Microrna hsa-mir-665 in cardiac hypertrophy

Abstract

To provide a human microRNA for the treatment or prevention of heart diseases associated with cardiac hypertrophy and consequent pathological remodeling of the heart, in particular for preventing and / or treating heart failure (HF).SOLUTION: Provided is a microRNA hsa-miR-665 or a primary transcript of such microRNA or a precursor of such microRNA or a mimic of such microRNA or a DNA coding for such microRNA, for use for the prevention or the treatment of a cardiac pathology characterized by cardiac hypertrophy.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to the fields of medicine and biotechnology.

[0002] In particular, the present invention relates to the human microRNA hsa-miR-665 for the treatment of cardiac diseases associated with cardiac hypertrophy and subsequent pathological remodeling of the heart, in particular for preventing and / or treating heart failure (HF). [Background technology]

[0003] 2. Background of the Invention Despite recent advances in cardiovascular surgery and treatment, cardiovascular disease (CVD) is responsible for more than 17 million deaths each year, representing 31% of deaths worldwide (http: / / www.who.int / cardiovascular_diseases / en / ). 80% of these deaths occur in middle- and low-income countries, so the burden of CVD is no longer restricted to high-income populations.

[0004] Within the scope of CVD, heart failure (HF) represents an end state determined by various underlying pathological causes. The American College of Cardiology / American Heart Association guidelines define HF as "a complex clinical syndrome resulting from any structural or functional cardiac disorder that impairs the ability of the ventricles to fill or empty with blood" [1][2]. The syndrome affects more than 26 million people worldwide, 74% of whom suffer from at least one comorbidity that is significantly worsened by HF [3].

[0005] The adult heart is a dynamic organ and can remodel significantly in response to different extrinsic and intrinsic stimuli. Most pathological stimuli initially induce a stage of cardiac hypertrophy resulting from a compensatory increase in the size of individual cardiomyocytes. Later, this often develops into a form of decompensated hypertrophy, which ultimately determines the remodeling of the LV anatomy and leads to overt HF (reviewed in [4]-[5]). Compensatory hypertrophy can be determined by cardiomyocyte loss due to chronic or sudden ischemic injury (ischemic cardiomyopathy or myocardial infarction), chronic hypertension or valvular heart disease (reviewed in [5]). The same response has also been observed as a consequence of various genetic mutations in hereditary cardiomyopathies leading to a hypertrophic phenotype (reviewed in [5]).

[0006] Cardiomyocyte hypertrophy is also commonly associated with the development of HF with preserved left ventricular (LV) ejection fraction (EF), HFpEF, which currently accounts for approximately 50% of HF cases. As far as this condition is concerned, epidemiological observations suggest that the prevalence of HFpEF compared to HF with reduced EF (HFrEF is usually observed due to ischemic heart disease) is increasing by 10% per decade and becoming endemic [6, 7]. This trend is sustained by the increasing rate of comorbidities associated with HFpEF (especially hypertension and obesity), the increasing life expectancy of the population, and better management of patients with coronary artery disease (CAD). HFpEF patients have a mortality rate close to 60% at 5 years, a high rate of hospitalization, and a significantly impaired quality of life.

[0007] Currently, treatments for HFrEF due to CAD (particularly angiotensin-aldosterone system inhibitors, beta-blockers, LCZ696, and pacemaker / device therapy) are largely ineffective in patients with HFpEF.[8] Treatment with statins showed a relative benefit in a small clinical trial of 185 patients with HFpEF;[9] however, the mechanism of action of this drug remains missing, and the results have not been replicated in a larger number of patients.

[0008] The physiology and molecular correlates of HFpEF are complex and only partially understood. The disease progresses progressively from asymptomatic to symptomatic and is variably characterized by LV hypertrophy, slow LV relaxation, LV diastolic stiffness, reduced LV contractile performance, left atrial remodeling, peripheral vascular resistance, endothelial dysfunction, increased pulmonary arterial and venous resistance, neurohormonal activation and ventricular-arterial coupling

[10] . Although the underlying pathogenesis of HFpEF remains unclear, there is ample consensus that the two most prominent phenotypic correlates of this condition are increased cardiomyocyte stiffness

[11] and impaired cardiomyocyte contractility

[12] . Aging prolongs relaxation and enhances LV stiffness through collagen accumulation and crosslinking

[13] , cardiomyocyte loss and reactive hypertrophy

[14] .

[0009] Thus, there remains a need for therapeutic tools for the treatment of cardiac hypertrophy and its consequences, in particular heart failure, and more particularly for the treatment of currently incurable heart failure with preserved left ventricular (LV) ejection fraction (EF) (HFpEF).

[0010] Additionally, it would be desirable to intervene in cardiac hypertrophy conditions to prevent the onset of associated diseases.

[0011] MicroRNAs are evolutionarily conserved small non-coding RNAs that regulate gene expression at the post-transcriptional level by imperfect base pairing to complementary sequences present in target messenger mRNAs, more frequently in the 3'-UTR, but also in 5'UTRs and coding sequences. Pairing can result in translational repression, mRNA degradation, or both

[15] . The microRNA seed sequence, essential for microRNA binding to mRNA, is a conserved sequence and is most often located 2-8 positions from the microRNA 5' end

[16] .

[0012] MicroRNAs are genome-encoded sequences that are generally transcribed by RNA polymerase II into primary microRNAs (pri-microRNAs). The pri-microRNAs are sequentially processed by different cellular endonucleases, ultimately generating duplexes containing two strands of approximately 19–23 nucleotides. The microRNA duplex is then unwound and the mature microRNA is incorporated into the RNA-induced silencing complex (RISC) to mediate microRNA-mediated silencing (reviewed in

[17] ).

[0013] The role of different microRNAs in cardiac development, hypertrophy and heart failure has been extensively studied (

[18] ; reviewed in

[19] ). In particular, it has been shown that microRNAs are involved in proper cardiac development. Ablation of the entire microRNA network by knockout of Dicer leads to severe heart failure and death in both the developing and adult heart

[20] . Moreover, many gain- and loss-of-function studies have highlighted microRNA families or single microRNAs involved in pathological cardiac remodeling, raising the possibility of using these microRNAs either as biomarkers or targets for novel therapeutics (reviewed in references:

[21] and

[22] ). Several microRNAs have been described to regulate specific processes in cardiac ecology, including cardiomyocyte proliferation (miR-1

[23] , miR-133a

[24] , miR-199a-3p

[25] ), cardiomyocyte hypertrophy (miR-208a

[26] , microRNA-212 / 132 family

[27] , miR-378

[28] ), cardiac fibrosis (miR-29

[29] , miR-21

[30] , miR-378

[28] ), and cardiomyocyte contractility (miR-208a

[26] ).

[0014] Given the role of microRNAs in almost every human disease, many of them are considered as targets for innovative therapeutic approaches, and some even as therapeutic agents themselves.

[0015] However, only a few of these potential applications have made it to clinical trials so far, and none of these have been used to treat heart disease.

[0016] Therefore, there is a strong desire for microRNAs that are involved in heart failure and can be used to treat heart failure and related diseases, with particular reference to the currently incurable form of HFpEF.

[0017] A previously unknown role for the microRNA hsa-miR-665 in regulating cardiomyocyte function has been discovered.

[0018] Indeed, this microRNA has been shown to effectively reduce the size of neonatal cardiomyocytes in prehypertrophic rats and mice.

[0019] Previously published evidence has reported that this microRNA is increased in the blood of patients with heart failure.

[31] Conversely, another report showed that patients with heart failure have even lower cardiac levels of the same microRNA.

[32]

[0020] However, the prior art does not demonstrate or even suggest a causative or beneficial role for this microRNA in the development, let alone the treatment, of heart failure.

[0021] We have now discovered a previously unknown pharmacological role for exogenously administered hsa-miR-665 in cultured cardiomyocytes and the heart in vivo.

[0022] Notably, in hypertrophic conditions, microRNAs have been found to block and reduce cardiac hypertrophy and pathological cardiac remodeling while simultaneously enhancing cardiac function, and they have been found to specifically downregulate the expression of genes involved in regulating the biomechanical stress response, further supporting their therapeutic use in hypertrophic conditions.

[0023] These results, obtained in a well-validated and accepted animal model, allow the development of medicinal products containing hsa-miR-665 for the treatment of cardiac diseases in human subjects.

[0024] Hsa-miR-665 may be used as a therapeutic option to counter pathological hypertrophy and prevent and / or treat heart failure.

[0025] MicroRNAs may also be used to prevent and / or treat pathological cardiac hypertrophy, in particular any condition characterized by increased cardiomyocyte stiffening, such as post-ischemic pathological hypertrophy, HFpEF and genetically determined cardiomyopathies with a hypertrophic phenotype. Summary of the Invention

[0026] One subject of the present invention is the microRNA hsa-miR-665 for the prevention and / or treatment of cardiac pathologies characterized by cardiac hypertrophy.

[0027] In particular, the disease may be selected from the group consisting of pathological cardiac hypertrophy, heart failure with preserved ejection fraction (HFpEF), heart failure with reduced ejection fraction (HFrEF) and cardiomyopathies of genetic and non-genetic origin.

[0028] In particular, one subject of the present invention is the microRNA hsa-miR-665 for the prevention and / or treatment of heart failure, especially when heart failure is a consequence of or associated with cardiac hypertrophy.

[0029] More particularly, the microRNAs may be used to restore cardiac function after the onset of hypertrophy.

[0030] It may also be used in conditions where there is a risk of developing cardiac hypertrophy, such as, for example, myocardial infarction and cardiomyopathies of ischemic or non-ischemic origin.

[0031] Also within the scope of the invention is a method for modulating, preventing and / or treating cardiac hypertrophy in vivo comprising administration of hsa-miR-665 to a subject in need thereof.

[0032] Compositions, vectors and formulations for the administration of hsa-miR-665 for the above-mentioned uses are also within the scope of the present invention.

[0033] Detailed Description of the Invention definition Within the meaning of the present invention, by heart failure is intended a complex clinical syndrome that may result from any structural or functional cardiac disorder that impairs the ability of the ventricles to fill or empty with blood.

[0034] Within the meaning of the present invention, by cardiac hypertrophy, this is intended an abnormal enlargement or thickening of the myocardium due to an increase in the size of cardiomyocytes and changes in other myocardial components such as the extracellular matrix, which leads to a decrease in cardiac function. [Brief description of the drawings]

[0035] [Figure 1] High-content screening for microRNAs that regulate cardiomyocyte cell size. A. Screening workflow. B. Correlation between CM size across screening replicates, Spearman coefficient = 0.84. Each dot represents the effect of an individual microRNA. The dotted line indicates the average area of ​​normal control CM. CM area fold change relative to control (CTRL) after transfection with 875 microRNA; ~2500 cells were analyzed per microRNA / replicate. The effect of hsa-miR-665 is specifically shown. CM area fold change relative to control (CTRL) after transfection with 875 microRNA; ~2500 cells were analyzed per microRNA / replicate. The effect of hsa-miR-665 is specifically shown. [Diagram 2]Validation of hsa-miR-665 anti-hypertrophic effect in rat CM. A, Experimental model. B. Expression levels of the indicated genes assessed by qRT-PCR after treatment of rat CM with phenylephrine PE (40 μM) or in rat CM treated with phenylephrine PE (40 μM) and transfected with hsa-miR-665. Results are the mean ± sd of at least three different experiments. **P<0.01 for hsa-miR-665 vs. untreated cardiomyocytes. [Diagram 3] Effect of overexpression of hsa-miR-665 in the TAC model of chronic pressure overload. A. Experimental model: sham operation or thoracic aortic constriction (TAC) was performed in 10-week-old female CD1 mice. Mice (n=8) that underwent TAC surgery at the same time were injected with AAV9-hsa-miR-665 or AAV9 control (1×1011vg / animal). Cardiac function was analyzed by echocardiography at 15, 30 and 60 days after TAC, and mice were sacrificed at 30 or 60 days after TAC for histological analysis. B. Heart mass reported as the ratio of heart weight to tibia length for sham, AAV9-empty, AAV9-hsa-miR-665 at 30 and 60 days after TAC. **P<0.01; ***P<0.001. C. Measurement of CM cross-sectional area after Periodic Acid Schiff (PAS) staining of heart sections in mice with the same treatment as in panel B. D and E. Echocardiographic assessment of LV anterior wall thickness (LVAW) during diastole and ejection fraction (EF) at 30, 45, and 60 days after intracardiac injection of AAV-control or AAV-miR-665 (1 × 1011 vg / animal) (panels D and E, respectively). *P<0.05; **P<0.01; ***P<0.001. [Figure 4]Effect of hsa-miR-665 overexpression on the established hypertrophic phenotype. A. Experimental model: Sham surgery or thoracic aortic constriction (TAC) was performed in 10-week-old female CD1 mice. 15 days after TAC, mice (n=8) that underwent TAC were injected with AAV9-hsa-miR-665 or AAV9 control (1x1011vg / animal), cardiac function was analyzed by echocardiography 30, 45 and 60 days after TAC, and mice were sacrificed 45 or 60 days after TAC for histological analysis. Echocardiographic measurements of LVAWd-left ventricular anterior wall thickness (B), EF-ejection fraction (C), LVID-left ventricular internal diameter (D) during diastole 30, 45 and 60 days after TAC. [Diagram 5] hsa-miR-665 directly targets FHl1, Enah and Xirp2. A. In vivo mRNAseq of mouse samples upon hsa-miR-665 overexpression under TAC compared to control; Log2 fold change of up / downregulated mRNAs in TAC-mice injected with AAV9-hsa-miR-665 (n=3) versus TAC-mice injected with AAV9-Ctrl (n=3). B. 3'-UTR luciferase reporter assays were performed in HeLa cells transfected with cel-miR-67 (non-targeting control) or hsa-miR-665. Results are shown for the 3'-UTR of Fhl1, Enah, and for the 5'UTR of Xirp2, as well as for empty vector (vector); Renilla luciferase activity was normalized to the activity of firefly luciferase. Values ​​are presented as mean ± sd. *p<0.05, **P<0.01

[0036] The sequence of hsa-miR-665 is up to date and available under the ID hsa-miR-665 (www.mirbase.org; Database Release 21; stem-loop sequence accession number MI0005563, mature sequence accession number MIMAT0004952).

[0037] In particular, hsa-miR-665 may have any one of the following sequences: 5'-UCUCCUCGAGGGGUCUCUGCCUCUACCCAGGACUCUUUCAUGACCAGGAGGCUGAGGCCCCUCACAGGCGGC-3' (SEQ ID NO: 1), corresponding to accession number MI0005563; 5'-ACCAGGAGGCUGAGGCCCCU-3' (SEQ ID NO: 2), corresponding to mature sequence accession number MIMAT0004952. According to the present invention, any one of the above sequences may be used.

[0038] The microRNAs of the present invention were identified for the claimed uses by a high-content, fluorescence microscopy-based, high-throughput screen performed in neonatal rat cardiomyocytes (CMs) using a library of 875 microRNA mimics.

[0039] The present invention also includes primary transcripts, precursors and mimics of hsa-miR-665 for use as disclosed herein for hsa-miR-665. The concepts of microRNA primary transcripts, precursors and mimics are well known in the art and do not require further explanation. In particular, for microRNA mimics, RNA molecules that are intended to "mimic" the activity and function of native microRNA are intended. The use of DNA encoding hsa-miR-665 is also within the scope of the present invention. Such DNA, for example, cDNA, can be designed according to the general knowledge of the art.

[0040] The microRNA hsa-miR-665 has been shown to have anti-hypertrophic effects, in fact it can reduce the size of cardiomyocytes.

[0041] More specifically, it was found that hsa-miR-665 effectively reduced the size of rat and mouse neonatal cardiomyocytes in a strong prehypertrophic state and negatively regulated the expression of genes related to pathological cardiac remodeling after pathological hypertrophy, such as atrial natriuretic peptide (ANP), α-skeletal actin (sk-α actin) and brain natriuretic peptide (BNP), while positively regulated genes involved in normal cardiac function, such as Serca2a, ryanodine receptor (Ryr) and α-myosin heavy chain (Myh6). It was also found that hsa-miR-665 blocked cardiac hypertrophy and pathological cardiac remodeling, while increasing cardiac function after intracardiac injection of a viral vector expressing this microRNA in a mouse model of LV pressure overload.

[0042] Thus, said microRNAs are able to prevent the pathological heart that can occur after pathological hypertrophy.

[0043] Furthermore, it was found that hsa-miR-665 could rescue the pathological phenotype and extend the life span of mice exposed to pathological LV pressure overload by preventing LV dilation and HF.

[0044] Therefore, said microRNAs can be used to treat cardiac hypertrophy and prevent heart failure.

[0045] Finally, without being limited to a specific mechanism of action, hsa-miR-665 activity was found to be involved in post-transcriptional regulation involved in cardiomyocyte contractility and, in particular, modulates cardiomyocyte response to pressure overload by regulating cellular stiffness and elasticity, thereby further strengthening the role of this microRNA in regulating cardiac hypertrophy.

[0046] Therefore, hsa-miR-665 can be used to treat any condition characterized by cardiac hypertrophy, particularly one that is accompanied by increased stiffness of cardiomyocytes.

[0047] In particular, it may be used in the prevention or treatment of any cardiac disease characterized by cardiac hypertrophy.

[0048] A person skilled in the art, for example a physician specializing in the field of cardiology, can identify pathologies characterized by cardiac hypertrophy according to the common knowledge in the field, see also the last edition of Hurst's "The Heart" by Faster and other authors, McGrowth Hill.

[0049] The microRNAs may also be used advantageously in any condition where there is a risk of developing cardiac hypertrophy.

[0050] Indeed, for prevention, administration of microRNAs to subjects susceptible to or at risk of developing a condition of cardiac hypertrophy and thus associated cardiac disease due to the presence of some condition or pathology, such as, for example, genetic predisposition, environmental conditions or hypertension, is contemplated.

[0051] For example, they may be used in conditions characterized by cardiomyocyte loss due to chronic or sudden ischemic insults such as ischemic cardiomyopathy and myocardial infarction, chronic hypertension, valvular heart disease, inherited cardiomyopathies causing a hypertrophic phenotype and cardiomyopathies of unknown etiology, particularly those characterized by increased diastolic dysfunction.

[0052] Thanks to its activity on cardiomyocyte size and stiffness, hsa-miR-665 may be advantageously used especially for the prevention or treatment of heart failure with preserved left ventricular ejection fraction (HFpEF).

[0053] Diseases which may be prevented or treated by the microRNA of the present invention are preferably selected from the group consisting of pathological cardiac hypertrophy, in particular post-ischemic pathological hypertrophy, cardiomyopathies of genetic and non-genetic origin, myocardial infarction, cardiomyopathies of ischemic or non-ischemic origin, myocardial ischemia and heart failure, including heart failure with preserved ejection fraction (HFpEF) and heart failure with reduced ejection fraction (HFrEF).

[0054] The use of hsa-miR-665 in gene therapy is also a preferred embodiment of the present invention.

[0055] Gene therapy contemplates the therapeutic delivery of nucleic acid polymers to cells of a patient as a medicine to treat a disease. According to the present invention, hsa-miR-665 can be delivered to cells of a subject in need thereof to treat such a disease, for example, a subject suffering from or at risk of developing cardiac hypertrophy or heart failure or any of the above-mentioned diseases.

[0056] A further subject of the present invention is an RNA stretch, including a microRNA. The concept of an RNA stretch as a continuous region of RNA is generally known in the art. The RNA stretch can be obtained in vitro by cell-free transcription methods, produced synthetically, or expressed in cells by transfer of the relevant DNA coding sequence, or introduced or expressed in cells by administration of a plasmid, virus or other type of vector.

[0057] The present invention provides stretches of RNA for use as medicaments for the prevention and / or treatment of cardiac hypertrophy and any of the aforementioned diseases.

[0058] In an embodiment of the invention, the microRNA hsa-miR-665 may be administered to a subject as a pharmaceutical by conventional methods.

[0059] Conveniently, the medicament is in the form of a formulation for parenteral, intracoronary, intravenous or intracardiac administration, although other forms are equally suitable for carrying out the invention. The skilled person will determine the effective administration time depending on the condition of the patient, the severity of the disease, the patient's response, and any other clinical parameters within his general knowledge of the matter.

[0060] Another subject of the present invention is a pharmaceutical composition comprising the microRNA hsa-miR-665 as active ingredient for the prevention and / or treatment of cardiac hypertrophy and any associated conditions, in particular heart failure.

[0061] The pharmaceutical compositions of the invention contain at least one of the following: a synthetic RNA corresponding to a microRNA of the invention or its primary transcript or precursor, a DNA encoding the microRNA, a DNA encoding the primary transcript or a precursor for an RNA such as a microRNA produced inside a cell containing this DNA.

[0062] The microRNA (or primary transcript or precursor) of the present invention or the corresponding coding DNA may be administered in conjunction with lipid molecules or peptides, such as cationic lipids, or in the context of a polymer scaffold, which are known in the art to facilitate delivery.

[0063] When the miRNA is administered using lipid molecules such as liposomes as vectors, the sequence of the miRNA is preferably SEQ ID NO: 2 (Accession No. MIMAT0004952).

[0064] Another method of administering such microRNA or its corresponding DNA is by suitable vectors known for the administration of RNA or DNA.

[0065] Preferred vectors are adeno-associated vectors (AAV) of any capsid serotype, either natural (such as but not limited to AAV1, AAV2, AAV8, AAV9) or artificial, viral vectors known for administration of DNA in vivo

[33] .

[0066] When a viral vector is used as the vector to administer the miRNA, the sequence of the miRNA is preferably SEQ ID NO: 1 (Accession No. MI0005563).

[0067] Vectors for use according to the invention comprising at least the microRNA hsa-miR-665 and / or DNA encoding at least said microRNA and / or DNA encoding at least the primary transcript or precursor or a mimic of said microRNA, or combinations thereof, are also subject of the present invention.

[0068] All these methods and formulations for administering synthetic RNA corresponding to the microRNA of the invention, DNA encoding the microRNA, DNA encoding the primary transcript or precursors for RNA such as microRNA produced inside cells containing this DNA are conventional and well known in the art and need not be described further.

[0069] In particular, a person skilled in the art knows how to select appropriate modes of administration and vectors, eg for human administration, according to general knowledge in the art.

[0070] Intracardiac injection and systemic injection are the preferred routes of administration.However, those skilled in the art can decide to administer microRNA by any conventional pharmaceutical composition.See Remington's Pharmaceutical Sciences, final edition.

[0071] The administration schedule, dosage and povidity will be determined by the physician according to the physician's experience, the disease being treated and the condition of the patient.

[0072] Depending on the chosen route of administration, the compositions may be in solid or liquid form suitable for oral, parenteral, intravenous or intraarterial administration.

[0073] The compositions according to the invention contain, together with the active ingredient, at least one pharma- ceutically acceptable vehicle or excipient, which may be particularly useful formulation auxiliaries, such as solubilizers, dispersants, suspending agents and emulsifiers.

[0074] The average amount of active agent may vary and should be based on the recommendations and prescriptions of a specifically qualified physician. EXAMPLES

[0075] The following examples further illustrate the invention. Example 1 A high-content, fluorescence microscopy-based, high-throughput screen identifies microRNAs capable of altering cardiomyocyte size.

[0076] A high-content, fluorescence microscopy-based, high-throughput screen was performed in neonatal rat cardiomyocytes (CMs) using a library of 875 microRNA mimics (988 mature microRNAs, 875 unique sequences, miRBase release 13.0 (2009, http: / / mirbase.org). Cultures of CMs from neonatal rat ventricles were reverse transfected with the library of microRNA mimics. After 72 h, cells were stained with sarcomeric α-actinin to specifically measure the size and number of CMs (Experimental schematic in Figure 1, panel A). Screens were performed in duplicate; replicates showed very good reproducibility (Spearman coefficient 0.84; Figure 1B). Figure 1, panel C, shows the effect of each microRNA on CM cell size, expressed as fold change relative to mock-transfected CMs. On average, approximately 2500 cells per experimental condition were analyzed and replicated.

[0077] We found that 11 microRNAs were able to reduce CM cell size by more than 1 Log2-fold versus control (miR-cel67-transfected CM); hsa-miR-665 was the most effective anti-hypertrophic microRNA.

[0078] Example 2 Validation of screening hits Based on the screening results, we decided to validate and characterize the effects of the selected top microRNAs under anti- and prehypertrophic conditions with the specific aim of evaluating the ability of each microRNA to counter or induce a hypertrophic phenotype. First, we examined the effect of each microRNA on the expression of a set of genes that were previously associated with pathological hypertrophy (fetal cardiac gene program: atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), β-myosin heavy chain (MYH7) and α-skeletal actin (sk-α-actin) [4] or physiological hypertrophy (Serca2a, α-myosin heavy chain (MYH6) and ryanodine receptor (RYR) [4]). In particular, to examine the effect of microRNAs that can reduce CM cell size, we stimulated these cells with phenylephrine (PE), a known prohypertrophic stimulus, and simultaneously stimulated individual microRNAs with PE. MicroRNAs were transfected into rat CM. Total RNA was extracted 72 h after transfection and analyzed by qRT-PCR using TaqMan probes specific for the gene subsets investigated (experimental schematic, Figure 2, panel A). The pure effect of PE (40 μM) on rat CM cultured in 0.1% FBS was assessed by qRT-PCR after 72 h of treatment in the absence of microRNAs. PE strongly upregulated a subset of genes typical of the fetal cardiac gene program (ANP, BNP and sk-α-actin) and significantly increased CM size compared to controls (Figure 2, panel B).

[0079] Hsa-miR-665 was able to revert the signature of pathological hypertrophy induced by PE by reducing the levels of ANP and BNP, while upregulating all known markers of good contractility, Serca2a, RyR and MYH6 (Figure 2, Panel B) [34, 35].

[0080] Example 3 Hsa-miR-665 counteracts the development of hypertrophy and preserves cardiac function after transverse aortic constriction in mice. To evaluate the antihypertrophic effect of hsa-miR-665 in vivo, we investigated whether this microRNA could prevent hypertrophy in a transverse aortic constriction (TAC) model of chronic cardiac pressure overload. Eight-week-old CD1 mice were subjected to TAC or sham surgery and simultaneously injected into the LV anterior wall (LVAW) with an adeno-associated virus (AAV) serotype 9 (AAV9) vector expressing hsa-miR-665 or a control vector (1 × 10 11 vg / animal; n = 8 per group; Figure 3, Panel A). Our previous experience has shown that this procedure results in efficient myocardial transduction and expression of the transgene for up to one month

[25] .

[0081] As reported in Figure 3 panel B, overexpression of hsa-miR-665 was able to counteract cardiac hypertrophic remodeling at both 30 and 60 days and maintained cardiac mass at physiological levels (30 days after TAC, cardiac mass (gr) normalized to tibia length (mm) was 0.09 ± 0.020; for animals receiving control vector, 0.013 ± 0.019, P < 0.001; 60 days after TAC, for AAV9-miR-665-injected animals, 0.010 ± 0.0006 and for animals receiving control vector, 0.015 ± 0.0008, P < 0.01).

[0082] Periodic acid-Schiff (PAS) staining of cardiac sections from treated and control mice revealed a significant decrease in cardiomyocyte cross-sectional area 30 days after TAC (242.26 ± 76.020 μm2 for AAV9-miR-665-injected animals compared with 487.26 ± 104.27 μm2 for animals receiving the control vector, P<0.001) (Figure 3, Panel C).

[0083] Diastolic LV anterior wall thickness (LVAW), as assessed by echocardiography 15, 30, and 60 days after TAC, was significantly reduced in hsa-miR-665-treated mice compared with controls at both 30 and 60 days. As assessed by echocardiography at 15, 30, and 60 days after TAC, LV anterior wall thickness (LVAW) in diastole was significantly reduced in hsa-miR-665-treated mice compared to controls at both 30 and 60 days. At 30 days after TAC, AAV9-miR-665-injected animals had LVAW thickness-d of 1.01 ± 0.05 mm compared to 1.36 ± 0.65 mm for animals receiving the control vector, P < 0.01, and this effect was maintained up to 60 days after TAC, 1.02 ± 0.15 mm for AAV9-miR-665 and 1.44 ± 0.8 mm for control-treated animals; P < 0.01 (Figure 3, panel D).

[0084] Notably, at 30 and 60 days after TAC, left ventricular ejection fraction (LVEF) was also significantly preserved in stenotic mice treated with AAV9-miR-665 compared with control animals (Figure 3, panel E) (30 days after TAC, LVEF in AAV9-miR-665-injected animals was 56.5 ± 5.4, compared with 42.54 ± 5.90% in control-treated animals, P < 0.05; at 60 days after TAC, LVEF in AAV9-miR-665-injected animals was 51.3 ± 5.8, compared with 34.82 ± 0.77% in control-treated animals, P < 0.01).

[0085] Taken together, these results indicate that overexpression of hsa-miR-665 after aortic banding has a favorable effect on reducing cardiac hypertrophy and preserving cardiac function in a model of chronic cardiac overload, consistent with the demonstrated effect of this microRNA in reducing pathological cardiomyocyte hypertrophy in vitro.

[0086] Example 4 Hsa-miR-665 delays the onset of cardiac dilation and dysfunction in the hypertrophied heart. Focusing on the possible translational application of these results, we next decided to test whether hsa-miR-665 could rescue the development of pathological hypertrophy and revert to a normal cardiac phenotype.

[0087] In this rescue experiment, mice were injected with AAV9-hsa-miR-665 or AAV9 control by LVAW (1 × 10 11 vg / animal; 10-week-old female CD1 mice, n=8 per group). Animals were followed for 30, 45 and 60 days (experimental schematic in FIG. 4, panel A). Although no significant rescue of the mild hypertrophy phenotype was observed during follow-up (15 / 30 days after AAV injection - 45 and 60 days after TAC, LVAW-d thickness: 0.88±1.13 / 0.91±0.08 mm for AAV9-miR-665-treated animals and 0.8±0.11 / 0.96±0.33 mm for controls), LVID-d and EF were strongly maintained in the normal range in treated animals versus controls (LVEF at end time point 57.5%±5.60 vs. 28.4%±15 in treated and control animals, respectively, p<0.001; LVID-d at end time: 4.05±0.16 mm, 4.8±0.69 mm in treated and control animals, respectively, p<0.001) (Figure 4 Panels B, C, and D).

[0088] Taken together, these results indicate that hsa-miR-665 exerts profound beneficial effects on the maintenance of cardiac function after aortic constriction-induced pressure overload, which seems consistent with the ability of this microRNA to prevent cardiomyocyte hypertrophy both ex vivo and in vivo.

[0089] Example 5 hsa-miR-665 targets Four and Half Lim domain 1 (FHL1) and promotes LV compliance To identify relevant targets of hsa-miR-665, we assessed global transcriptome changes in the heart by RNA deep sequencing after TAC and transduction with hsa-miR-665 or AAV-control. This analysis identified 90 downregulated transcripts (FPKM > 20, threshold set at 2-fold downregulated) and 47 upregulated mRNAs (FPKM >20, threshold set at 2-fold upregulation) (Figure 5, panel A). We next looked for downregulated transcripts that were functionally coupled to sarcomeric I-band mechanotransduction and myofibrillar remodeling.

[0090] This approach identified three sarcomeric proteins (i.e., Enah, Fhl1, and Xirp2) that were shown to be direct targets of hsa-miR-665 by UTR-luciferase reporter assays (Figure 5, panel B).

[0091] All these transcripts encode proteins localized near the intercalated disc (ID), where they integrate and transmit multiple mechanical stimuli, thereby promoting cardiac hypertrophic remodeling at both the transcriptional and structural levels [36-39]. Furthermore, Sheikh F. et al. (

[40] ) showed that FHL1 binds to the N2B region of titin, a spring-like domain responsible for myofibrillar passive tension during diastolic function

[41]

[42] . Indeed, hearts from FHL1- / - mice exhibit reduced myocardial stiffness, increased contractile function, and a blunted hypertrophic phenotype after TAC

[40] .

[0092] These results indicate that the antihypertrophic effects of hsa-miR-665 are exerted, at least in part, through downregulation of genes known to mediate the biomechanical stress response in cardiomyocytes.

[0093] References [Table 1] [Table 2] [Table 3] [Table 4]

Claims

1. MicroRNA hsa-miR-665 or a primary transcript of such a microRNA or a precursor of such a microRNA or a mimic of such a microRNA or a DNA encoding such a microRNA for use in the prevention or treatment of a cardiac disease characterized by cardiac hypertrophy.

2. 2. The microRNA for use according to claim 1, wherein said cardiac disease is selected from the group consisting of heart failure, cardiomyopathies, pathological cardiac hypertrophy, in particular post-ischemic pathological hypertrophy, cardiomyopathies of genetic and non-genetic origin, in particular cardiomyopathies with a hypertrophic phenotype, myocardial infarction, cardiomyopathies of ischemic or non-ischemic origin, cardiomyopathies of unknown etiology characterized by myocardial ischemia and increased diastolic dysfunction.

3. The microRNA for use according to claim 1, wherein the cardiac disease is heart failure with preserved ejection fraction (HFpEF).

4. The microRNA for use according to claim 1, wherein the microRNA is used in a condition at risk of developing cardiac hypertrophy.

5. 5. The microRNA for use according to claim 4, wherein the risk conditions for developing cardiac hypertrophy are selected from the group consisting of chronic hypertension, cardiac valve disease, inherited cardiomyopathies leading to a hypertrophic phenotype, myocardial infarction, cardiomyocyte loss due to chronic or sudden ischemic injury and cardiomyopathies of ischemic or non-ischemic origin.

6. The microRNA of claim 1 for use in gene therapy.

7. A vector for use in the prevention or treatment of a cardiac disease characterized by cardiac hypertrophy comprising at least the microRNA described in claim 1 and / or a primary transcript of said microRNA and / or a precursor of said microRNA and / or a mimic of said microRNA and / or a DNA encoding said microRNA and / or a DNA encoding the primary transcript, a precursor or mimic of said microRNA, or a combination thereof.

8. 8. The vector for use according to claim 7, which is an adeno-associated vector (AAV) of any capsid serotype, either natural or artificial.

9. A pharmaceutical composition comprising at least the microRNA of claim 1 and / or a primary transcript of said microRNA, and / or a precursor of said microRNA, and / or a mimic of said microRNA and / or a DNA encoding said microRNA, a primary transcript or precursor or mimic therefor, or a combination thereof, and at least one pharma- ceutical acceptable vehicle or excipient, for use in the prevention or treatment of a cardiac disease characterized by cardiac hypertrophy.

10. A composition for use according to claim 9 for the treatment of heart failure.

11. 2. An RNA stretch comprising a microRNA, a primary transcript, a precursor or a mimic thereof according to claim 1 for use in the prevention or treatment of a cardiac disease characterized by cardiac hypertrophy, said stretch being obtained in vitro by cell-free transcription methods, produced synthetically, expressed in a cell by transfer of a relevant DNA coding sequence or introduced or expressed in a cell by administration of a plasmid, virus or other type of vector.

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