Field of the invention

The invention relates to the field of molecular biology and medicine, more specifically the invention is directed towards the treatment, delay and amelioration of heart diseases. More in particular, microRNAs are provided that induce cardiac regeneration by inducing cell cycle proliferation of cardiomyocytes, thereby treating or ameliorating heart diseases associated with a loss of cardiomyocytes or cardiomyocyte function. Such diseases include myocardial infarction, cardiomyopathy of ischemic or non-ischemic origin, myocarditis and heart failure.

Background of the invention

Cardiovascular diseases, including hypertension, coronary artery disease and genetic forms of cardiomyopathies may result in heart failure, which is associated with pathological remodelling of the myocardium, pump failure and sudden death.

Epidemiological analysis in Western countries indicates that cardiovascular disorders are among the first causes of morbidity and mortality among people over 60 years. There are approximately 600,000 deaths per year in Europe from myocardial infarction and, even more relevant, heart failure is estimated to affect over 15 million people worldwide, representing one of the leading causes of death. This number is likely going to increase as a consequence of the ageing of the global population. While conventional

pharmacological treatment strategies (e.g., β-blockers and ACE-inhibitors) have shown effectiveness in prolonging survival of heart failure patients [1], the prognosis of affected individuals remains poor, leaving a need for new concepts.

In mammals, enlargement of the heart during embryonic development is primarily dependent on the increase in cardiomyocyte number, but early after birth cardiac myocytes stop proliferating and further growth of the myocardium occurs through hypertrophic enlargement of existing myocardial cells [2]. In the mouse, the stop in cell cycle activity of heart muscle cells occurs shortly after birth; whereas human

cardiomyocytes show a striking reduction in the proliferative capacity after 7 months of age. Recent evidences obtained by dating of cardiomyocyte DNA in humans has indicated that cardiomyocytes physiologically renew at a rate of 1 % at the age of 25 and 0.45% at the age of 75, and that fewer than 50% of the cardiomyocytes are exchanged during the normal life span [3].

As a consequence of this limited proliferation capacity of adult cardiomyocytes, the ability of the mammalian adult heart to repair itself following injury is very restricted [4]. In particular, the loss of cardiomyocyte that occurs after various types of myocardial damage, typically after myocardial infarction, is not repaired by the generation of new contractile tissue but by the formation of a scar, which compromises cardiac function and often tends to worsen over time, leading to heart failure. Thus, the identification of novel means to promote the regeneration of contractile cardiac tissue after injury appears mandatory. However, the exact molecular mechanisms controlling the cell cycle in the adult cardiomyocyte remain largely unknown.

MicroRNAs (MiRs) are a family of small (19-25 nucleotide) single-stranded non-coding RNA molecules that regulate gene expression at the post-transcriptional level. Inhibition of gene expression occurs through complementary base pairing with sequences mainly located in the 3' untranslated region (3' UTR) of the target mRNA [5], leading to translational repression or mRNA degradation. Key recognition elements comprise nucleotides 2-8 at the 5' end of the microRNA and are known as seed sequences [6]. MicroRNAs are often represented as families, defined by conservation of their seed region, with conservation of sequences from nematodes through to humans, implying importance of function during evolution. Between 10-40% of human mRNAs are regulated by microRNAs whereby single microRNA species can regulate multiple mRNA targets and single microRNAs may contain several microRNA recognition sites in their 3'UTR [7]. Such complex regulatory networks can control key biological functions and alterations in microRNA expression are associated with numerous human pathologies including cardiovascular diseases [8, 9].

In contrast to many cellular factors involved in disease, which are difficult to modulate therapeutically, microRNA levels can be easily modulated in vivo by using microRNA mimics which provide a surrogate microRNA action and antimiRs which are RNA molecules comprising sequences complementary to the mature microRNA sequence. Through this complementarity, the antimiRs hybridize to the microRNAs and thereby block their activity. In fact, the efficient use of antimiRs has been demonstrated in non-human primates [10, 1 1], and these studies have been advanced to human clinical trials [12].

Only a few microRNAs have been so far clearly implicated in cardiomyocyte proliferation, including miR-1 , miR-133, miR-199a, miR-590 and members of the miR-15 family. Overexpression of miR-1 in the embryonic heart was shown to inhibit

cardiomyocyte proliferation, which was linked to the repression of Hand2, a transcription factor required for cardiac growth during embryogenesis [13]. miR-133 inhibits proliferation of cardiomyocytes through the repression of SRF and cyclin D2, two essential regulators of muscle cell differentiation [14]. The miR-15 family was shown to regulate the post-natal mitotic arrest of mouse cardiomyocytes, through the downregulation of the expression of Chekl [15]. Exogenous administration of miR-590 and miR-199a) were shown to promote cell cycle re-entry of adult cardiomyocytes ex vivo [16].

Despite the above detailed advances, there remains a need in the art for better and more effective and efficient treatments of heart diseases.

Summary of the invention

The invention relates to a composition comprising a microRNA selected from the group consisting of microRNA 106b, microRNA 93 or microRNA 25 and the

complements thereof, for use in the treatment, prevention, delay or amelioration of a heart disease. More in particular, the invention relates to a composition comprising microRNA microRNA 106b for use in the treatment, prevention, delay or amelioration of a heart disease.

In other terms, the invention provides a method of treating, preventing, delaying, or ameliorating a heart disease wherein a composition comprising a microRNA selected from the group consisting of microRNA 106b, microRNA 93 or microRNA 25 and the complements thereof is administered to a subject in need thereof.

Legend to the figures

Figure 1. Genomic localization of the MicroRNA-106b~25 cluster.

Panel (a) The miR-106b~25 microRNA cluster is located within the minichromosome maintenance deficient 7 (Mcm7) gene on chromosome 5.

Panel (b) Graph showing the expression levels of individual members of the miR-106b~25 microRNA cluster, miR-106b, miR-93 and miR-25 in hearts from mice at distinct time points after birth. P1 indicates I week after birth, P2 two weeks etcetera

Panel (c) Graph showing the expression levels of individual members of the miR-106b~25 microRNA cluster, miR-106b, miR-93 and miR-25 in adult hearts from mice under control conditions, after transverse aortic constriction (TAC) or from transgenic mice with cardiac-specific overexpression of calcineurin (MHC-CnA).

Figure 2. Overexpression of the MicroRNA-106b~25 cluster induces cardiomvocvte proliferation in vitro and in vivo.

Panel (a) Overexpression of the miR-106b~25 microRNA cluster in cultured cardiomyocytes by transfection with precursor molecules for each cluster member results in proliferation as measured by Edu (5-ethynyl-2'-deoxyuridine) incorporation.

Panel (b) Graph showing the quantification of the number of proliferating, cultured cardiomyocytes.

Panel (c) Graph showing the set-up of the experiment. Ten days before sacrifice, the animals received an injection with Edu to mark proliferating cells in vivo. Twelve weeks after the injection, the adult hearts were removed for further analysis.

Panel (d) Graph showing the overexpression of the miR-106b~25 microRNA cluster in vivo by adeno-associated virus 9 (AAV9) gene therapy resulting in proliferation as measured by Edu incorporation.

Panel (e) Quantification of the number of proliferating cardiomyocytes in vivo. Confocal microscopy of the free left ventricular wall stained for alpha-actinin to identify cardiac muscle, Hoechst to identify nuclei and Edu for proliferating myocytes.

Panel (f) Graph showing that EdU positive cardiomyocytes following AAV9- 106b~25 gene therapy demonstrated an approximate doubling of the number of adult proliferating cardiomyocytes as compared to AAV9-MCS therapy (EdU+ CM%).

Figure 3. MicroRNA-106b~25 gene therapy results in regeneration of the infarcted heart in vivo.

Panel (a) Schematic representation of the study setup. Mice were randomized to receive a sham surgery or myocardial infarction (Ml). Immediately after the infarct, animals received either a control AAV9 (AAV9-MCS) or AAV9-106b~25, designed to overexpress the microRNA-106b~25 cluster members.

Panel (b) demonstration that AAV9-106b~25 increases myocardial expression of miR-106b, miR-93 and miR-25.

Panel (c) Schematic representation of the plane of section through the infarcted heart. Sirius red staining indicates a large infarct (red staining) in infarcted animals that received AAV9-MCS gene therapy, and a substantially reduced infarct size in infarcted animals that received AAV9-106b~25 gene therapy.

Panel (d) Infarcted animals that received AAV9-106b~25 gene therapy have greatly increased left ventricular mass.

Panel (e) Infarcted animals that received AAV9-106b~25 gene therapy have maintained left ventricular posterior wall thickness.

Panel (f) Infarcted animals that received AAV9-106b~25 gene therapy have reduced left ventricular internal dimension. Panel (g) Infarcted animals that received AAV9-106b~25 gene therapy have maintained ejection fraction.

Detailed description of the invention

In mammals, during embryogenesis and early after birth, the heart retains an ability to grow through cardiac muscle proliferation, including a preadolescent burst in myocyte proliferation, shortly before adulthood. In the mouse, the stop in cell cycle activity of heart muscle cells occurs shortly after birth; whereas human cardiomyocytes show a striking reduction in the proliferative capacity after 7 months of age. Recent evidences obtained by dating of cardiomyocyte DNA in humans has indicated that cardiomyocytes physiologically renew at a rate of 1 % at the age of 25 and 0.45% at the age of 75, and that fewer than 50% of the cardiomyocytes are exchanged during the normal life span [3]. As a consequence of this limited proliferation capacity of adult cardiomyocytes, the ability of the mammalian adult heart to repair itself following myocardial infarction is very restricted [4], tissue injury is not repaired by the generation of new contractile tissue but by the formation of a scar, which compromises cardiac function and often tends to worsen over time, leading to heart failure. Thus, the identification of novel means to promote cell cycle reactivation in adult cardiac myocytes may have regenerative consequences for the myocardium.

We discovered that microRNAs 160b, microRNA 93 and microRNA 25, herein together referred to as the microRNA cluster miR-106b~25, display high expression in early stages after birth and low expression in the adult heart. The intronic miR-106b~25 cluster is part of the minichromosome maintenance deficient 7 (Mcm7) gene and located in mice on chromosome 5 and in human on chromosome 7 in the same genetic organization (Figure 1 a).

The miR-106b~25 cluster harbors 3 microRNAs, miR-106b, miR-93 and miR- 25 and are co-transcribed. First, we tested the expression of miR-106b, miR-93 and miR- 25 in 5 hearts from wildtype mice sacrificed at postnatal (P) day 1 (P1 ), P3, P5, P7 (or 1 week after birth), P10, P12, P15, P18, P21 (or 3 weeks after birth) and at P56 (or 8 weeks after birth; Figure 1 b). The data show that compared to adult hearts (P56), miR-106b, miR-93 and miR-25 display an expression level of at least 2 fold higher than adult expression levels with a characteristic additional elevation between 5 - 7 fold around P15. This characteristic additional elevation corresponds to a recently described brief but intense proliferative burst of predominantly binuclear cardiomyocytes in preadolescent hearts in mice and humans [19]. Finally, we also evaluated expression of miR-106b, miR-93 and miR-25 in healthy adult myocardium compared to adult heart failure myocardium resulting from sustained pressure overloading by transverse aortic constriction (TAC) or from transgene expressing a constitutively activated form of the pro-hypertrophic phosphatase calcineurin in the myocardium (MHC-CnA). In line with the very limited ability of the mammalian adult heart to induce cardiomyocyte proliferation and to repair itself, the expression of miR- 106b, miR-93 and miR-25 was even lower in failing mouse hearts compared to the healthy myocardium. In conclusion, these data show that members of the microRNA cluster miR- 106b~25 are elevated in expression in the myocardium at time points that correlate with activity of the cell cycle and proliferation of cardiac muscle cells.

Next, to evaluate whether the increased expression of the microRNA cluster miR-106b~25 would result in re-entry of the cell cycle, we cultured neonatal rat cardiomyocytes and stimulated the expression of either miR-106b, miR-93 or miR-25 by precursor microRNA transfection for 24 hrs. Following fixation, we treated the cultures with an antibody for alpha-actinin to detect individual myocytes, counterstained the nuclei with the nuclear stain TOTO-3 and using the nucleoside analogue EdU (5-ethynyl-2'- deoxyuridine) for thymidine substitution to detect chromosomal DNA synthesis

characteristic of the S1 cell cycle phase.

Only myocytes with EdU positivity were considered in the proliferation assay and 1500 cell where counted in each condition (Figure 2a). The data demonstrate that cultured myocytes transfected with a control precursor for scrambled microRNA (pre-scr- miR) demonstrated that 10% of myocytes were proliferating. In contrast, transfection with either miR-106b, miR-93 or miR-25 elevated the proliferation rate of myocytes to around 30% without any apparent difference between the individual members of the microRNA cluster miR-106b~25 (Figure 2b).

To verify these results in vivo, we induced elevated expression of miR-106b, miR-93 and miR-25 by construction of a recombinant adeno-associated virus engineered to overexpress the microRNA cluster miR-106b~25 (AAV9-106b~25). AAV9 has the advantage that it has a natural cardiac tropism for the myocardium resulting in myocardial restricted gene therapy characteristics when injected in animals or humans. To accomplish cardiac gene therapy with overexpression of miR-106b, miR-93 and miR-25, mice were injected at P1 with either 10 ¹¹ particles of a control AAV9 with only the empty multiple cloning site (AAV9-MCS) or AAV9-106b~25 in the tail vein. Ten days before sacrifice, the animals received an injection with Edu to mark proliferating cells in vivo. Twelve weeks after the injection, the adult hearts were removed for further analysis (Figure 2c). The data show that miR-106b, miR-93 and miR-25 were elevated between 4 and 6 fold in mice that received AAV9-106b~25 gene therapy compared to gene therapy with the control AAV9-MCS vector (Figure 2d). Confocal microscopy of the free left ventricular wall stained for alpha-actinin to identify cardiac muscle, Hoechst to identify nuclei and Edu for proliferating myocytes demonstrated a dramatic elevation of myocyte proliferation in vivo following AAV9-106b~25 gene therapy (Figure 2e). Quantification of EdU positive cardiomyocytes following either AAV9-MCS or AAV9-106b~25 gene therapy demonstrated an approximate doubling of the number of adult proliferating

cardiomyocytes (EdU+ CM; Figure 2f).

In conclusion, the combined data demonstrate that elevation of miR-106b, miR-93 and miR-25 evoked cell cycle re-entry and proliferation of cardiomyocytes both in short term cultures in vitro as well as proliferation of cardiomyocytes in the adult myocardium in vivo.

Next we assessed whether elevated expression of miR-106b~25 might boost the normally ineffective myocardial repair that takes place after myocardial infarction.

Adult CD1 mice (8-12-weeks old) underwent permanent left anterior descending coronary artery ligation to induce myocardial infarction (Ml) or sham operation and were injected, in the peri-infarcted area, with AAV9 vectors expressing the three miRNAs (AAV9-106b~25) or a control vector (Figure 3a). In line with the above presented results, this treatment results in efficient myocardial transduction and month-long transgene expression with 4 to 6-fold elevation of individual miRNA cluster members (Figure 3b). Sirius red staining of cross sections just below the ligation indicated a large and thinned infarct in mice subjected to Ml and injected with the AAV9 control vector, while the myocardial integrity was much better preserved and infarct was much smaller in mice subjected to Ml and receiving the AAV9 vector designed to overexpress the miR-106b~25 cluster. As evaluated by echocardiography, left ventricular mass was increased in infarcted mice receiving AAV9-106-25, indicating preserved muscularity (Figure 3d). Functionally, left ventricular posterior wall thickness (LVPW, Figure 3e), left ventricular internal diameter (LVID; Figure 3f) and left ventricular ejection fraction (EF, Figure 3g) were significantly preserved over time in the infarcted mice injected with AAV9-106b~25.

In conclusion, these data show that the expression of miR-106b~25 after infarction exerts a marked beneficial effect in reducing infarct size and improving cardiac function, consistent with the effect of these miRNAs in actively stimulating cardiomyocyte proliferation and regenerative capacity of the heart. Examples

Example 1 : Primary cardiomvocvtes cultures, immunocytochemistry.

Cardiomyocyte cultures were isolated by enzymatic dissociation of 1 - 2- day- old neonatal rat hearts and processed for immunofluorescence as described previously [17]. Neonatal cardiomyocytes were transfected with precursors (Ambion) of microRNAs (10mM) using Oligofectamin (Invitrogen). For visualization of cardiomyocyte size and sarcomeric organization the cells were stained for a2-actinin with mouse monoclonal anti- sarcomeric oactinin antibody (Sigma-Aldrich, A781 1 clone EA-53, 1 :500) followed by rat anti-mouse monoclonal antibody Oregon green-conjugated antibody (Life Technologies, A-889, 1 :1000). Nuclear staining was performed with VECTASHIELD Mounting Medium (Vector Laboratories) with 4',6-diamidino-2-phenylindole (DAPI).

Example 2: Luciferase 3'-UTR reporter assays

Constructs bearing the murine 3'-UTR of Cdknl a, Cdknl c or E2f5 were obtained PCR amplification and subcloned into psiCHECK2 vector (Promega). HL-1 cells were transfected with a scrambled precursor or precursor for mmu-miR1 -106b, mmu-miR- 93 or mmu-miR-25 (Exiqon) at a final concentration of 10 nM in 48-well plates using oligofectamine (Invitrogen). The luciferase reporters were transfected using Fugene-6 reagent (Roche). Twenty-four hours after transfection of the reporters and miRNAs, Firefly and Renilla luciferase activities were measured 48 h after plasmid transfection using the Dual Luciferase Reporter Assay System (Promega), according to the manufacturer's instructions.

Example 3: Production and purification of recombinant AAV vectors

The murine miR-106b~25 cluster plus upstream and downstream flanking sequences (total approximating 300 base pairs) were amplified from human genomic DNA isolated from HeLa cells, using the QIAamp DNA mini kit (Qiagen), according to the manufacturer's instructions. The amplified sequences were cloned into the pZac2.1 vector (Gene Therapy Program, Penn Vector core, University of Pennsylvania, USA), which was used to produce recombinant AAV vectors. Recombinant AAV vectors were prepared in the AAV Vector Unit at ICGEB Trieste, as described previously [18]. Briefly, AAV vectors of serotype 9 were generated in HEK293T cells, using a triple-plasmid co-transfection for packaging. Viral stocks were obtained by CsCI ₂ gradient centrifugation. Titration of AAV viral particles was performed by real-time PCR quantification of the number of viral genomes, as described previously; the viral preparations had titres between 1 ^χ 10 ¹³ and 3 x 10 ¹³ viral genome particles per ml.

Example 4: Injection of AAV vectors in neonatal and adult mice

In the experiments using AAV vectors, neonatal CD1 mice (post-natal day 1 ) were intra-peritoneally injected with AAV9-MCS (AAV9-control) or AAV9-106b~25 at a dose of 1 x 10 ¹¹ viral genome particles per animal, using an insulin syringe with incorporated 30-gauge needle. The hearts of the injected mice were collected 12 weeks after AAV injection.

Adult mice intracardiac injection of AAV vectors (AAV9-control or AAV9- 106b~25), at a dose of 1 ^χ 10 ¹¹ viral genome particles per animal, was performed as described below for animals that underwent myocardial infarction. The hearts of the injected animals were collected 12 days after AAV injection.

Example 5: Myocardial infarction

Myocardial infarction was produced in adult CD1 mice (12 weeks old), by permanent left anterior descending (LAD) coronary artery ligation. Briefly, mice were anesthetized with an intraperitoneally injection of ketamine and xylazine, endotracheally intubated and placed on a rodent ventilator. Body temperature was maintained at 37 °C on a heating pad. The beating heart was accessed via a left thoracotomy. After removing the pericardium, a descending branch of the LAD coronary artery was visualized with a stereomicroscope (Leica) and occluded with a nylon suture. Ligation was confirmed by the whitening of a region of the left ventricle, immediately post-ligation. Recombinant AAV vectors, at a dose of 1 ^χ 10 ¹¹ viral genome particles per animal, were injected immediately after LAD ligation into the myocardium bordering the infarct zone (single injection), using an insulin syringe with incorporated 30-gauge needle.

Example 6: Echocardiography analysis

To evaluate left ventricular function and dimensions, transthoracic two- dimensional echocardiography was performed on mice sedated with 5% isoflurane at 12, 30 and 60 days after myocardial infarction, using a Visual Sonics Vevo 770 Ultrasound (Visual Sonics) equipped with a 30-MHz linear array transducer. M-mode tracings in parasternal short axis view were used to measure left ventricular anterior and posterior wall thickness and left ventricular internal diameter at end-systole and end-diastole, which were used to calculate left ventricular fractional shortening and ejection fraction. Example 7: Heart collection and histological analysis

At the end of the studies, animals were anaesthetized with 5% isoflurane and then killed by injection of 10% KCI, to stop the heart at diastole. The heart was excised, briefly washed in PBS, weighted, fixed in 10% formalin at room temperature, embedded in paraffin and further processed for histology or immunofluorescence. Haematoxylin-eosin and Sirius red staining were performed according to standard procedures, and analysed for regular morphology and extent of fibrosis.

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