-497 eg Angiotensin II type 1 receptor (AGTRl) or other genes that are predicted by bioinformatic programs or proven by experiments to be regulated by these microRNAs.

In a further preferred embodiment the use of microRNA hsa-miR-152, hsa-miR-155 or hsa-miR-497 is combined with the use of one or more clinical biomarkers which are comprised in a group of biomarkers consisting of blood pressure, heart rate, pulmonary artery pressure, or systemic vascular resistance.

In a further preferred embodiment the use of microRNA hsa-miR-152, hsa-miR-155 or hsa-miR-497 is combined with the use of one or more diagnostic imaging methods which are comprised in a group of methods consisting of PET (Positron Emission Tomography), CT (Computed Tomography), ultrasonic, SPECT (Single Photon Emission Computed Tomography), Echocardiography, or Impedance Cardiography.

In a further preferred embodiment the use of microRNA hsa-miR-152, hsa-miR-155 or hsa-miR-497 is combined with the use of one or more diagnostic methods which are comprised in a group of methods consisting of PET (Positron Emission Tomography), CT (Computed Tomography), ultrasonic, SPECT (Single Photon Emission Computed Tomography), Echocardiography, Impedance Cardiography, blood pressure, heart rate, pulmonary artery pressure and systemic vascular resistance.

A further preferred embodiment is a kit for identifying an individual risk for developing a disease, for predicting a disease or an adverse outcome in a patient diagnosed with a disease, or for guiding a therapy in a patient with a disease, the kit comprising one ore more antibodies which specifically binds microRNA hsa-miR- 152, hsa-miR-155 or hsa-miR-497, detection means, one or more containers for collecting and or holding the biological sample, and an instruction for its use.

Another preferred embodiment is a kit for identifying an individual risk for developing a disease, for predicting a disease or an adverse outcome in a patient diagnosed with a disease, or for guiding a therapy in a patient with a disease, the kit comprising one or more probes or primers for detecting microRNA hsa-miR-152, hsa-miR-155 or hsa-miR-497, detection means, one or more containers for collecting and or holding the biological sample, and an instruction for its use.

hi a preferred embodiment the disease is a cardiovascular disease, in a more preferred embodiment the disease is heart failure.

Examples

Example 1: Expression analysis by micro-fluid cards (Taqman human microarray v 1.0)

TaqMan is a recently developed technique, in which the release of a fluorescent reporter dye from a hybridisation probe in real-time during a polymerase chain reaction (PCR) is proportional to the accumulation of the PCR product. Quantification is based on the early, linear part of the reaction, and by determining the threshold cycle (CT), at which fluorescence above background is first detected.

Gene expression technologies may be useful in several areas of drug discovery and development, such as target identification, lead optimization, and identification of mechanisms of action. The TaqMan technology can be used to compare differences between expression profiles of normal tissue and diseased tissue. Expression profiling has been used in identifying genes, which are up- or downregulated in a variety of diseases. An interesting application of expression profiling is temporal monitoring of changes in gene expression during disease progression and drug treatment or in patients versus healthy individuals. The premise in this approach is that changes in pattern of gene expression in response to physiological or environmental stimuli (e.g., drugs) may serve as indirect clues about disease-causing genes or drug targets. Moreover, the effects of drugs with established efficacy on global gene expression patterns may provide a guidepost, or a genetic signature, against which a new drug candidate can be compared.

Expression analysis of microRNAs can be done by using micro-fluid cards (Taqman human microarrays v 1.0 - Applied Biosystems) which enables simultaneously analysis of hundreds microRNAs in one sample. The Human Taqman microarray consists of 365 microRNAs and of small nucleolar RNAs (snoRNAs) which can be used as endogenous control for data normalization. The principle behind this Taqman array is that the different RNAs samples are reverse transcribed into cDNAs by using unique hairpin-loop RT primers that specifically detect only the mature form of the microRNAs. In particular, all 365 microRNAs are reverse transcribed in eight separate reverse transcribe (RT) reactions. Each RT reaction consists of a pool of primers containing up to 48 primers each. Then, the 8 RT reactions are loaded into the eight filling ports of the Taqman arrays. hi more details, total RNA was isolated from heart tissues with Trizol (a mono- phasic solution of phenol and guanidine Isothiocyanate) according to the manufacturer's specifications (Invitrogen). The isolated RNA was further treated with DNAse I to remove genomic DNA contamination and a part of it was run on the Bioanalyzer to confirm that the RNA was mainly intact and thus can be used for further profiling.

For the relative quantification of microRNAs in heart tissues, the total RNA was first reverse transcribed into 8 separate RT reactions using 8 predefined Multiplex RT primer pools (Applied Biosystems). For each reaction, lOOng of total RNA was reverse transcribed using the Multiplex RT primer pool (consist up to 48 primers),

0.1U MultiScribe™ Reverse Transcriptase (50 U/mL), Ix RT Buffer, 2.5mM dNTPs (with dTTP) and 0.3U RNase Inhibitor. The reaction was incubated at 16°C for 30 minutes, at 42°C for 30 minutes, at 85°C for 5 minutes and then cooled on ice. Then the reaction was further diluted 62,5 times. 50ul of the diluted samples were added into 50 ul of the TaqMan 2x Universal PCR Master Mix (No AmpErase UNG) and this lOOul were further added on each port of the micro fluid card. For relative quantitation of the microRNAs in heart tissues the Applied Bioscience 7900HT Sequence Detection system was used according to the manufacturer's specifications and protocols.

Calculation of relative expression

The analysis of the microfluid card and thus the expression profile of the microRNAs were done by using the comparative Ct method. This method uses an arithmetic formula rather than a standard curve to measure the relative expression of the microRNAs. For this analysis, all the microRNAs data were normalized to the geometric mean of the snoRNAs that are present on the micro fluid card. The formula that was used is the following: 2^"ΔΔCt.

- where ΔCt: Ct _of microRNA - Ct _Of endog control (Target microRNAs - Endogenous Control)

- whereΔΔCt: ΔCt of sample - ΔCt of calibrator (sample refers to microRNAs detected in the heart failure samples where calibrator refers to the microRNAs detected in the non failing samples)

Ihe-abo ve-formula_calcul ates_the_fold_ofjiifference in microRNAs expression in the heart failure samples in comparison to the non failing samples. Therefore, with these calculations always the non failing samples has the value of 1 (2^A° = 1). The relevance of the results from the microRNA microfluid card where further verified by using t-test with p value <0.1 Any Ct value more than 33 cycles were not taken into account.

Example 2: Expression analysis by real time PCR

Expression analysis of microRNAs can be done by using SYBR technology eg miScript System (Qiagen). To this end, total isolated RNA can be transcribed into cDNA by using the miScript Reverse Transcription Kit. The miRNAs are not polyadenylated in nature, however during the reverse transcription step performed by the miScript RT kit, the microRNAs are polyadenylated by poly(A) polymerase, while reverse transcriptase converts the RNA to cDNA using oligo-dT and random primers. The oligo-dT primers have a universal tag sequence on the 5' end, which allows amplification in the real — time PCR step. The generated cDNA can be then used for detection of multiple miRNAs in the real-time PCR step when different miRNA primers are used.

In more details, total RNA was isolated from heart tissues with Trizol according to the manufacturer's specifications (Invitrogen). The isolated RNA was further treated with DNAse I to remove genomic DNA contamination and a part of it was run on the Bioanalyzer to confirm that the RNA was mainly intact and thus can be used for further profiling. For the relative quantification of microRNAs in heart tissues, the total RNA was first transcribed using the miScript Reverse Transcription kit as follows : For a 20ul final reaction, lOOng RNA were mixed with Ix miScript RT

2+ buffer (contains Mg , dNTPs, oligo-dT primers, and random primers) and 1 μl of miScript Reverse Transciptase mix. The micture were incubated at 37°C for 60min, 95 °C for 5 min and then cooled on ice. For the real-time PCR reaction, the following were added in a 384-well plate: template cDNA, Ix QuantiTect SYBR Green PCR Master Mix, Ix miScript Universal Primer and Ix miScript Primer assay (microRNA specific primer).

Calculation of relative expression

The analysis of the real-time PCR using SYBR green for expression profile of the microRNAs were done by using the comparative Ct method.

Example 3: Identification of hsa-miR-152, hsa-miR-155 and hsa-miR-497 regulated genes as biomarker or targets for cardiovascular diseases

Different computational approaches have been developed in order to predict the genes that are regulated from microRNAs eg miRanda, PicTar and TargetScan algorithms. Such computational approaches usually predict around 1000-3000 target mRNAs for each microRNA and therefore further experimental validation for identification of the true mRNA targets is needed. For example, using the miRanda prediction algorithm, the predicted number of mRNA targets is 2.774 for hsa-miR- 152, 2.421 for hsa-miR-155 and 3.806 for hsa-miR-497. However, from these predicted mRNA targets few have been experimentally validated. One such example is with miR-155 where it has been shown that negatively regulates the expression of

Angiotensin II type 1 receptor (13). Therefore, such genes are expected to be regulated according to the microRNA regulation in the cardiovascular diseases eg overexpression of microRNA 155 will have an affect in the levels of the Angiotensin π type 1 receptor. Example 4: In vivo use of microRNAs

Inhibitors or mimics of microRNAs can be administrated in vivo for various purposes e.g. elucidating the physiological or pathophysiological role of microRNAs, for therapeutic applications or for determination of the effective dose. These oligonucleotides can be delivered by known routes of administration including but not limited to intradermal, subcutaneous, intramuscular, intravenous and intraperitoneal-inj ection — CurrentLy_modified_anti;miRNA oligonucleotides (AMOs) or otherwise 'antagomirs' have been used in vivo for miRNA inhibition. These

AMOs can be mainly divided into three categories: modified 2-OH residues of the ribose by 2'-O-methyl (2'-0Me), 2'-O-methoxyethyl (2'-MOE) and locked nucleic acid (LNA). One such example is the inhibition of the microRNA -122 using PBS- formulated LNA-modified oligonucleotide by intravenous injection in non-human primates, where it was shown that it can efficiently silencing the targeted microRNA thus affecting the signaling pathway (14). hi vivo targeted of this microRNA was shown to regulate a number of downstream genes (15).

Restoring the levels of the altered miRNA expression can be beneficial in the case whereas miRNAs expression is downregulated in a disease status eg heart failure, hi this case restoring the mature miRNA leves in the disease tissue could have theurapeutic benefits as this could potential restore the alter expression of the target gene (s). Restoration of the microRNAs levels can be done by using a vector that overexpresses the specific miRNA, by transient transfection of double-stranded miRNAs, by artificial microRNA-shRNA.

Example 5: Production of transgenic Animals

Animal model systems which elucidate the physiological and behavioral roles of the hsa-miR-152, hsa-miR-155 and hsa-miR-497 are produced by creating nonhuman transgenic animals in which the expression of the hsa-miR-152, hsa-miR-155 and hsa-miR-497 is either increased or decreased by a variety of techniques. Examples of these techniques include, but are not limited to: 1) Insertion of normal or mutant versions of DNA encoding a hsa-miR-152, hsa-miR-155 and hsa-miR-497 by microinjection, electroporation, retroviral transfection or other means well known to those skilled in the art, into appropriately fertilized embryos in order to produce a transgenic animal or 2) homologous recombination of mutant or normal, human or animal versions of these genes with the native gene locus in transgenic animals to alter the regulation of expression or the structure of these hsa-miR-152, hsa-miR-155 and hsa-miR-497 sequences. The technique of homologous recombination is well known-in-the-artτ— It-replaces-the-native-gene -with-the-mserted-gene-and-hence_is useful for producing an animal that cannot express native hsa-miR-152, hsa-miR-155 and hsa-miR-497 but does express, for example, an inserted mutant hsa-miR-152, hsa-miR-155 and hsa-miR-497, which has replaced the native hsa-miR-152, hsa- miR-155 and hsa-miR-497 in the animal's genome by recombination, resulting in underexpression of the transporter. Microinjection adds genes to the genome, but does not remove them, and the technique is useful for producing an animal which expresses its own and added hsa-miR-152, hsa-miR-155 and hsa-miR-497, resulting in overexpression of the hsa-miR-152, hsa-miR-155 and hsa-miR-497.

One means available for producing a transgenic animal, with a mouse as an example, is as follows: Female mice are mated, and the resulting fertilized eggs are dissected out of their oviducts. The eggs are stored in an appropriate medium such as cesiumchloride M2 medium. DNA or cDNA encoding hsa-miR-152, hsa-miR-155 and hsa-miR-497 is purified from a vector by methods well known to the one skilled in the art. Inducible promoters may be fused with the coding region of the DNA to provide an experimental means to regulate expression of the transgene. Alternatively or in addition, tissue specific regulatory elements may be fused with the coding region to permit tissue-specific expression of the transgene. The DNA, in an appropriately buffered solution, is put into a microinjection needle (which may be made from capillary tubing using a piper puller) and the egg to be injected is put in a depression slide. The needle is inserted into the pronucleus of the egg, and the DNA solution is injected. The injected egg is then transferred into the oviduct of a pseudopregnant mouse which is a mouse stimulated by the appropriate hormones in order to maintain false pregnancy, where it proceeds to the uterus, implants, and develops to term. As noted above, microinjection is not the only method for inserting DNA into the egg but is used here only for exemplary purposes.

Example 6: Expression analysis of human heart in HF patients

Implantation of left ventricular assist devices (LVAD) often is the only possible means of supporting patients with end-stage heart failure in the form of bridging to transplantation. Like the heartTthe LVAD is a "pump. One end-hooks up to the^~lefr ventricle - that's the chamber of the heart that pumps blood out of the lungs and into the body. The other end hooks up to the aorta, the body's main artery. A tube passes from the device through the skin. The outside of the tube is covered with a special material to aid in healing and allow the skin to regrow. The LVAD is implanted during open-heart surgery. Recent reports demonstrate that LVAD support may be associated with adaptive remodeling of the ventricular myocardium, including reduced LV mass, wall thickness and myocyte diameter, changes in LV pressure- volume relationships and reversal of LV chamber dilation.

References

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3. Lee Y, Jeon K, Lee JT, Kim S, Kim VN (2002) MicroRNA maturation: stepwise processing and subcellular localizationrEMBO-J-^i :4663-4670

4. Jackson RJ, Standart N (2007) How do microRNAs regulate gene expression? Sci STKE 23:243-249

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170:1831-1840

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Profiling MicroRNA Expression in Biological Samples Angew. Chem. Int. Ed. 47: 644 - 652

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A, Hedtjarn M, Hansen JB, Hansen HF, Straarup EM, McCullagh K, Kearney P, Kauppinen S. (2008) Antagonism of microRNA-122 in mice by systemically administered LNA-antimiR leads to up-regulation of a large set of predicted target mRNAs in the liver. Nucleic Acids Res. 2008 Mar;36(4):l 153-62.

Claims

1. A method of use of microRNAs comprised in a group of microRNAs consiting of miR-152, miR-155 and miR-497 as a biomarker for a miR-152, miR-155 and miR-497 related disease comprising :

(a) measuring the level of microRNAs miR-152, miR-155 or miR-497 in a biological sample taken from a mammal,

(b)-measuring the_ level of microRNAs miR-152, miR-155 or miR-497, respectively, in a control sample taken from a mammal,

(c) comparing the level of microRNAs miR-152, miR-155 or miR-497 in the biological sample with the level of microRNAs miR-152, miR-155 or miR- 497, respectively, in a control sample, and

(d) diagnosing a disease based upon the microRNAs miR-152, miR-155 or miR-497 level of the biological sample in comparison to the control sample.

2. A method of use of microRNAs comprised in a group of microRNAs consiting of miR-152, miR-155 and miR-497 as a biomarker for guiding a therapy of a miR-152, miR-155 and miR-497 related disease comprising:

(a) obtaining a baseline level of microRNA miR-152, miR-155 or miR-497 in biological sample taken from a diseased mammal,

(c) measuring the level of microRNA miR-152, miR-155 or miR-497, respectively, in the one or more subsequent biological samples,

(d) comparing the level of microRNA miR-152, miR-155 or miR-497, respectively, in the one or more subsequent biological samples with the baseline sample, and (e) determining whether increased dosages, additional or alternative treatments are necessary based on microRNA miR-152, miR-155 or miR-497 levels obtained from one or more subsequent biological samples compared to the baseline microRNA miR-152, miR-155 or miR-497 level, respectively.

3. A method according to one of the foregoing claims, wherein the biological sample is comprised in a group of samples consiting of a blood, plasma, serum, tissue, oral mucosa, saliva, interstitial fluid, and urine sample.

4. A^~methδd according^{^}tcT^oneπjf^'the^'foregoing^'claims^wherein-the^" biological sample is a heart tissue sample.

5. A method according to one of the foregoing claims, wherein the microRNA is combined with one or more other biomarkers.

6. A method of screening for therapeutic agents useful in the treatment of a miR- 152, miR-155 and miR-497 related disease in a mammal comprising the steps of

(a) determining the activity of a microRNA hsa-miR-152, hsa-miR-155 or hsa- miR-497 at a certain concentration of a test compound or in the absence of said test compound,

(b) determining the activity of said microRNA at a different concentration of said test compound, and

(c) determining compounds that lead to a difference in the activity of the microRNA hsa-miR-152, hsa-miR-155 or hsa-miR-497 comparing step (a) and (b).

7. A method according to claim 6 wherein the following step is added:

(a) determining which of these compounds have an effect on symptoms or diseases related to microRNA miR-152, miR-155 or miR-497 in an in vivo assay.

8. A method according to one of the foregoing claims, wherein the rήiR-152, miR- 155 and miR-497 related disease is a cardiovascular disease.

9. A method according to one of the foregoing claims, wherein the mammal is human and the microRNA is comprised in a group of microRNAs consiting of hsa-miR-152, hsa-miR-155 and hsa-miR-497.

10. A kit for identifying an individual risk for developing a miR-152, miR-155 and miR-497 related disease, for predicting a miR-152, miR-155 and miR-497 reTated^~disease^~or^"an^~adverse^~outcome-in^"a^"patient-diagnosed-with-a-miR-l-52₇ miR-155 and miR-497 related disease, or for guiding a therapy in a patient with a miR-152, miR-155 and miR-497 related disease, the kit comprising one or more probes or primers for detecting microRNAs comprised in a group of microRNAs consiting of miR-152, miR-155 and miR-497, one or more containers for collecting and or holding the biological sample.

11. A kit according to claim 10, wherein the mammal is human and the microRNA is comprised in a group of microRNAs consiting of hsa-miR-152, hsa-miR-155 and hsa-miR-497.

12. A pharmaceutical composition comprising a therapeutic agent identified by the methods of claim 6 or 7.

13. A pharmaceutical composition comprising a microRNA. comprised in a group of microRNAs consiting of miR- 152, miR- 155 and miR-497.

14. A pharmaceutical composition comprising a microRNA comprised in a group of microRNAs consiting of hsa-miR-152, hsa-miR-155 and hsa-miR-497.

15. A pharmaceutical composition according to claims 12-14 for the treatment of a cardiovascular disease.