Compounds for treatment of ischemic injury

Abstract

The present invention relates to microRNA compounds and pharmaceutical composition comprising them for use in a prophylaxis and / or treatment of cells, tissues and / or organs to protect them against ischemia- reperfusion injury in a patient predisposed to or affected by ischemia, use of the miRNA compounds and nucleic acids encoding them in diagnosis and in the preparation of pharmaceutical compositions and methods for treatment of a patient in need of protecting cells or tissues against consequences of acute ischemia-reperfusion injury.

Description

[0001] Compounds for treatment of ischemic injury

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to microRNA compounds and pharmaceutical composition comprising them for use in a prophylaxis and / or treatment of cells or tissues to protect them against ischemia-reperfusion injury in a patient predisposed to or attacked by ischemia, use of the miRNA compounds and nucleic acid encoding them in diagnosis and in the preparation of pharmaceutical compositions and methods for treatment of a patient in need of protecting cells or tissues against consequences of acute ischemia-reperfusion injury.

[0004] BACKGROUND ART

[0005] Ischemic conditioning stimuli have been shown to induce cardioprotective signaling in experimental animal models of acute ischemia / reperfusion (I / R) injury. However, translation of cardioprotection to clinical practice to treat acute myocardial infarction and its consequences has failed so far. Indeed, clinical trials showed the lack of infarct size sparing effect of several drug candidates targeting cardioprotective mechanism [Botker et al., 2018; Ferdinandy et al., 2014; Hausenloy et al., 2017; Lecour et al.; 2014]. Targeting a single molecular mechanism of myocardial I / R injury may be an insufficient strategy to produce a strong and robust cardioprotective effect [Davidson et al., 2019]. However, unbiased, comprehensive analysis of gene expression fingerprints in normal and protected conditions of the ischemic myocardium may lead to identification of novel molecular targets for cardioprotection and better understanding of its complex molecular mechanisms [Perrino et al., 2017; Schreckenberg et al., 2020; Varga et al., 2015].

[0006] Micro RNAs are small non-coding RNAs (typically about 18 to about 25 nucleotides in length, preferably 19-23 nucleotides in length, preferably about 22 nucleotides) acting as posttranscriptional regulators of gene expression in many cardiac physiological and pathological processes [Colpaert et al., 2019]. Micro RNAs also play an important role in myocardial infarction (MI). The first study demonstrating miRNA dysregulation in MI reported upregulation of miR-15b, miR-21, miR-199, miR-214 and down-regulation of miR-29c and miR-150 in the border zone of infarct in mouse and human samples 3 and 14 days after MI [van Rooij et al., 2008]. MicroRNAs can also mediate protective signals [Moghaddam et al., 2019; Varga et al., 2014], and deciphering their role in cardioprotection can provide novel therapeutic targets [Ong et al., 2018; Perrino et al., 2017]. MicroRNA modulators may result in significant multitarget effects, as one microRNA can modulate the expression of multiple target genes [Lim et al., 2005]. miRNA mimics as well as inhibiting miRNAs’ action with modified antisense oligonucleotides (miRNA inhibitors, antagomiRs) have great potential for novel treatments of cardiac diseases and multitarget therapeutics [Davidson et al., 2019; Roberts et al., 2020; Taubel et al., 2021].

[0007] Cardioprotective miRNAs, that the present inventors have termed protectomiRs previously, were identified with the comparative analysis of their expression in MI and in ischemic conditionings showing protective phenotype [Varga et al., 2014]. The present inventors and others have reported several miRNAs (e.g. miR-139- 5p, miR-125b*, let-7b, miR-487b, miR- 144 / 451 cluster, miR-107 and miR-210) involved in cardioprotection [Kim et al., 2012; Varga et al., 2014; Wang et al., 2012]. Moreover, miR-125b* and other microRNAs were reported previously as common effectors in ischemic pre- and postconditioning [Varga et al., 2014] and miR-21 can play a role in ischemic post- and remote conditioning [Jia P. et al., 2017; Jia Z. et al., 2017]. These results show that certain microRNAs play a role in cardioprotection by ischemic conditioning. Large animal models have a high translational value and therefore should be involved in the preclinical phase of drug development process before entering clinical trials according to regulatory guidelines and position papers [Botker et al., 2018; Lecour et al., 2014]. In porcine AMI models, critical endpoints, including infarct size and microvascular obstruction as well as edema, have been established, allowing correlation of preclinical outcomes with future clinical endpoints [Ellenbroek et al., 2016; Koudstaal et al., 2014]. Therefore, in the present work, according to the 3R animal ethical considerations, the present inventors utilized myocardial samples from one of their previous study [Baranyai et al., 2017], where they carefully analyzed the cardioprotective phenotype according to the recommendations of European Society of Cardiology Working Group of Cellular Biology of the Heart [Botker et al., 2018; Sluijter et al., 2014].

[0008] In EP2768959B1 and in EP3399039B1, both granted to PharmaHungary Ltd., miRNA compounds and pharmaceutical compositions are disclosed for use in a prophylaxis or treatment of cells or tissues to protect them against consequences of acute ischemic and reperfusion injury in a patient predisposed to or affected by ischemia. In EP2768959B1, said miRNA compound is a miRNA agonist of miR-125b* (used in itself or in combination with other miRNA compounds). In EP3399039B1, said miRNA compound is a miRNA antagonist of miR-487b (used in itself or in combination with other miRNA compounds).

[0009] In EP2584040A1, microRNA compounds and pharmaceutical compositions are disclosed for use in a treatment to protect cells, tissues and / or organs against consequences of ischemic injury and / or reperfusion injury in a patient. In the disclosure, miR-450a is identified as a post-ischemic cytopathic miRNA species. It was considered deleterious rather than cytoprotective, i.e. the disclosure states that antagonists or inhibitors against it should have cytoprotective effect against ischemia-reperfusion injury.

[0010] Varga et al. [Varga et al., 2014] aimed to characterize early changes in microRNA expression in acute cardioprotection by ischemic pre- and postconditioning in rat hearts. They identified microRNA mimics or antagonists that may have pre- and postconditioning-like cardioprotective effects: mimics of microRNA- 139-5p, microRNA-125b*, microRNA let-7b, and inhibitor of microRNA-487b showed a significant cytoprotective effect when tranfected into cardiac myocytes subjected to simulated ischemia-reperfusion. They do not mention any effect of miR-450a.

[0011] Li, W. et al. [Li, W. et al., 2019] investigated the role of microRNA-451 (miRNA-451) on cerebral ischemia-reperfusion. According to their results, the expression of miRNA-451 was downregulated in rats with cerebral ischemia-reperfusion. In an in vitro model of cerebral ischemia-reperfusion, the upregulation of miRNA- 451 decreased inflammation. However, they did not discuss whether this inflammatory phenomenon has any relevance in cardiac ischemia-reperfusion injuries.

[0012] Yu-mei Li et al. [Li, Y-m. et al., 2019] investigated the molecular mechanism underlying the cardioprotective effect of propofol against myocardial I / R injury. They demonstrated that propofol-mediated cardioprotection against myocardial I / R injury is dependent of microRNA-451 / HMGBl. They did not mention that miR-451 agonist therapy may have a protective effect.

[0013] Jianlei Cao et al. [Cao J. et al., 2020] investigated the role of microRNA-451 and HMGB1 in the pathological process of myocardial I / R injury. They hypothesized that miR-451 could improve this injury by inhibiting HMGB 1. After their experiment, they concluded that upregulation of miR-451 could prevent myocardial I / R injury by suppressing HMGB 1. Chakraborty, Chiranjib et al. [Chakraborty et al., 2020] reviewed the preclinical / clinical trials about miRNA therapeutics. However, they did not disclose any effect of miR-450a.

[0014] The present inventors aimed (i) to identify cardioprotective miRNA mimics and antagomiRs (protectomiRs) by a systematic analysis of microRNA expression changes due to ischemia / reperfusion with or without ischemic conditioning stimuli according to their previously described approach [Varga et al., 2014] in a clinically relevant closed-chest porcine model of acute myocardial infarction and (ii) to validate their cardiocytoprotective effect in cardiac myocytes subjected to simulated ischemia-reperfusion injury.

[0015] In a broader sense, the aim of the invention was to provide miRNA compounds for use in a treatment to protect cells, tissues and / or organs against consequences ischemia, in particular ischemic injury and / or reperfusion injury in a patient.

[0016] BRIEF DESCRIPTION OF THE INVENTION

[0017] The invention relates to a miRNA compound for use in a treatment to protect cells, tissues and / or organs of a subject against consequences of ischemic injury and / or reperfusion injury in a patient predisposed to or affected by ischemia, wherein said miRNA compound is a miRNA agonist of a miRNA species miR-450a, said miRNA agonist having the same target specificity as miR-450a or overlapping target specificity with miR-450a.

[0018] Preferably, said subject is a vertebrate animal, preferably a mammal, highly preferably a human. In particular the subject is an animal having a cardiovascular system. In particular the subject is a patient.

[0019] Preferably, said miRNA agonist comprises an oligonucleotide moiety, wherein optionally said oligonucleotide moiety is a nucleic acid analogue, wherein preferably one or more nucleotides of said oligonucleotide moiety is / are chemically modified nucleotides, and / or said miRNA agonist comprises one or two tag(s) at either or both end of the oligonucleotide moiety.

[0020] Preferably said miRNA compound comprises a a double stranded oligonucleotide or a single stranded oligonucleotide.

[0021] In a preferred embodiment said miRNA compound shows a cell protective effect in cellular test as provided herein. In a preferred embodiment said miRNA compound shows a cell protective effect in a cardiomyocyte cell viability assay.

[0022] In an embodiment the oligonucleotide is 15 to about 30 nucleotides in length, in particular about 18 to about 25 nucleotides in length, preferably 19 to 23 nucleotides in length, preferably about 22 nucleotides.

[0023] In a preferred embodiment said miRNA agonist comprises a nucleotide sequence identical to the seed region of the corresponding miRNA species or is different at most in 1, 2, 3, 4 or 5 nucleotide(s) from said sequence and / or said miRNA agonist comprises a 17 to 27 nucleotides long nucleic acid segment, the sequence of which is identical to the sequence of said miRNA species miR-450a, or is different at most in 1, 2, 3, 4, 5, 6 or 7 nucleotide(s) from said sequence, respectively, with the proviso that chemically modified nucleotides of natural nucleotides or base analogs of natural bases are not considered as being different in terms of sequences.

[0024] In an embodiment, said miRNA agonist of miR-450a consist of 15 to about 30 nucleotides, in particular of about 18 to about 25 nucleotides, preferably of 19 to 23 nucleotides, preferably of about 22 nucleotides; and comprises the nucleotide sequence AUUGGGAACAUUUUGCAU (SEQ ID NO: 248), or a nucleotide sequence which has at least 75%, preferably at least 80%, preferably at least 85%, more preferably at least 90% sequence identity to SEQ ID NO: 248.

[0025] Preferably, the treatment is to protect cells, tissues and / or organs against ischemic and reperfusion injury. Preferably ischemic and reperfusion injury is acute ischemic and reperfusion injury. In a particular embodiment, the treatment is to protect cells, tissues and / or organs against short term and / or direct consequences of acute ischemic and reperfusion injury.

[0026] In a preferred embodiment, said miRNA compound is administered to the patient within two days, preferably within one day, in particular within 24 hours after the ischemic attack in said tissue and / or organ, or simultaneously with reperfusion therein.

[0027] In a highly preferred embodiment, said miRNA compound is administered to the patient less than two days, preferably less than one day, in particular less than 24 hours before surgery or intervention in said tissue and / or organ.

[0028] In particularly preferred embodiments, the treatment is to protect tissues and / or organs, preferably heart tissues or the heart, against acute ischemic and reperfusion injury in the heart of said patient.

[0029] In particularly preferred embodiments, the treatment is to protect tissues and / or organs, preferably heart tissues or the heart, against acute ischemic and reperfusion injury in the heart of said patient, wherein said miRNA compound is administered to the patient within 6 hours after the ischemic attack or simultaneously with reperfusion and / or said miRNA compound is administered to the patient within 4 or 3 hours after the ischemic attack or simultaneously with reperfusion.

[0030] In a highly preferred embodiment, said miRNA compound is administered to the patient less than one day before surgery or intervention.

[0031] In further embodiments, the miRNA compound of the invention is used in combination with one or more further miRNA compound(s) useful to protect the same cell, tissue and / or organ against ischemic and reperfusion injury, preferably against acute ischemic and reperfusion injury, in particular against short term and / or direct consequences of acute ischemic and reperfusion injury.

[0032] In further embodiments, the miRNA compound of the invention is used in combination with one or more further cardioprotective miRNA compound(s). Preferably in this embodiment the tissue is a cardiovascular tissue, preferably a heart tissue, and / or the organ is an organ of the cardiovascular system, preferably the heart.

[0033] In a particular embodiment, the miRNA compound of the invention is used in combination with a miRNA agonist of a miRNA species miR-451, as defined herein.

[0034] In a further aspect, the invention relates to combination of miRNA compounds for use in a treatment as defined above, wherein said combination comprises a miRNA compound according to the invention and a miRNA agonist of a miRNA species miR-451, wherein the miRNA agonist of miRNA species miR-451 is defined the same way as (analogously to) the miRNA agonist of miRNA species miR-450a defined herein, mutatis mutandis.

[0035] Preferably in the combination according to the invention, said miRNA agonist of miRNA species miR-451 comprises a nucleic acid sequence identical with the seed region of said miRNA species and / or which comprises a 17 to 27 nucleotides long nucleic acid segment, the sequence of which is identical to the sequence of said miRNA species or is different at most in 1, 2, 3, 4, 5 or 6 nucleotide(s) therefrom, preferably while maintaining the agonizing (agonist) effect, as defined herein.

[0036] The invention also relates to a pharmaceutical composition for use in a treatment to protect cells, tissues and / or organs against short term and / or direct consequences of acute ischemic and reperfusion injury in a patient predisposed to or affected by ischemia, in the heart of a patient predisposed to or attacked by acute ischemia, said composition comprising a miRNA compound for use as defined herein or a combination for use as defined herein, and pharmaceutically acceptable excipient and / or carrier.

[0037] In particular, the combination and / or the pharmaceutical composition is for use in a treatment to protect tissues and / or organs, preferably heart tissues or the heart, against acute ischemic and reperfusion injury in the heart of said patient; said miRNA compound is administered to the patient within 24 hours after the ischemic attack or simultaneously with reperfusion and / or said miRNA compound is administered to the patient within 6 or 4 or 3 hours after the ischemic attack or simultaneously with reperfusion.

[0038] In a highly preferred embodiment, said miRNA compound is administered to the patient less than 24 hours before surgery or intervention.

[0039] The invention relates to a method of treatment of a subject in need of a miRNA compound for use as defined herein or a combination for use as defined herein, or a pharmaceutical composition for use as defined herein wherein said miRNA compound, combination or pharmaceutical composition is administered to said subject in a method as defined herein for said miRNA compound, combination or pharmaceutical composition. As defined herein is preferably or in particular as defined in the paragraphs above in the Brief description section or paragraphs below in the Brief description section.

[0040] The invention relates to a miRNA compound for use in a treatment to protect cells, tissues and / or organs against consequences of acute ischemic injury and / or reperfusion injury in a patient predisposed to or affected by ischemia, wherein said miRNA compound is a miRNA agonist of a miRNA species miR-450a, which comprises a nucleotide sequence identical to the seed region of the corresponding miRNA species and / or which comprises a 17 to 27 nucleotides long nucleic acid segment the sequence of which is identical to the sequence of said miRNA species miR-450a, or is different at most in 1, 2, 3, 4, 5 or 6 nucleotide(s) from said sequence, respectively, wherein said miRNA compound shows a cell protective effect in a cardiomyocyte cell viability assay.

[0041] In an embodiment of the invention, acute myocardial infarction is treated or prevented.

[0042] In a preferred embodiment, an additional miRNA compound is used in combination with the miRNA agonist of miR-450a, wherein the additional miRNA compound is a miRNA agonist of a miRNA species miR- 451.

[0043] Preferably, any further miRNA compound for use in a treatment to protect cells, tissues and / or organs against consequences of ischemic injury and / or reperfusion injury in a patient predisposed to or affected by ischemia may be selected from a miRNA compound obtainable by a method of the invention as disclosed herein. The invention also relates to the miRNA compound of the invention for use in a treatment to protect cells, tissues and / or organs against short term and / or direct consequences of acute ischemic and reperfusion injury in a patient predisposed to or affected by ischemia, in the heart of said patient, wherein said miRNA compound is for administration to the patient within 24, preferably 12, 8, 6, 5, 4, 3, 2 or 1.5 hours after the ischemic attack or simultaneously with reperfusion and / or less than one day or less than 24, preferably 12, 8, 6, 5, 4, 3, 2 or 1 hour(s) before surgery or intervention associated with a risk of ischemia, wherein said miRNA compound is a miRNA agonist of a miRNA species miR-450a, wherein the miRNA agonist is a nucleic acid moiety comprising a nucleotide sequence identical to the seed region of the corresponding miRNA species and / or which comprises a 17 to 27 nucleotides long nucleic acid segment the sequence of which is identical to the sequence of a mature miRNA species, or is different at most in 1, 2, 3, 4, 5 or 6 nucleotide(s) therefrom, wherein said miRNA compound shows a cell protective effect in a cardiomyocyte cell viability assay.

[0044] In a highly preferred embodiment, said miRNA compound is administered to the patient within 4 or 3 hours after the ischemic attack or simultaneously with reperfusion and / or less than 6 hours before surgery or intervention.

[0045] In a preferred embodiment the invention also relates to a combination for use of more than one miRNA compounds for use in a treatment to protect cells, tissues and / or organs against consequences of acute ischemic and reperfusion injury in a patient affected by ischemia or against consequences of ischemic injury and / or reperfusion injury in a patient predisposed to ischemia, wherein preferably said combination comprises at least two miRNA agonists, wherein the two miRNA agonists are a miRNA agonist of miR-450a and a miRNA agonist of miR-451, and optionally further miRNA compounds, preferably said agonists comprising a nucleotide sequence being identical with the seed region of said miRNA species and / or which comprises a 17 to 27 nucleotides long nucleic acid segment the sequence of which is identical to the sequence of said miRNA species or is different at most in 1, 2, 3, 4, 5 or 6 nucleotide(s) therefrom, wherein said miRNA agonist compound shows a cell protective effect in a cardiomyocyte cell viability assay. Preferably said further miRNA compounds also show a cell protective effect in a cardiomyocyte cell viability assay. Preferably said further miRNA compound is identified by the method for identifying a micro-RNA (miRNA) species described hereinbelow.

[0046] In a preferred embodiment, the treatment is to protect cells, tissues and / or organs against consequences of acute ischemic and reperfusion injury in the heart of said patient affected by ischemia. In another preferred embodiment, the treatment is to protect cells, tissues and / or organs against consequences of ischemic injury and / or reperfusion injury in the heart of said patient predisposed to ischemia.

[0047] In a preferred embodiment the invention also relates to a method of treatment wherein said miRNA compound or combination for use, preferably said combination for use is administered to a subject, in particular a patient.

[0048] In a preferred embodiment, said miRNA compound is for use in cytoprotection in a tissue endangered by ischemia in a patient predisposed to ischemia, said miRNA compound being administered to said patient before or during surgery or during intervention associated with a risk of ischemia, wherein the miRNA compound is a miRNA agonist of miR-450a. In another preferred embodiment, the miRNA compounds are miRNA agonist of miR-450a and a miRNA agonist of miR-451. The cells, the tissue or the organ may be endangered by ischemia e.g. due to surgery or a therapeutic intervention or due to patient condition. The surgery or intervention is preferably one wherein the ischemic event can be predicted in advance, e.g. in which blood vessels, e.g. an artery or arteries are or may be blocked or blood flow is reduced or there is a risk of occlusion thereof or a risk of sudden hypoperfusion.

[0049] The surgery or intervention may be an emergency intervention or elective intervention, cardiac surgery, e.g. percutaneous coronary intervention (PCI) or angioplasty, placement of stents, removal of ill-functioned blood vessels, e.g. varicose veins, lung surgery, transplantation of the heart, lung, kidney, liver, skin, muscle etc., surgery of the brain or nervous system, removal of tumors from the heart, brain, lung, kidney, liver, bowels, peripheral vessel diseases. Preferably the miRNA compounds are applied typically for preventive purposes.

[0050] Preferably, the miRNA compound is for use in a patient predisposed to ischemia wherein said patient is under a lingering risk of ischemia and the miRNA compound is administered to said patient regularly for a given period of time, preferably for at least one week, more preferably for at least two weeks, three weeks, one month, two months or more, for cytoprotection in a tissue endangered by ischemia.

[0051] In any embodiment of the invention, preferably, the patient predisposed to ischemia is a patient predisposed to acute ischemia and / or acute reperfusion injury. Preferably, the patient is a patient having high risk or at least medium risk of cardiovascular disease or of ischemic heart disease, preferably of acute ischemia and / or acute reperfusion injury, or preferably ischemic heart attack or myocardial infarction. Preferably the patient is at risk of dying in one or more of these conditions within the subsequent 15, 12, 10, 8, 5, 3 or 1 year(s).

[0052] Preferably the miRNA compound is administered before less than one week, less than one day or less than 12, 8, 6, 5, 4, 3, 2 or 1 hour(s) before surgery or intervention.

[0053] In a further preferred embodiment, the miRNA compound is for use in cytoprotection in a tissue of a patient having acute ischemic injury and / or acute reperfusion injury and / or a patient receiving reperfusion therapy, and is administered

[0054] - between the ischemic attack and reperfusion, or immediately before reperfusion, after the ischemic attack, preferably within three days, one day or half day, more preferably within 6, 5, 4, 3, 2 or 1.5 hours, more preferably within 60, 50, 40 or 30 minutes after the ischemic attack, during reperfusion or after reperfusion, preferably within three days, 60 hours, two days, one day, 24 or 12 hours, more preferably within 6, 5, 4, 3, 2 or 1.5 hours, more preferably within 60, 50, 40, 30, 20 or 10 minutes after reperfusion or simultaneously with reperfusion, wherein the miRNA compound is a miRNA agonist of miR-450a, preferably miRNA agonist of miR-450a and a miRNA agonist of miR-451.

[0055] In a preferred embodiment the miRNA compound comprises at least 16, 17, 18 or 19 nucleotide units, preferably about 18 to about 25 nucleotide units having or corresponding to the sequence of the miRNA species or a sequence different in 1 , 2 or 3 nucleotide unit(s) therefrom.

[0056] Preferably, the miRNA agonist is a miRNA mimic of said miRNA species.

[0057] In a preferred embodiment assessing the expression level of miRNA species is carried out by RNA array or miRNA array or "chip" technology or by quantitative RT-PCR technology. The invention also relates to a pharmaceutical composition for use in the treatment of a patient predisposed to or affected by ischemia to protect cells or tissues against consequences of ischemic injury and / or reperfusion injury in said patient, said composition comprising a miRNA agonist of miR-450a and optionally a miRNA agonist of miR-451, or a combination of a miRNA agonist of miR-450a and miR-451, and pharmaceutically acceptable excipient(s) and / or carrier(s).

[0058] Application or administration of multiple compounds or compositions can take place either sequentially or simultaneously.

[0059] In a preferred embodiment the pharmaceutical composition comprises multiple compounds. In a further embodiment the invention relates to a pharmaceutical kit comprising multiple compounds.

[0060] It is disclosed herein that the up-regulation or down-regulation of said miRNA agonist or miRNA antagonist, respectively, in said mammalian heart tissue sample during preconditioning or postconditioning or remote preconditioning can be assessed by the following method. Thus, the invention relates to or discloses a method for identifying a micro-RNA (miRNA) species as a target for treatment to protect cells or tissues against consequences of acute ischemic injury in a patient predisposed to or attacked by ischemia, said method comprising the following steps: i) providing a set of biological samples comprising expressed miRNA species, said set of samples comprising a control sample, a first sample and a second sample and / or a third sample and / or a fourth sample, wherein the samples can be subjected to ischemia, ii) exposing each of the samples to aerobic perfusion for a time-period, and within this time period the control sample is exposed neither to ischemia nor to preconditioning nor to postconditioning nor to remote perconditioning, thereby obtaining a non-ischemic control sample, and exposing the first sample to ischemia (preferably to ischemia and reperfusion), thereby obtaining an ischemic sample, and preconditioning the second sample by exposing it to a preconditioning protocol and then to ischemia, thereby obtaining a preconditioned sample, and / or postconditioning the third sample by exposing it to ischemia and to a postconditioning protocol, thereby obtaining a postconditioned sample, and / or remote perconditioning the fourth sample by exposing it to ischemia and to a remote perconditioning protocol, thereby obtaining a perconditioned sample, iii) assessing the expression level of said miRNA species

[0061] - in the non-ischemic control sample

[0062] - in the ischemic sample as compared to the non-ischemic control sample and

[0063] - in the preconditioned sample as compared to the non-ischemic control sample and / or

[0064] - in the postconditioned sample as compared to the non-ischemic control sample, and / or

[0065] - in the remote perconditioned sample as compared to the non-ischemic control sample, and iv) calculating the ratio of the expression levels of said miRNA species

[0066] - in the preconditioned sample as compared to the ischemic sample and / or

[0067] - in the postconditioned sample as compared to the ischemic sample and / or

[0068] - in the remote perconditioned sample as compared to the ischemic sample, v) identifying said miRNA species the expression level of which is up-regulated by at least 1.3 fold, preferably by at least 1.5 fold or down-regulated by at least 1.3 fold, preferably by at least 1.5 fold, in the preconditioned sample and / or in the postconditioned sample and / or in the remote perconditioned sample relative to the ischemic / reperfused sample.

[0069] Preferably, the miRNA species are termed as defined above dependent on their up -or down-regulation in the samples.

[0070] In the method disclosed herein the calculated ratios of the expression levels are obtained by a) assessing the expression level of miRNA species

[0071] - in the non-ischemic control sample

[0072] - in the ischemic / reperfused sample and

[0073] - in the preconditioned sample and

[0074] - in the postconditioned sample and

[0075] - in the remote perconditioned sample, b) calculating the ratio of the expression levels of said miRNA species

[0076] - in the ischemic / reperfused sample relative to non-ischemic control sample

[0077] - in the preconditioned sample relative to non-ischemic control sample

[0078] - in the postconditioned sample relative to non-ischemic control sample

[0079] - in the remote perconditioned sample relative to non-ischemic control sample

[0080] - in the preconditioned sample relative to the ischemic / reperfused sample and / or

[0081] - in the postconditioned sample relative to the ischemic / reperfused sample and / or

[0082] - in the remote perconditioned sample relative to the ischemic / reperfused sample.

[0083] Unless specifically indicated otherwise, in the present disclosure one or more embodiments or features described in respect of a given aspect or embodiment of the invention can be combined with any other aspect or embodiment disclosed herein provided that it is technically reasonable for a person skilled in the art and provides the contribution to the prior art according to the present invention.

[0084] DEFINITIONS

[0085] A "miRNA (microRNA) species" is a naturally occurring ribonucleic acid (RNA) molecule which is a small, about 18 to about 25 nucleotides long non-coding RNA molecule that is an endogenous physiological regulator of gene expression [Bartel, 2004]. MiRNAs play an essential role in the posttranscriptional regulation of gene expression by repression or activation of translation / transcription ("RNA interference").

[0086] The miRNA sequences, unless defined otherwise herein, are from the miRBase database release 20 (ftp: / / mirbase.org / pub / mirbase / 20 / database_files / ) [Kozomara A, 2019; Kozomara A and Griffiths-Jones, Sam, 2014],

[0087] A "miRNA compound" is a synthetic or artificial nucleic acid compound which is an agonist (miRNA agonist) or an antagonist of a miRNA species. A "nucleic acid compound" is an organic molecule comprising a nucleic acid consisting of nucleotide units having a specific sequence which can be determined by chemical or biochemical method and optionally comprising one or more further moieties e.g. for improved biological function, stability, targeting, reporting or detectability.

[0088] A "miRNA agonist" of a miRNA species is a nucleic acid compound which, once introduced into a biological material, e.g. cells or tissue, due to its nucleotide sequence, produces the same type of a biological effect or pattern of effects, e.g. induce the regulation of the same gene or genes as the corresponding miRNA species in the same biological material.

[0089] MiRNA compounds may comprise natural nucleotides or nucleotide analogs.

[0090] As far as the bases in the miRNA compound are naturally occurring bases like A, T, G, C or U, or an analog thereof having unchanged base-pairing properties, in particular if the base is selected from A, T, G, C or U, and the base is unchanged in the position given, the sequence is considered as identical with the original miRNA sequence even if the sugar moiety or the phosphate backbone is altered or chemically modified (e.g. stabilized).

[0091] A "miRNA mimic " is a miRNA agonist which is an artificial (man-made) double-stranded RNA or RNA derivative which mimics mature endogenous miRNAs after transfection into cells and / or copy the functionality of mature endogenous miRNA upon transfection. The miRNA mimic may be chemically synthesized or recombinantly produced and processed from a precursor. Optionally, a miRNA mimic may comprise one or more modified or artificial base(s), sugar unit(s) or internucleotide linkage(s)

[0092] A „miRNA agomir” is a miRNA mimic with chemical modifications to prevent degradation and / or increase transfection efficiency. miRNA mimics and agomirs bind to the 3'UTR of genes to knock down native gene expression in cells. They can be used for functionality assessments and serve as useful exogenous tools for gain-of-function studies.

[0093] A "miRNA antagonist" of a miRNA species is, in a broader sense, any compound which is capable of reducing, inhibiting or blocking the regulatory effect of a miRNA. Preferably, it is a nucleic acid compound which, once introduced into a biological material, e.g. cells or tissue, due to its nucleotide sequence, antagonizes the effect of the corresponding miRNA species in the same biological material, e.g. inhibits or silences the regulation of said miRNA species on a gene or genes.

[0094] In a preferred embodiment, the miRNA compound has a nucleic acid moiety comprising a nucleotide sequence identical with the seed region of the corresponding miRNA species.

[0095] In a preferred embodiment, a miRNA compound is a nucleic acid compound which comprises a 17 to 27 nucleotides long, preferably 18 to 24 or 19 to 24 or 19 to 23 nucleotides long nucleic acid segment, the sequence of which ("core sequence") is identical to the sequence of a mature miRNA species, or is different at most in 1, 2, 3, 4, 5 or 6 nucleotide(s) therefrom, while maintaining the agonizing effect. A miRNA compound may also comprise modified bases (in this case in the sequence a modified base is to be considered as the starting base from which it is modified), or artificial bases; if it is not evident which of the four natural RNA bases correspond to the artificial base then the artificial base is to be considered in the sequence as a difference or mutation. miRNA compound may also comprise any modified internucleotide linkage or linkages. Such modified linkages are well known in the art and suitable, for example for stabilizing oligonucleotides. Moreover, the miRNA compound may comprise flanking regions which flank the core sequence. "Ischemia" is a restriction, i.e. an absolute or relative shortage in blood supply to a tissue or cells of a tissue or to a whole organ with resultant damage or dysfunction of the tissue with a consequence of reduced oxygen delivery to the tissue (hypoxia). Insufficient blood supply leads to hypoxic tissue (anoxic in case of no oxygen supply at all) with the consequence of necrosis, apoptosis or autophagy which all determine the cell-death.

[0096] Ischemia is "acute ischemia" if it is due to an ischemic attack, preferably a sudden shortage of blood supply due to e.g. embolism, thrombosis, thromboembolism (blood clots), sudden obstruction of a blood vessel, e.g. by surgery, occurrence of foreign bodies in the circulation or vascular injury. "Acute ischemia" is to be treated within 3 or 2 days, preferably within 24 or 12 hours, more preferably within 6, 5, 4, 3, 2 or 1.5 hours from the onset of symptoms. Short term consequences of acute ischemia are directly the consequences of cell death. In case of cardiac ischemia, cell death results in pumping failure of the heart, causing hypotension / hypoperfusion of other organs (brain, kidney, bowels, etc.), this is known as cardiogenic shock. The cell death also results in altered electrical activity of myocardial cells, very often resulting in life threatening arrhythmias (tachycardia, fibrillation, bundle branch block), causing sudden cardiac death. Death of papillary muscles often results in cardiac valve dysfunction, thereby causing regurgitation of blood and ineffective pump function. Death of the cardiac cell may cause aneurysm formation and rupture of the myocardial wall.

[0097] Acute ischemia of the brain is "stroke".

[0098] In "chronic ischemia" the shortage in blood supply is developed gradually and is partial, due to e.g. atherosclerosis (lipid-laden plaques obstructing the lumen of arteries), hypotension (e.g. in sepsis or heart failure), etc.

[0099] Ischemia may be cardiac ischemia, e.g. ischemia of the myocardium.

[0100] In a "transient ischemic attack" (TIA) the ischemic symptoms disappear within a few minutes. TIAs are usually caused by a blood clot blocking one of the blood vessels leading to the given tissue (e.g. brain, heart, liver or kidney).

[0101] A "patient predisposed to ischemia" is a patient who is inclined to, susceptible to, is endangered by an ischemic attack, for example a patient who

[0102] - has experienced an ischemic attack [e.g. a patient at risk for postoperative myocardial infarction (PMI)], shows any genetic or metabolic risk factor of metabolic syndrome, diabetes, obesity, hypertension, atherosclerosis, high level of low-density lipoprotein cholesterol, elevated serum homocysteine, increased platelet function or deteriorated platelet function, smoking, low level of daily physical exercise, in particular aerobic exercise, or multiple risk factors, is or is to be subjected to a therapeutic intervention increasing the risk of ischemia, e.g. an intervention resulting in a hypoxic state, e.g. a surgical intervention, in particular a surgery in which the cardiovascular system is involved, etc. For example, the intervention can be an elective cardiac interventions, such as elective PCI, heart surgery, coronary artery bypass graft surgery (CABG) or (early) surgical revascularization e.g. after an acute myocardial infarction (AMI).

[0103] "Reperfusion" is the restoration of blood supply to a tissue which is ischemic due to decrease in normal blood supply. The decrease may result from any source including atherosclerotic obstruction, narrowing of the artery, or surgical clamping. Reperfusion may occur spontaneously or may be effected as a part of a treatment. It is primarily a procedure for treating infarction or other ischemia, by enabling viable ischemic tissue to recover, thus limiting further necrosis and therefore infarct size. However, reperfusion can itself further damage the ischemic tissue, causing reperfusion injury.

[0104] "Ischemic and reperfusion injury" or "reperfusion injury" or "ischemic -reperfusion injury" (I / R injury) is understood herein as any functional, metabolic and / or structural changes, including necrosis, apoptosis, and autophagy, in ischemic tissues which are consequences of reperfusion, or ischemia and reperfusion together, to any area of the tissue affected by ischemia.

[0105] "Acute ischemic-reperfusion injury" or "acute reperfusion injury" refers to ischemic-reperfusion injury, i.e. any consequences, preferably short term and / or direct consequences of reperfusion, or ischemia and reperfusion together. In a sense, in acute reperfusion injury, as a consequence, cell death occurs, at least pre-eminently, via a necrotic and / or apoptotic mechanism and / or via autophagy. In a sense, acute reperfusion injury is insensitive to anti-inflammatory treatment. In a sense, any consequence, optionally cell death, occurs during or soon after or immediately after reperfusion, preferably within one day, more preferably within 5, 4, 3, 2 or 1.5 hours, more preferably within 60, 50, 40 or 30 minutes after reperfusion.

[0106] Much of the consequences and damage that occur after an acute ischemia-reperfusion injury is caused by acute inflammatory responses during reperfusion, the restoration of blood flow to the affected organ.

[0107] "Acute myocardial infarction" (AMI) is myocardial infarction occurring during the period when circulation to a region of the heart is obstructed and myocardial cell death due to necrosis, apoptosis or autophagy is occurring. AMI is to be treated within 3 or 2 days, preferably within 12 hours or 6 hours, more preferably within 5, 4, 3, 2 or 1.5 hours from the onset of symptoms. AMI is characterized by acute ischemia and acute reperfusion injury.

[0108] "Ischemic heart disease" (IHD) or myocardial ischemia is a disease characterized by ischemia of the heart muscle. IHD includes acute myocardial infarction that causes death of myocardial tissue thereby acute heart failure and arrhythmias, as well as chronic myocardial infarction causing cardiac tissue remodeling and heart failure.

[0109] "Ischemic preconditioning" is the exposure of the tissue (e.g. myocardium, kidney or nervous tissue) endangered by ischemia to brief, repeated periods of hypoxia, preferably ischemia (e.g. by vascular occlusion). In an embodiment, ischemic preconditioning includes exposure of the tissue by an external effect having the same result in the tissue as said repeated periods of hypoxia; this can be achieved e.g. by treatment with pharmaceutical, physical, and chemical agents mimicking the preconditioning effect. Preconditioning has a cardioprotective effect, renders the tissue resistant to the deleterious effects of ischemia or reperfusion and lessens myocardial infarct size and dysfunction and arrhythmias after ischemia.

[0110] A "preconditioning protocol" is the sequence of a given number of time-periods of hypoxia, preferably with a given time-period of normal oxygen supply, e.g. by reperfusion between them. For example, the time-period of pre-exposure may be e.g. 1 to 10, minutes, 2 to 8, 7 or 6 minutes, more preferably about 3 to 6 or 5 minutes and the number of times the tissue is exposed to ischemia and reperfusion may vary.

[0111] "Ischemic postconditioning" is the exposure of the tissue (e.g. myocardium, kidney or nervous tissue) attacked by ischemia to brief, repeated periods of hypoxia, preferably ischemia (e.g. by vascular occlusion) briefly after ischemia. In an embodiment ischemic preconditioning includes exposure of the same tissue or any remote tissue in the body by an external effect having the same result in the tissue as said repeated periods of hypoxia; this can be achieved e.g. by treatment with pharmaceutical, physical, and chemical agents mimicking the preconditioning effect. Postconditioning has a cardioprotective effect, renders the tissue resistant to the deleterious effects of ischemia-reperfusion injury and lessens myocardial infarct size and dysfunction and arrhythmias after ischemia.

[0112] A "postconditioning protocol" is the sequence of a given number of time-periods of hypoxia, preferably with a given time-period of normal oxygen supply, e.g. by reperfusion between them after an ischemic attack. For example, the time-period of pre-exposure may be e.g. half minute to 10 minutes, more preferably about 1 -2 minutes and the number of times the tissue is exposed to ischemia and reperfusion may vary from 1 to 10, or preferably from 1 to 6.

[0113] “Ischemic remote perconditioning” is the exposure of a tissue different from the tissue of interest, i.e. the tissue which is considered to be treated, to repeated periods of hypoxia, preferably ischemia (e.g. by vascular occlusion). In an embodiment ischemic remote perconditioning includes exposure of any remote tissue in the body by an external effect having the same result in the tissue as said repeated periods of hypoxia; this can be achieved e.g. by treatment with pharmaceutical, physical, and chemical agents mimicking the perconditioning effect.

[0114] A “remote perconditioning protocol” is the sequence of a given number of time-periods of hypoxia, preferably with a given time-period of normal oxygen supply, e.g. by reperfusion between them after an ischemic attack. Here specification of other protocols apply, mutatis mutandis.

[0115] A "cytopathic" effect is understood herein as an effect pertaining to, relating to or characterized by any adverse change or effect at least at the cellular level either detectable or not. A cytopathic effect may be the consequence of an intracellular or extracellular effect, e.g. contacting the cells by a cytopathic agent or hypoxia, ischemia, e.g. acute myocardial infarction or stroke, or ischemia-reperfusion injury, e.g. acute ischemiareperfusion injury.

[0116] A "cytotoxic" effect is understood herein as an effect pertaining to, relating to or characterized by detectable damaging, degenerative or pathologic changes at the cellular level or the effect of a disease or disorder having an effect at the cellular level.

[0117] A "cytoprotective" effect is understood herein as an effect of a miRNA species or miRNA compound which reduces or inhibits the cellular damage, caused by an external or internal e.g. biological effect, e.g. a cytotoxic effect.

[0118] A "biological sample" is to be understood herein as any biological material which comprises biological material in which miRNAs can be up- and / or down-regulated due to an external effect and the biological sample can be subjected experimentally (i.e. in a test to any condition selected from a test ischemia-reperfusion, hypoxia, preconditioning, post-conditioning, remote perconditioning).

[0119] The biological sample can be a cell, a group or aggregate of cells, a cell culture, a tissue sample, organ, or a model animal body.

[0120] A "tissue sample" is a biological sample which is a tissue culture, a sample taken by biopsy, or an isolated organ. In a preferred embodiment of the invention an isolated organ is applied.

[0121] "Administration of" an effective amount of miRNA compound to a mammal is understood herein to include any way of administration of a compound which results in the presence of said miRNA compound in said mammal even if not the miRNA compound itself is introduced into said mammal. In particular, this term covers administration of a precursor miRNA or of a composition from which said miRNA compound is released or administration of a vector e.g. an expression vector resulting in the presence of said miRNA at a level providing an effective amount. Route of administration includes oral, intravenous, intracutaneous, subcutaneous, intramuscular, topical, inhaling.

[0122] "Percutaneous Coronary Intervention" (PCI), also known as angioplasty involves the insertion of a balloontype device into a patient’s artery to widen and open the blocked artery.

[0123] ABBREVIATIONS

[0124] AMI acute myocardial infarction

[0125] IPostC ischemic postconditioning

[0126] IPreC ischemic preconditioning

[0127] I / R ischemia / reperfusion

[0128] LAD left anterior descending

[0129] MI myocardial infarction

[0130] RIPerC ischemic remote perconditioning

[0131] Sham sham operation sI / R simulated ischemia-reperfusion

[0132] BRIEF DESCRIPTION OF THE FIGURES

[0133] Fig. 1 a Experimental protocol for closed-chest coronary occlusion / reperfusion and different ischemic conditionings in pigs (Sham = sham operated control, Isch = ischemia / reperfusion, IPreC = ischemic preconditioning, IPostC = ischemic postconditioning, RIPerC = ischemic remote conditioning group), b Experimental flow-chart.

[0134] Fig. 2 MicroRNA expression pattern analysis: Number of microRNAs associated with cardioprotection induced by ischemic pre- and / or post- and / or remote conditioning based on the HT-qPCR results. The three circles of the Venn-diagram are representing the miRNA expression patterns of the three different conditioning maneuvers compared to the ischemia-reperfusion (Isch) group. Number of microRNAs associated with either IPreC or IPostC or RIPerC are indicated in the outer sections of the diagram. (List of microRNAs presented in Table 4.) The number of microRNAs with the same expression pattern in two conditionings are indicted in the intersections of the diagram. (List of microRNAs is presented in Table 5.) The central intersection shows the number of microRNAs affected by all the three ischemic conditioning protocols (Expression pattern of microRNAs is presented in Figure 3). Isch: ischemia-reperfusion, IPreC: ischemic preconditioning, IPostC: ischemic postconditioning, RIPerC: ischemic remote conditioning.

[0135] Fig. 3 MicroRNA expression pattern analysis of the selected protectomiR candidate microRNAs associated with cardioprotection by ischemic pre-, post- and remote conditioning, a Four miRNAs (miR-199a, miR-450a, miR-450c-3p and miR-451) were up-regulated in all conditionings (IPreC, IpostC and RIPerC) vs Isch. b, c Ten miRNAs (miR-29a, miR-34a, miR-105-2, miR-127, miR-142, miR-181a, miR-193a, miR-204a, miR-339a and miR-424-3p) were down-regulated in both IPreC, IPostC and RIPerC vs Isch. Sham = sham operated control, Isch: ischemia-reperfusion, IPreC: ischemic preconditioning, IPostC: ischemic postconditioning, RIPerC: ischemic remote conditioning, N=3, technical replicates n=2, values are logj expression changes ± SD. Criteria for significant change is logj change is greater than or equal to ±0.585 and * p < 0.05 vs. Sham. #p < 0.05 vs. Isch.

[0136] Fig. 4 Selection of the appropriate modulation with protectomiR mimic or protectomiR antagomiR candidates based on the direction of the observed expression changes in. Fig. 5 Effect on cell viability of mimic protectomiRs after simulated ischemia-reperfusion (sI / R) in primary neonatal rat cardiomyocytes. Cell viability is expressed as percent of simulated ischemia-reperfusion + negative control group. Transfection of primary cardiomyocytes with a miR-451-5p or b miR-450a-3p miRNA mimic showed cardiocytoprotective effect, c MiR-450b-5p mimic transfection showed toxic effect after simulated ischemia-reperfusion injury, d MiR-199a-3p mimic transfection did not affect the cell viability. N=4-6, technical replicates n=6-12, values are mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. sI / R Negative Control, oneway ANOVA with Dunnett’s post-hoc test or Kruskal- Wallis test with Dunn’s post-hoc test.

[0137] Fig. 6 Effect on cell viability of antagomiR protectomiRs after simulated ischemia-reperfusion (sI / R) in primary neonatal rat cardiomyocytes. Cell viability is expressed as percent of simulated ischemia-reperfusion + negative control group. Transfection of primary cardiomyocytes with b miR-34a-5p, c miR-127-3p, f miR-142-5p and h miR-29a-3p antagomiRs showed toxic effect after simulated ischemia-reperfusion injury. Meanwhile transfection with a miR-339-5p, d miR-204-3p, e miR-181a-5p and g miR-193a-3p antagomiRs did not affect cell viability of cardiomyocytes after simulated ischemia-reperfusion injury. N=4-6, technical replicates n=6-12, values are mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. sI / R Negative Control, one-way ANOVA with Dunnett’s post-hoc test or Kruskal- Wallis test with Dunn’s post-hoc test.

[0138] Fig. 7 a Effect of simulated ischemia-reperfusion injury (sI / R) on the cell viability of naive neonatal rat cardiac myocytes, b Fluorescent images of un-transfected neonatal cardiac myocytes or transfected with 50 nM Dy-546 fluorescently labelled transfection control, c Effect of simulates ischemia-reperfusion (sI / R) on transfected neonatal cardiac myocytes. Values are mean ± SEM, * p < 0.05 vs. SI / R Negative Control, one-way ANOVA with LSD post-hoc test.

[0139] Fig. 8 Experimental protocol figure for in vitro validation of miR-450a on human cardiomyocyte cell line AC16.

[0140] Fig. 9 Effect on cell viability of miR-450a-3p after simulated ischemia-reperfusion (sI / R) in human AC 16 cardiomyocytes. Cell viability is expressed as the percent of normoxia + negative control group. N=5, values are mean ± SEM, Kruskal-Wallis test with Dunn’s post-hoc test, *p < 0.05, ** p < 0.01, *** p < 0.001 vs. sI / R + Negative Control.

[0141] DETAILED DESCRIPTION OF THE INVENTION

[0142] MicroRNAs (microRNAs), also known as "mature microRNA", are small (approximately 18-25 nucleotides in length), endogenously expressed RNA oligonucleotides taking part in a multitude of regulation processes, typically by regulating the expression of genes by binding to the 3 ’-untranslated regions (3’-UTR) of specific mRNAs.

[0143] The present inventors have previously identified cardioprotective microRNAs, termed protectomiRs, by a systematic analysis of microRNA expression pattern in myocardial infarction and cardioprotection induced by ischemic conditioning in rats.

[0144] Here the present inventors aimed to identify protectomiRs in a translational porcine model of reperfused acute myocardial infarction (AMI) and cardioprotection by ischemic conditioning and validate their cardiocytoprotective effect.

[0145] The present inventors used cardiac tissue samples from their previous study in closed-chest AMI model in domestic pigs. Pigs were subjected to sham operation (Sham), ischemia / reperfusion to induce AMI (AMI) or preconditioning (IPreC), postconditioning (IPostC), and remote perconditioning (RIPerC). Tissue samples were collected from the infarcted region of the left ventricles. MiRNA expression pattern was detected by high- throughput qRT-PCR. Potential protectomiRs were selected by systematic comparison of significant expression changes due to different conditioning stimuli vs. AMI. To validate the cardiocytoprotective effect of potential protectomiRs, isolated rat cardiomyocytes were transfected with specific miRNA mimics or inhibitors (antagomiRs) of the selected protectomiRs with cross -species sequence homology, and the survival of cells was measured after simulated ischemia / reperfusion injury.

[0146] Expression of 220 miRNAs was assessed. Expression of 57 microRNAs were changed by IPreC, 54 by IPostC and 68 by RIPerC as compared to AMI (min. 1.5*log2 fold-change, -logiop> 1.31 vs. AMI). Expression of 14 microRNAs changed significantly due to all three conditionings vs. AMI (4 miRNAs were upregulated and 10 downregulated). Rat homologs of these 14 protectomiR candidates were identified and 12 showed 100% sequence homology with the original pig miRNAs. The selected miRNAs (4 mimics and 8 antagomiRs) were transfected into isolated rat cardiomyocytes. Mimics of miR-450a and miR-451 significantly improved the survival of cells after ischemia / reperfusion injury.

[0147] This is the first demonstration that miR-450a and miR-451 is associated with cardioprotection by ischemic pre-, post- and remote conditioning in a clinically relevant porcine model and also show direct cardiocytoprotective effect. These protectomiRs are potential therapeutics for cardioprotection.

[0148] In the present work, the inventors identified potential cardioprotective microRNAs (protectomiRs) in a porcine model of reperfused acute myocardial infarction (AMI) and cardioprotection and validated their cardiocytoprotective effect. In this clinically relevant closed-chest porcine model of acute myocardial infarction, pre-, post-, and remote conditioning maneuvers regulated the expression of 14 different miRNAs: miR-199a-3p, miR-450a, miR-450c-3p and miR-451 were upregulated, whereas miR-181a, miR-339, miR-142, miR-193a-3p, miR-29a, miR-204a, miR-424-3p, miR-127, miR-34a and miR-105-2 were down regulated as compared to ischemia / reperfusion. The present inventors validated the cardiocytoprotective effect of these respective protectomiR mimics or antagomiRs and found that out of the 14 protectomiR candidates, mimic of miR-450a and mimic of miR-451 exerted direct cardiocytoprotection in vitro. This is the first demonstration that miR-450a mimic is an effective protectomiR and that both miR-450a and miR-451 may be involved in the mechanism of ischemic pre-, post and remote conditioning.

[0149] In this work, the present inventors searched for cardioprotective microRNAs, which are differentially expressed in cardiac tissue, that were subjected to ischemic conditioning stimuli versus ischemia-reperfusion in a pig model of myocardial infarction. The present inventors have previously identified cardioprotective microRNAs, termed protectomiRs in a rat model, by a systematic analysis of microRNA expression pattern in myocardial infarction and cardioprotection induced by ischemic conditioning [Varga et al., 2014]. Although the search for novel therapeutic targets with high translational value is a focus of preclinical research, the application of unbiased high throughput omics approaches after myocardial ischemia-reperfusion injury is still an emerging need [Perrino et al., 2017]. Therefore, in the present work, the inventors utilized myocardial samples from their previous porcine study. In that model, ischemic preconditioning reduced infarct size, whereas ischemic post- and remote perconditioning reduced microvascular obstruction and edema formation [Baranyai et al., 2017]. miRNAs associated with all the 3 conditioning stimuli are likely contribute to both cardioprotection and vasculoprotection, thereby increasing the chance to be an important player of protection. Porcine MI models are in use for translational studies to validate therapeutic concepts before human application [Huang et al., 2020]. Although annotation of the porcine genome is still behind the human or mouse, omics tools are available for high throughput analysis in pigs as well [Chilukoti et al., 2018]. In the literature, there are no studies aimed at such high-throughput analysis of transcriptome in pig model of MI or ischemic conditioning pig. In the present work, the inventors analyzed the expression pattern of 221 different known pig microRNAs in ischemia-reperfusion with or without three different conditioning maneuvers. The present inventors’ microRNA pattern analysis included some microRNAs, which were described previously in porcine ischemic conditioning models. Baars et al. concluded that miRNA-29b, - 133a, and -146b as miRNAs are associated with postconditioning [Baars et al., 2014]. In the present inventors’ samples miR-29b expression was also increased after conditioning stimuli in line with the previous findings, however miR-133a and miR-146b did not show altered expression, probably due to the different postconditioning protocol or longer myocardial ischemia. Expression of miR-199a showed significant change in the present inventors’ samples, in line with the study by Rane et al., who found it to be associated with preconditioning alone in a pig model [Rane et al., 2009]. Exogenous miR-199a has great therapeutic value in cardiac regeneration therapies [Lim, 2019].

[0150] Next, the present inventors further selected the miRNAs to choose protectomiR candidates. The present inventors took miRNAs associated with significant reduction in ischemia / reperfusion induced myocardial injury in preconditioning and selected 57 miRNAs. To increase the chance of finding effective cardioprotective microRNAs, the present inventors selected miRNAs also involved in mainly vasculoprotection shown by post- and remote conditioning in the pig model and narrowed the number of potential protectomiR candidates to 14 miRNAs. Mimics of miR-199a, miR-450a, miR-450c-3p and miR-451 and antagomiRs of miR-29a, miR-34a, miR-105-2, miR-127, miR-142, miR-181a, miR-193a, miR-204a, miR-339a and miR-424-3p were determined by the direction of expression changes of microRNAs in ischemic conditionings versus ischemia / reperfusion alone. These miRNA mimics and antagomiRs are considered as potential protectomiRs.

[0151] To test the cardiocytoprotective effect of the selected mimic and antagomiR protectomiR candidates, the present inventors used an in vitro neonatal rat cardiac myocyte simulated ischemia / reperfusion test system [Gorbe et al., 2010; Makkos et al., 2019]. The present inventors utilized neonatal rat cardiac cells due to their ability to be transfected with miRNA mimic and antagomiRs [Varga et al., 2014]. Although pig ventricular cardiomyocytes can be freshly isolated from adult pig heart, transfectability and seeding for long-term culturing of those cells raise technical difficulties and not in regular use in preclinical studies [Gadeberg et al., 2016; Louch et al., 2011; Voigt et al., 2015]. To bridge the potential cross species differences in miRNA sequences, the present inventors performed cross-species pig-rat microRNA sequence similarity matching and tested mimic or antagomiR protectomiRs with 100% homology to pig miRNAs.

[0152] Transfection of cardiac myocytes with two of all the selected protectomiRs (miR-450a and miR-451 mimic) significantly increased cardiac myocyte cell survival after simulated I / R injury. miR-451 have been studied in hypoxia and considered as a hypoxia-related microRNA in human hearts [Chouvarine et al., 2019]. In a recent publication miR-45 la upregulation reduced the hypoxia induced MMP2 and MMP9 expression in human cardiac myocytes [Scrimgeour et al., 2020], and alleviated myocardial ischemia / reperfusion injury in rats by suppression of MAPlLC3B-mediated autophagy [Lv et al., 2021]. Furthermore, preconditioning failed to protect against I / R injury in the absence of miR-451 in mice [Wang et al., 2012]. On the other hand, the protective effect of miR-450a in cardiomyocytes against simulated I / R injury was not investigated before. MiR-450a was previously shown to be enriched in exosomes in the pericardial fluid of patients with atrial fibrillation and its effect on the cellular activity of cardiac fibroblasts was further validated in primary rat cells [Liu et al., 2020; Liu et al., 2021]. However, in mouse embryonic fibroblasts, the overexpression of miR-450a-3p increased cell apoptosis and suppressed cell proliferation via the downregulation of Bubl protein level [Luo et al., 2012]. In this perspective, the present inventors’ results further emphasize the translational potential of miR-451 miRNA mimic to induce cardioprotection and this is the first demonstration of miR-450a to be included in the cardioprotective mechanism induced by ischemic pre-, post- and remote conditioning.

[0153] The cardioprotective effect of miR-450a was also validated in the human cardiocyte cell line AC 16 (see Example 3). The results showed that miR-450a mimic enhanced the survival of AC16 cells from simulated ischemia-reperfusion injury. This result further improves the translational value of the inventors’ findings.

[0154] The sequence of rat miR-450a-3p (SEQ ID NO: 247) is identical to a pig miR sequence (SEQ ID NO: 246), and is very similar to the sequence of the human miR-450a-l-3p (SEQ ID NO: 249). In fact, there is an 18- nucleotide long core sequence (AUUGGGAACAUUUUGCAU, SEQ ID NO: 248), which is present in the corresponding miRNA sequences of the aforementioned species.

[0155] Other protectomiR mimic or antagomiR candidates did not show cardiocytoprotective effect in the present inventors’ test system.

[0156] The present inventors have utilized high throughput analysis to describe the miRNA expression pattern of the porcine myocardium. Although the present inventors were unable to access all known pig microRNAs, they based their choices on careful literature search and selected 221 miRNAs that had known sequences in pigs. While the primary measurement of miRNA expression pattern was performed on porcine samples, the in vitro experimental validation of cytoprotective modulation of selected protectomiRs was performed in rat cardiac myocytes also due to ethical (3R) and feasibility causes. The present inventors utilized neonatal rat cardiac cells due to their high translational value [Onodi et al., 2022] and their ability to be transfected with miRNA mimic and antagomiRs, which showed 100% sequence homology to the detected pig miRNAs. The inventors also performed an in vitro experimental validation of the cardiocytoprotective effect of miR-450a in a human cell line, as mentioned previously.

[0157] The miR-450a protectomiR is thus found to be cardioprotective as being probably involved in the mechanism of ischemic pre-, post and remote conditioning in a clinically relevant pig model of cardioprotection.

[0158] Therefore, the miRNA agonist of a miRNA species miR-450a is a useful drug candidate and is further developed as therapeutic for cardioprotection.

[0159] Methods to develop microRNA into medicaments are well known in the art.

[0160] While miRNA may be applicable, oligonucleotides composed only of natural nucleotide units are rapidly degraded in a biological system by endo- and exo-nucleases and may fail to connect effectively with the target in mammalian cells or large amount may be required. Non-natural nucleic acids may provide a solution.

[0161] Several types of chemical modifications have been studied in the art.

[0162] A review on non-natural nucleic acids is given by Daniel H. Appella disclosing method for providing synthetic analogues of nucleic acids which potentially have the same effect [Appella, 2009]. Molecule types alternative to nucleic acids are known in the art such as peptide nucleic acids (PNA), locked nucleic acids (LNA), morpholino oligomers, glycol nucleic acid (GNA), threose nucleic acid (TNA) and hexitol nucleic acids (HNA), a part of which is discussed below.

[0163] Modification can be grouped in general as sugar modifications, backbone modifications and base modifications. Examples are briefly discussed below to show that such modifications are known in the art and are at hand of the skilled person to carry them out.

[0164] Sugar modifications

[0165] An option is to introduce modification in the sugar ring, especially at the C2’ position. Modifications at this position affect the sugar moiety conformation which modifies, possibly improves binding affinity. Several modifications are described by Lima et al. [Lima et al., 2018]. For example, 2’-OMe, 2’-M0E and 2’-F are modifications often used at this position.

[0166] Examples of modifications in the sugar moiety include threose nucleic acids (TNA), wherein the phosphates are linked to oxygens on the 3' and 2' positions of the furanose and there is no methylene group present between the sugar ring and an oxygen atom on the phosphate; TNA sequences interact with complementary sequences of DNA or RNA (intersystem cross-pairing); locked nucleic acid (LNA) derived from RNA, which have a 2'-oxygen, and an extra methylene group that is covalently linked to C4' and the oxygen of C2' of the furanose ring; and effectively binds natural nucleic acids, beta-hexitol nucleic acids (HNA), wherein a nucleobase is present in the 0 position (0-HNA); and are capable of both intra- and intersystem cross -pairing, and bind effectively RNA [Appella DH, 2009].

[0167] In certain embodiments, sugar moiety substitutions include 2’ substitutions or 2’ O-substitutions, e.g. by halogen, pseudohalogen or alkoxy substituent(s) or O ->X sugar oxygen substitutions wherein X is selected from S, NH, CHj, preferably S.

[0168] Normally sugar modifications are not considered as mutations of the sequence itself.

[0169] Backbone modifications

[0170] Backbone modifications may include even the substitution of the entire backbone to result in RNA or DNA analogue molecules. In most cases, however, substitutions include internucleotide linkage modifications.

[0171] Phosphodiester bond (PO) is the natural internucleotide linkage present in DNA and RNA and are more prone to be cleaved by endo- and exonucleases in mammalian cells. Chemical strategies have been developed to improve nuclease resistance. Using phosphate analogs is a strategy widely applied in the field.

[0172] Specific examples are phosphorothioate (PS) linkages which are resistant to the activity of exo- and endonucleases and can be placed at strategic points within the sequence.

[0173] Alternative approaches are using boranophosphate or phosphonate linkages.

[0174] Internucleotide modifications may be combined with sugar modifications to improve potency and resistance to the oligo- nucleotide sequence.

[0175] Further examples are described in the art [Lima et al., 2018; Lennox & Behlke, 2011]. While the authors discuss antisense modifications, in principle such modifications may be applied in miRNA agonists as well. Table 2 in Lima et al. (2018) lists a number of modifications.

[0176] Normally backbone modifications are not considered as mutations of the sequence itself.

[0177] Base analogs

[0178] In principle another option is to use base analogs that can substitute for normal bases in nucleic acids. A careful design would be necessary here as base analogs may result in altered base pairings. A few examples of base analogs are 2-aminopurine, 5 -bromouracil, 6-mercaptopurine etc.

[0179] A more detailed description of possible base analogs can be found e.g. in Chung Eun Ha, N.V. Bhagavan, in Essentials of Medical Biochemistry (Third Edition), 2023, Chapter 20 “Structure and properties of DNA” and in Chang-Hui Shen, Diagnostic Molecular Biology, Academic Press, 2019, Chapter 2 - “Nucleic Acid-Based Cellular Activities”.

[0180] Base replacements with analogs which do not alter binding properties in an embodiment can be considered as alternatives to the natural bases and therefore in a sense not to be a mutation. Such bases are for example isoguanine, isocytosine and 2,6-diaminopurine for adenine.

[0181] Alternatively base replacements can be considered as mutations, in particular if binding properties are changed.

[0182] Typical single base replacements are e.g.

[0183] Thimine: 2-thio-T, psiT, yT

[0184] Cytosine: yC, xC, isocytosine

[0185] Adenine: 2,6-diaminopurine xA

[0186] Guanin: isoguanine, xG.

[0187] Thus, using nucleic analogs in the form of oligonucleotides is contemplated in the present invention.

[0188] Such nucleic acid analogs comprise chemically modified or artificial nucleotides, exemplified herein. It will be understood by a person skilled in the art that other options for the preparation of nuclecic acid analogs are known and useful in the present invention. In general, such techniques are at hand of the skilled person.

[0189] There are techniques which are well developed and which can reliably provide essentially the same effect as the miRNA agonist of the present invention. For example, miRNA mimic preparation is a routine task and such technology (available from e.g. ThermoScientific, Dharmacon) may in itself be suitable.

[0190] While state of the art techniques allow a prediction on whether such a nucleic acid analogs would be sufficiently analogous to a miRNA agonist of the present invention, as is always the case in medical treatments, there is a certain unpredictability as to the level of the effect and actual usefulness in a real life clinical setting.

[0191] In particular, a nucleic analog may be part of a larger molecule considered as a miRNA compound. For example it may comprise flanking tags and sequences for stabilization, targeting or reporting. Such flanking tags and sequences may be removed in the patient’s body e.g. by nucleases or may be pharmaceutically tolerable.

[0192] As a miRNA agonist, both double-stranded and single stranded variants may work. In particular, a stranded variants, i.e. an RNA analogue is applied.

[0193] Delivery systems for miRNA compounds

[0194] In order to target organs and / or tissues and preferably to get miRNA across cellular membranes, several approaches have been developed. Independent of the delivery method, typically endocytosis is the mechanism by which miRNA get into the cells.

[0195] Applying miRNA as naked, albeit modified molecules has been possibly the most often applied strategy so far. One approach is to modify the molecule to withstand cellular enzymes. Natural nucleic acids with a phosphodiester backbone typically require a complex with a carrier or a ligand to which they are bound to.

[0196] Conjugation of derivatized sugars like N-acetylgalactosamine (GalNAc) has also been used as an approach for miRNA delivery (RG-101) and proved to be promising in preclinical models [Baek J., 2014]. Further examples are taught e.g. by Shah, AM and Giacca M [Shah, AM and Giacca M, 2022] and references cited by them.

[0197] A further approach, lipid-based delivery, e.g. by liposomes is one of the further most accepted approach for targeting miRNA to the site of the injury. From mixing lipids, e.g. cationic lipid and other, stabilizing molecules like PEG or a further helper lipid, is an option, e.g to form complexes called lipoplexes. As examples, helper lipids are phosphocholine-containing lipids, e.g. phosphatidylcholine ester, and which stabilize cell membrane. Nanoparticles formed by amphoteric lipids is also an option [Baumann V, and Winkler J, 2014 and Shah, AM and Giacca M, 2022].

[0198] Artificial liposomes are another well-studied delivery form which may also be suitable to package miRNA.

[0199] Lipid bilayer-enclosed extracellular structures, i.e. membrane vesicles like extracellular vesicle (EV), including exosomes are also a promising field to package miRNA. Exosomes, showing a marked stability in body fluids, contain a variety of intracellular components. These methods, however, lack cell-specifity and targeting is to be solved e.g. by means of administration. [Lee TJ et al., 2020 and Shah A M and Giacca M, 2022]

[0200] Polymers, like polyethylene imine (PEI) has been known as a means for delivering nucleic acid based medicinal agents. Various alternatives have been developed like copolymers. Another potential option is Poly(lactic-co-glycolic acid) (PLGA) which has been proposed for oligonucleotide delivery. For example, an antagomiR-92 encapsulated in PLGA microspheres was shown to improve cardiac function and angiogenesis after MI in pigs [Bellera N et al., 2014].

[0201] Other type of nanoparticles pharmaceutically tolerable and suitable to comprise non-covalently an oligonucleotide cargo may also be useful for such delivery. Various inert, inorganic carriers (including nanocrystals, silica- and calcium-based nanoparticles, gold nanoparticles, etc.) have also been studied for formulating miRNA compositions [Baumann V, and Winkler J, 2014 and Lee TJ et al. 2020].

[0202] Coding molecules like bacteriophages and viruses may be suitable to produce particles and have been used to develop virus-like particles for oligonucleotide and drug delivery.

[0203] Other formulation and delivery methods are known for a person skilled in the art and disclosed e.g. in review papers as listed herein and references cited therein [Baumann V, and Winkler J, 2014, Shah A M and Giacca M, 2022 and Lee TJ et al. 2020].

[0204] Treatment protocols with miRNA compounds of the invention

[0205] Recently, therapies with non-coding RNA have been applied in several fields. Thus, setting up such a therapy is well within the skills of a person skilled in the art.

[0206] In the present invention such therapies include administration of a miRNA of the invention to a patient in need of such treatment.

[0207] Exemplary administration times are disclosed in the Brief description of the invention.

[0208] Administration of a formulation, e.g. a formulation as disclosed herein is carried out depending on the target tissue. In case of cardiovascular conditions in a preferred embodiment it is administered intravenously (iv.) to the patient.

[0209] Further exemplary method for administrations and treatment of disesases by non-coding RNA such as miRNA are disclosed by Ajay M Shah and Mauro Giacca [Shah, A M and Giacca M, 2022]. In conclusion, this is the first demonstration that miR-450a and miR-451 mimics are effective protectomiRs and may be involved in the mechanism of ischemic pre-, post and remote conditioning in a clinically relevant pig model of cardioprotection and that agonist of these cardioprotective miRNAs are useful as potential therapeutics for cardioprotection.

[0210] EXAMPLES

[0211] Example 1: Animal models and assays - Methods

[0212] 1. Experimental Design

[0213] Myocardial tissue samples were obtained from the present inventors’ previous study in a clinically relevant, closed-chest porcine model of acute myocardial infarction and cardioprotection where cardioprotective phenotype was carefully evaluated [Baranyai et al., 2017]. In their previous porcine study, cardioprotection was assessed by measurement of infarct size / area at risk by TTC staining, as well as infarct size, edema, and microvascular obstruction by cardiac MRI. While ischemic preconditioning reduced infarct size, postconditioning and remote ischemic conditioning did not affect infarct size but reduced edema and MVO, signs of vascular protection. Myocardial tissue samples were collected from the ischemic region of the left ventricle after 3 hours of reperfusion (Fig. la). Samples were snap frozen in liquid nitrogen immediately and stored at -80°C. In the present work, the inventors used tissue samples from the ischemic zone of left ventricle for miRNA high-throughput qRT-PCR analysis of the following groups:

[0214] (i) Sham operated control (Sham): balloon catheter was placed into the coronary artery but remained deflated.

[0215] (ii) Ischemia-reperfusion group (Isch): 90 min myocardial ischemia was induced by the occlusion of the left anterior descending (LAD) coronary artery followed by 3 hours of reperfusion.

[0216] (iii) Ischemic preconditioning group (IPreC) : 3 * 5 min myocardial ischemia was applied before the 90 min LAD occlusion.

[0217] (iv) Ischemic postconditioning group (IPostC): 6^30 sec myocardial ischemia was applied at the start of reperfusion after the 90 min LAD occlusion.

[0218] ( v) Ischemic remote perconditioning (RIPerC) : 4 x 5 min hind limb ischemia was applied during the last 40 min of the 90 min LAD occlusion.

[0219] Then, miRNA expression pattern was assessed with high-throughput qRT-PCR. Potential cardioprotective miRNAs, protectomiR candidates, were selected by systematic analysis of their expression pattern. MiRNAs associated with I / R injury or with specified ischemic conditionings were identified (Table 2A-2B, 3, 4, 5). First, protectomiR candidates associated with cardioprotection by IPreC were selected. Then, the present inventors narrowed the selection further with miRNAs also associated with vasculoprotection by IPostC and RIPerC. ProtectomiR mimic or protectomiR antagomiR candidates were selected based on the direction of the expression change of the specific miRNA. Selected miRNAs were then analyzed for cross-species pig-rat miRNA sequence similarity. All protectomiR candidates showing pig -rat sequence similarity were validated for their cardiocytoprotective effect. Validation was performed in isolated neonatal rat cardiac myocytes transfected by the protectomiR mimics or antagomiRs then subjected to simulated ischemia-reperfusion injury (Fig. lb).

[0220] 2. Micro RNA Expression by High-Throughput qRT-PCR

[0221] MicroRNA was purified using High Pure miRNA isolation Kit (Roche; Cat# 5080576001) according to manufacturer’s instruction from the ischemic left ventricle of three randomly selected animals is each group. The quantity of miRNA was measured by NanoDrop 1000 spectrophotometer (Thermo Fischer Scientific, RRID:SCR_016517). 1000 ng miRNA was converted into cDNA with MicroRNA Reverse Transcription Kit (Thermo Fisher Scientific; Cat# 4366597) in a total volume of 20 pL. Identical reaction volumes were prepared by the Agilent Bravo Liquid Handling Platform (Agilent Technologies) in a 16^96 well design according to Agilent and Roche recommendations. Each 2 pL reaction mixture contained 4 ng cDNA, 10 pmol gene specific primers and 1 pL 2x LightCyclerl536 Green Master (Roche Applied Science; Cat# 05573092001). To measure specific microRNA expression pattern in the pig myocardium, 221 pig miRNAs were selected based on the miRBase Release 20 (June 2013, ftp: / / mirbase.org / pub / mirbase / 20 / database_files / ), that were further confirmed to be expressed in pig myocardium with literature search in PubMed until 2016 January. The following search string was utilized for PubMed search (pig OR swine OR porcine OR sus) AND (miRNA OR microRNA OR miR OR (micro AND RNA)) AND (heart OR cardi* OR myocardi*) and resulted in 90 hits, which were manually curated.

[0222] All primers used in quantitative real-time PCR were purchased from Bioneer (Daedong, Korea). Primer sequences are listed in Table 1. Table 1: miRNA HT-qPCR primer list

[0223] Data was collected and processed using the LightCycler® 1536 SW 1.0 software (Roche Applied Science, RRID:SCR_012155) and the following amplification protocol was used: 95 °C for 1 minute (activation), 60 cycles of 95 °C for 10 seconds and 60 °C for 10 seconds, followed by 40 °C for 10 second final cooling. Curves were analyzed by using dynamic tube and slope correction methods. Results were calculated with AACp evaluation method. For visual comparison and representation of both down- and upregulation, data were shown as logj changes. Averages of logj changes were plotted with SD values in Table 2.

[0224] Table 2A: miRNA HT-qPCR data table

[0225] Table 2B: miRNA HT-qPCR data table (cont.)

[0226] 3. Pattern Analysis and Selection of ProtectomiR Candidate miRNAs

[0227] The miRNA expression pattern associated with cardioprotection can be identified with the collection of miRNAs differentially expressed in one or multiple ischemic conditioning combined with ischemia -reperfusion versus ischemia-reperfusion alone.

[0228] First, miRNAs differentially expressed in IPreC versus Isch were selected. Then, miRNAs differentially expressed in IPostC and RIPerC versus Isch were added and the overlaps in the miRNA expression patterns were further analyzed.

[0229] Finally, the present inventors selected the protectomiR candidate miRNAs associated with all three cardioprotective maneuver (IPreC, IPostC and RIPerC), the triple overlap. The present inventors consider that the miRNA expression pattern significantly altered by all three ischemic conditioning stimuli is the most likely to participate in the development of cardioprotection (Fig. 2).

[0230] 4. Selection of ProtectomiR Mimic or ProtectomiR AntagomiR Candidates

[0231] After the analysis of the expression pattern of the selected protectomiR candidate miRNAs, the present inventors selected appropriate modulation of these miRNAs based on the direction of the observed expression changes in cardioprotection (Fig. 3a, b, c). If the specific miRNA was up-regulated in cardioprotection by IPreC, IPostC and RIPerC versus ischemia-reperfusion, then miRNA mimic (protectomiR mimic) was assigned as potential protective modulation.

[0232] If the specific miRNA was down-regulated in cardioprotection by IPreC, IPostC and RIPerC versus ischemia-reperfusion then miRNA downregulation with antagomiR (protectomiR antagomiR) was assigned as potential protective modulation (Fig. 4).

[0233] 5. Cross-Species Pig-Rat MiRNA Sequence Similarity

[0234] NCBI RNA blast and miRBase databases were used to identify rat miRNAs with sequence similarity of the selected pig protectomiR candidate miRNAs. All proctectomiR candidates showing sequence homology were selected for further validation for their cardiocytoprotective effect in rat cardiac myocytes.

[0235] 6. Validation of ProtectomiR Mimics or ProtectomiR AntagomiRs in Simulated Ischemia-Reperfusion in Rat Cardiac Myocytes

[0236] Cardiac myocyte cultures isolated from new-born Wistar rats were transfected, then subjected to simulated ischemia / reperfusion injury to validate the cardio-cytoprotective effect of proctectomiR mimics or antagomiRs [Varga et al., 2013]. Cardiac myocytes were cultured in a standard cell culture incubator (5% COj) as described previously [Varga et al., 2013].

[0237] Cardiac myocytes were transfected with mature microRNAs or hairpin inhibitors two days after isolation according to a previously described transfection protocol [Varga et al., 2013]. Cells were incubated with 25 nM, 50 nM or 100 nM Dharmacon miRIDIAN microRNA mimics (Horizon Discovery, UK) or miRIDIAN microRNA hairpin inhibitors (Horizon Discovery, UK) for 10 h with DharmaFect (Horizon Discovery, Cat# T-2001-03) transfection reagent in antibiotic-free growth medium. This was followed by 10 h recovery period without treatment. As a negative control, 50 nM Dharmacon miRIDIAN mimic or antagomiR negative control (Horizon Discovery, Cat# CN-001000-01-05, IN-001005-01-05), cel-miR-67 (a non-targeting Caenorhabditis elegans microRNA) was used.

[0238] After the transfection protocol, the transfected cells were subjected to a simulated ischemia-reperfusion (sI / R) protocol as previously described [Makkos et al., 2019; Paloczi etal., 2020]. To simulate ischemia, the culture medium was changed to hypoxic solution, plates were placed into a hypoxic chamber, and cells were exposed to a constant flow of a mixture of 95% Nj and 5% COj at 37°C for 6 h. Simulated ischemia was followed by 2 h of simulated reperfusion using standard medium and normoxia. To assess cell viability, cardiac myocytes were incubated with 1 pM calcein-AM (PromoKine, Cat# PK-CA707-80011) at room temperature for 30 min. Fluorescence intensity was measured with a fluorescence plate reader, VarioscanLUX (Thermo Fisher Scientific) at 490-nm excitation and 520-nm emission. The cytoprotective effect of different microRNA modulators was compared with the negative control, i.e., cardiac myocytes transfected with a non-targeting microRNA mimic and subjected to simulated I / R. The present inventors expressed viability as a percentage of non-targeting microRNA- transfected normoxia treated control groups.

[0239] 7. Statistical Analysis

[0240] Statistical analyses were carried out using GraphPad Prism software version 8 (GraphPad Software, Inc., RRID:SCR_002798). Quantitative real-time PCR data are presented as mean ± SD; all other data are displayed as mean ± SEM. One-way ANOVA followed by Dunnett’s post hoc test or Kruskal- Wallis test followed by Dunn’s post-hoc test was used to evaluate differences in mean values of viability of cardiac myocytes between groups.

[0241] Each microRNA was measured in randomly selected N=3-4 samples from each group, in n=2 technical parallel. A microRNA was considered as detected if at least 1 parallel could be detected. Using a two-tailed two- sample unequal variance Student's t-test, the P value was determined to find significant gene expression changes. MicroRNA expression ratios with p values of < 0.05 and logj changes of less than or equal to -0.586 or logj changes of > 0.586 were considered as repression or overexpression, respectively.

[0242] In vitro cardiac myocyte experiments were performed in N=4-6 repetitions. Each repetition contained n=6-12 technical parallel in one experimental group. Values are expressed as mean ± SEM. First, values were tested for Gaussian distribution, then analyzed with one-way ANOVA followed by Dunnett’s post-hoc test or Kruskal-Wallis test followed by Dunn’s post-hoc test. P value was determined as *p < 0.05 vs. SI / R negative control.

[0243] 8. Ethical Approval

[0244] The present investigation was carried out according to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85-23, revised 1996) and according to the ARRIVE guidelines, to the EU Directive (2010 / 63 / EU) and was approved by the animal ethics committee of Hungarian National Food Chain Safety Office (SOI / 31 / 26-11 / 2014).

[0245] Example 2: Animal models and assays - Results

[0246] 1. MicroRNA Expression Changes in Ischemia-Reperfused and Ischemic Conditioned Pig Hearts

[0247] Expression of 221 different known pig microRNA sequences were measured by RT-PCR and the following numbers of miRNAs were detectable in at least one sample of the experimental groups: 186 miRNAs in Sham, 196 miRNAs in Isch, 199 miRNAs in IPreC, 194 miRNAs in IPostC and 195 miRNAs in RIPerC. 8 miRNAs (miR-10a-3p, miR-24-2, miR-146a, miR-148a, miR-202-5p, miR-374a, miR-1277, miR-1297) were not detectable in any of the measured myocardial samples.

[0248] Relative expression of miRNAs was calculated in case of 213 miRNAs in Isch vs. Sham, in IPreC vs. Isch, in IPostC vs Isch and in RIPerC vs. Isch relation (Table 2).

[0249] 2. Identification of MicroRNAs Associated with Cardioprotection by Ischemic Pre- and / or Post- and / or Remote Conditioning

[0250] Number of microRNAs differentially expressed by one or multiple ischemic conditionings versus Isch are presented on a Venn diagram (Fig. 2). In the ischemic preconditioned group altogether 29 microRNAs were up- and 28 microRNAs were down-regulated. Altogether 19 microRNAs were up- and 35 microRNAs were down- regulated by postconditioning and 33 microRNAs were up- and 35 microRNAs were down-regulated by ischemic remote conditioning. Differentially expressed miRNAs in only one ischemic conditioning or overlapping in two ischemic conditioning groups is detailed in Tables 4 and 5.

[0251] Table 3: MiRNAs (miR-) associated with I / R injury. Upper part of the table shows microRNAs significantly upregulated by Isch vs Sham, but neither pre-, post-, nor remote conditioning influenced their expression vs. Isch. Lower part of the table shows miRNAs significantly downregulated by Isch vs Sham but neither pre-, post-, nor remote conditioning influenced their expression vs. Isch. V alues are logj expression changes ± SD. Criteria for significant change is *p < 0.05 vs Sham and #p < 0.05 vs Isch and the logj change is greater than or equal to ±0.585. Arrows shows directions of miRNA changes and ns. indicates non-significant results.

[0252] Table 4: List of microRNAs with significant expression changes in only one groups of ischemic conditioning compared to the ischemic group (IPreC vs Isch, IPostC vs. Isch, RIPerC vs Isch). *p < 0.05 vs Sham and #p < 0.05 vs Isch and the logj change of greater than or equal to ±0.585. Arrows shows directions of miRNA changes and ns. indicates non-significant results.

[0253] Table 5: List of microRNAs with significant expression changes in two groups of ischemic conditioning compared to the ischemic group (IPreC and IPostC vs Isch, IPostC and RIPerC vs. Isch, IPreC and RIPerC vs Isch). *p < 0.05 vs Sham and #p < 0.05 vs Isch and the logj change of greater than or equal to ±0.585. Arrows shows directions of miRNA changes and ns. indicates non-significant results.

[0254] MiRNAs modulated by three conditionings versus Ischemia were considered to have the highest probability to play a role in cardioprotection. There were 4 miRNAs upregulated (miR-199a-3p, miR-450a, miR-450c-3p and miR-451) and 10 miRNAs down-regulated (miR-181a, miR-339, miR-142, miR-193a-3p, miR-29a, miR-204a, miR-424-3p, miR-127, miR-34a and miR-105-2) in the center of the Venn diagram plotted in Fig. 2 and detailed expression data of these microRNAs in all comparisons are presented in Fig. 3.

[0255] 3. Selection of ProtectomiR Candidate Micro RNAs

[0256] The 14 miRNAs altered by three conditionings were further selected according to the direction of changes by conditioning versus ischemia-reperfusion. Accordingly, the 14 protectomiR candidates were classified into two possible expression pattern categories.

[0257] Mimics of miR-199a-3p, miR-450a, miR-450c-3p and miR-451 were considered as protectomiR, since these miRNAs were upregulated by ischemic pre-, post- and remote conditioning vs Isch. AntagomiRs of miR-181a, miR-339, miR-142, miR-193a-3p, miR-29a, miR-204a, miR-424-3p, miR-127, miR-34a and miR-105-2 were considered as protectomiRs as these miRNAs were downregulated by ischemic pre-, post- and remote conditioning (Fig. 4).

[0258] 4. Cross-Species Pig-Rat MicroRNA Sequence Similarity Matching

[0259] Using NCBI RNA Blast tool and miRBase database, the present inventors were able to identify rat homologs of 12 out of 14 selected miRNAs. These rat miRNA homologs showed 100% sequence homology with the original pig miRNAs. Rat homolog of miR-105-2 showed less than 60% sequence similarity. In case of miR-424-3p no rat homolog miRNA could be identified. Therefore, miR-105-2 and miR-424-3p were not assessed in the validation step (Table 6).

[0260] Table 6: Cross-species pig-rat miRNA sequence similarity matching. First column lists the name of the protectomiR candidate porcine miRNAs. Second column shows the sequence similarity between pig (ssc-miR) and rat (mo-miR) microRNAs. In next columns the similarity is expressed in percentage and the name of the homologue rat miRNA is indicated.

[0261] 5. Cardioprotective effect of ProtectomiRs in Cardiac Myocytes Subjected to Simulated I / R

[0262] To test the cardiocytoprotective effect of the selected miRNAs, neonatal rat cardiac myocytes were transfected with miRNA mimics or antagomiRs and subjected to simulated I / R. Transfection of cardiac myocytes with two of the selected protectomiRs (miR-450a and miR-451 mimic) significantly increased cardiac myocyte cell survival after simulated I / R injury. Cells transfected with miR-451 and miR-450a mimic showed increased viability both at 25 nM concentration (93.71 ± 3.47% and 84.84 ± 2.61%, respectively) compared to cells transfected with negative control mimic and subjected also to sI / R (Fig. 5).

[0263] Transfection with the other miRNA mimics (miR-199a-3p and miR-450b-5p) and antagomiRs (miR-29a- 3p, miR-34a-5p, miR-127-3p, miR-142-5p, miR-181a-5p miR-193a-3p, miR-204-5p and miR-339-5p) did not increase cell viability significantly after sI / R. MiR-29a-3p, miR-34a-5p, miR-127-3p, and miR-142-5p antagomiRs and miR-450b-5p mimic even enhanced sI / R injury under different concentrations (Fig. 5, 6). Example 3: Evaluation of the efficacy of miR450a protectomiR on a simulated ischemia -reperfusion model of AC16 cell line

[0264] The present inventors have previously shown that certain microRNAs may have cardioprotective effect in in vitro rat models of ischemia-reperfusion injury (I / R) [Varga et al., 2014]. In the previous examples, the inventors identified new miRNAs with cardioprotective effect (protectomiRs), in a closed-chest porcine model of acute myocardial infarction (AMI) and validated their cardiocytoprotective effect in isolated rat cardiomyocytes. Here, the inventors aimed to validate the cardiocytoprotective effect of miR-450a, a novel protectomiR, in the human cardiomyocyte cell line AC16, to improve the translational value of their finding.

[0265] 3.1 Test articles miR-450a-3p mimic

[0266] Vehicle

[0267] Test Article Preparation The article has been previously prepared in 5 pM concentrations by adding 1 mL RNAse-free water to the lyophilized 5 nmol miRNA batch. After resuspension, the 5 pM miRNA solutions were aliquoted in 50 pL volumes.

[0268] 3.2 General test system parameters

[0269] Cell line requirements

[0270] The cells were purchased from Sigma-Aldrich (Cat# SCC109). Cells were maintained in a standard cell culture incubator at 37 °C, supplemented with 5% COj. The cell handling and all experimental procedures were performed under a laminar hood, in a sterile environment. All reagents, tools and equipment used for cell handling were sterilized before use. All media, buffers and treatment solutions that were to be added to the cells were warmed to 37 °C before use.

[0271] Guidelines

[0272] All procedures were performed in accordance with Hungarian legislation and permitted by the government (Ministry of Agriculture, Department for Biodiversity and Gene Conservation, approval number: BGMF / 683- 10 / 2022).

[0273] 3.3 Experimental design

[0274] Experimental model

[0275] AC16 cells, purchased from Sigma-Aldrich (Cat# SCC109) were maintained in DMEM-F12 medium (Capricorn Scientific, Cat# DMEM-12-A), with supplementation of 12.5% FBS (Corning, Cat# 35-079-CV), 10 mM HEPES (Gibco, Cat# 15630-056), 2 mM L-glutamine (Corning, Cat# 25-005-CI) and 1% Antibiotic- Antimycotic Solution (Coming, Cat# 30-004-CI). Cells were maintained for a maximum of 15 passages and kept at a 37 °C cell culture incubator with 5% COj. For the experimental procedure, cells were seeded on 96 well plates (Corning, Cat# CLS2592) at a density of 10000 cells / well. 24 hours after seeding, the cells were transfected with rno-miR-450a-3p mimic (Horizon Discovery, Cat# C-320808-00-0005) or Dharmacon miRIDIAN mimic negative control miRNA (Horizon Discovery, Cat# CN-001000-01-05) at 6.25, 12.5, 25, 50 and 100 nM with 0.02% DharmaFectl transfection reagent (Horizon Discovery, Cat# T-2001-03) in antibiotic-free growth medium for 24 hours according to the manufacturer’s protocol. The transfected cells were then subjected to simulated (I / R) as described before [Onodi et al., 2022]. To simulate ischemia, the cell culture medium was changed to a hypoxic solution, plates were placed into a hypoxic chamber with 1 % Oj, 94% N2, and 5% CO2 at 37°C for 16 h. Simulated ischemia was followed by 2 h of simulated reperfusion using antibiotic -free medium and normoxic conditions. Then, cell viability was measured with CellTiter-Glo Luminescent Viability Assay (Promega, Cat# G7571) according to the manufacturer’s protocol. Luminescence intensity was measured using VarioscanLUX (Thermo Fischer Scientific) plate reader. Time matched normoxic control groups were subjected to normoxia in normoxic medium, followed by 2h normoxic period using antibiotic -free medium. All experiments were performed at a sample size of N=5 with 6-8 technical replicates.

[0276] Experimental Groups

[0277] Figure 8 shows the experimental protocol for in vitro validation of miR-450a. As shown in Figure 8, there were 5 experimental groups: Group 1 was normoxia + vehicle, Group 2 was simulated ischemia + vehicle, Group 3 was normoxia + negative control miRNA, Group 4 was simulated ischemia + negative control miRNA, and Group 5 was simulated ischemia + rno-miR-450a-3p.

[0278] The experimental protocol for these 5 groups were the following.

[0279] Group 1: normoxia + vehicle

[0280] • Day 0: Cell seeding • Day 1: Transfection of cells with DharmaFectl and RNase-free water for 24 hours

[0281] • Day 2: Medium change to normoxic medium and placing plates in a normal cell culture incubator with 21% Oj for 16 hours

[0282] • Day 3: Medium change to antibiotic -free growth medium for 2 hours in normoxia and measurement of cell viability

[0283] Group 2: simulated ischemia + vehicle

[0284] • Day 0: Cell seeding

[0285] • Day 1: Transfection of cells with DharmaFectl and RNase-free water for 24 hours

[0286] • Day 2: Medium change to hypoxic medium and placing plates in a hypoxic chamber with 1% Oj for 16 hours

[0287] • Day 3: Medium change to antibiotic -free growth medium for 2 hours in normoxia and measurement of cell viability

[0288] Group 3: normoxia + negative control miRNA

[0289] • Day 0: Cell seeding

[0290] • Day 1: Transfection of cells with DharmaFectl and miRIDIAN mimic negative control miRNA at 6.25, 12.5, 25, 50 or 100 nM for 24 hours

[0291] • Day 2: Medium change to normoxic medium and placing plates in a normal cell culture incubator with 21% Oj for 16 hours

[0292] • Day 3: Medium change to antibiotic -free growth medium for 2 hours in normoxia and measurement of cell viability

[0293] Group 4: simulated ischemia + negative control miRNA

[0294] • Day 0: Cell seeding

[0295] • Day 1: Transfection of cells with DharmaFectl and miRIDIAN mimic negative control miRNA at 6.25, 12.5, 25, 50 or 100 nM for 24 hours

[0296] • Day 2: Medium change to hypoxic medium and placing plates in a hypoxic chamber with 1% Oj for 16 hours

[0297] • Day 3: Medium change to antibiotic -free growth medium for 2 hours in normoxia and measurement of cell viability

[0298] Group 5: simulated ischemia + rno-miR-450a-3p

[0299] • Day 0: Cell seeding

[0300] • Day 1: Transfection of cells with DharmaFectl and rno-miR-450a-3p mimic for 24 hours

[0301] • Day 2: Medium change to hypoxic medium and placing plates in a hypoxic chamber with 1% Oj for 16 hours

[0302] • Day 3: Medium change to antibiotic -free growth medium for 2 hours in normoxia and measurement of cell viability

[0303] All experiments were performed at a sample size of N=5 with 6-8 technical replicates.

[0304] 3.4 Evaluations

[0305] Statistical analysis

[0306] The difference between the above parameters measured in multiple groups were determined by one-way ANOVA, with Sidak’s post hoc test. Study limitations and risks

[0307] The study protocol used was the most suitable one for the purpose of the study according to the best of the inventors’ knowledge. A clinically more relevant transfection time would be after ischemia-reperfusion injury, however, transfection time is only feasible before simulated ischemia or normoxia. The effects of microRNA on mRNA expression resulting in a potential cardiocytoprotective effect might not be detected during the 2 hours of reperfusion since according to the literature [Hausser et al., 2013] the effects of microRNA on mRNA expression takes approx. 6 hours. Based on the inventors’ previous experience using similar cell culture models, there are no specific risks regarding this protocol.

[0308] 3.4 Results

[0309] The results are shown in Figure 9.

[0310] In line with the inventors’ findings in neonatal rat cardiac myocytes, rno-miR-450a-3p mimic could increase cell survival of AC16 cells after simulated I / R injury at 25 nM concentration (87.10 ± 5.03 %) compared to the negative control miRNA transfected cells.

[0311] 3.5 Conclusions

[0312] This is the first demonstration that rno-miR-450a-3p could show a cardiocytoprotective effect against I / R injury in the human cardiomyocyte cell line, AC16.

[0313] INDUSTRIAL APPLICABILTY

[0314] Any miRNA compound which is an agonist of miR-450a is an effective protectomiR and these cardioprotective miRNAs are useful as potential therapeutics for cardioprotection.

[0315] REFERENCES

[0316] Appella DH. (2009) Non-natural nucleic acids for synthetic biology. Curr Opin Chem Biol. 2009 Dec; 13(5 -6):687 - 96. doi: 10.1016 / j.cbpa.2009.09.030. Epub 2009 Oct 29. PMID: 19879178; PMCID: PMC3152792.

[0317] Baars T, Skyschally A, Klein-Hitpass L, Cario E, Erbel R, Heusch G, Kleinbongard P (2014) microRNA expression and its potential role in cardioprotection by ischemic postconditioning in pigs. Pflugers Archiv : European journal of physiology 466:1953-1961 doi:10.1007 / s00424-013-1429-3

[0318] Baek J, Kang S, Min H. MicroRNA-targeting therapeutics for hepatitis C. Arch. Pharm. Res. 2014

[0319] Baranyai T, Giricz Z, Varga ZV, Koncsos G, Lukovic D, Makkos A, Sarkozy M, Pavo N, Jakab A, Czimbalmos C, Vago H, Ruzsa Z, Toth L, Garamvblgyi R, Merkely B, Schulz R, Gybngybsi M, Ferdinandy P (2017) In vivo MRI and ex vivo histological assessment of the cardioprotection induced by ischemic preconditioning, postconditioning and remote conditioning in a closed-chest porcine model of reperfused acute myocardial infarction: importance of microvasculature. Journal of translational medicine 15:67 doi: 10.1186 / s 12967 -017- 1166-z

[0320] Bartel DP. (2004) MiRNAs: genomics, biogenesis, mechanism, and function Cell. 2004; 116: 281 -297.

[0321] Baumann V, Winkler J. miRNA-based therapies: strategies and delivery platforms for oligonucleotide and nonoligonucleotide agents. Future Med Chem. 2014;6( 17): 1967-84. doi: 10.4155 / fmc.l4.116. PMID: 25495987; PMCID: PMC4417715. Bellera N, Barba I, Rodriguez-Sinovas A, Ferret E, Asin MA, Gonzalez-Alujas MT, et al.. Single intracoronary injection of encapsulated antagomir-92a promotes angiogenesis and prevents adverse infarct remodeling. J Am Heart Assoc 2014;3:e000946.

[0322] Botker HE, Hausenloy D, Andreadou I, Antonucci S, Boengler K, Davidson SM, Deshwal S, Devaux Y, Di Lisa F, Di Sante M, Efentakis P, Femmind S, Garcia-Dorado D, Giricz Z, Ibanez B, Iliodromitis E, Kaludercic N, Kleinbongard P, Neuhauser M, Ovize M, Pagliaro P, Rahbek-Schmidt M, Ruiz-Meana M, Schluter KD, Schulz R, Skyschally A, Wilder C, Yellon DM, Ferdinandy P, Heusch G (2018) Practical guidelines for rigor and reproducibility in preclinical and clinical studies on cardioprotection. Basic research in cardiology 113:39 doi: 10.1007 / s00395 -018-0696-8

[0323] Cao J, Da Y, Li H, Peng Y, Hu X (2020) Upregulation of microRNA-451 attenuates myocardial I / R injury by suppressing HMGB1. PLOS ONE 15(7): e0235614. https: / / doi.org / 10.1371 / joumal.pone.0235614

[0324] Chakraborty, C., Sharma, A. R., Sharma, G., & Lee, S. S. (2020). Therapeutic advances of miRNAs: A preclinical and clinical update. Journal of advanced research, 28, 127-138. https: / / doi.Org / 10.1016 / j.jare.2020.08.012

[0325] Chang-Hui Shen (2019) Diagnostic Molecular Biology, Editor(s): Chang-Hui Shen, Academic Press, 2019, Pages 27-57, ISBN 9780128028230,

[0326] Chilukoti RK, Lendeckel J, Darm K, Bukowska A, Goette A, Suhling M, Utpatel K, Peters B, Homuth G, Volker U, Wolke C, Scharf C, Lendeckel U (2018) Integration of "omics" techniques: Dronedarone affects cardiac remodeling in the infarction border zone. Experimental biology and medicine (Maywood, N.J.) 243:895-910 doi:10.1177 / 1535370218788517

[0327] Chouvarine P, Legchenko E, Geldner J, Riehle C, Hansmann G (2019) Hypoxia drives cardiac miRNAs and inflammation in the right and left ventricle. Journal of molecular medicine (Berlin, Germany) 97: 1427-1438 doi:10.1007 / s00109-019-01817-6

[0328] Chung Eun Ha, N.V. Bhagavan (2023) Essentials of Medical Biochemistry (Third Edition), Editor(s): Chung Eun Ha, N.V. Bhagavan, Academic Press, 2023, Pages 453-476, ISBN 9780323885416,

[0329] Colpaert RMW, Calore M (2019) MicroRNAs in Cardiac Diseases. Cells 8 doi: 10.3390 / cells8070737

[0330] Davidson SM, Ferdinandy P, Andreadou I, Botker HE, Heusch G, Ibanez B, Ovize M, Schulz R, Yellon DM, Hausenloy DJ, Garcia-Dorado D (2019) Multitarget Strategies to Reduce Myocardial Ischemia / Reperfusion Injury: JACC Review Topic of the Week. Journal of the American College of Cardiology 73:89-99 doi:10.1016 / j.jacc.2018.09.086

[0331] Ellenbroek GH, van Hout GP, Timmers L, Doevendans PA, Pasterkamp G, Hoefer IE (2016) Primary Outcome Assessment in a Pig Model of Acute Myocardial Infarction. Journal of visualized experiments : JoVE doi:10.3791 / 54021

[0332] Ferdinandy P, Hausenloy DJ, Heusch G, Baxter GF, Schulz R (2014) Interaction of risk factors, comorbidities, and comedications with ischemia / reperfusion injury and cardioprotection by preconditioning, postconditioning, and remote conditioning. Pharmacological reviews 66:1142-1174 doi:10.1124 / pr.l 13.008300

[0333] Gadeberg HC, Bond RC, Kong CH, Chanoit GP, Ascione R, Cannell MB, James AF (2016) Heterogeneity of T- Tubules in Pig Hearts. PLoS One l l:e0156862 doi: 10.1371 / journal.pone.0156862 Gorbe A, Giricz Z, Szunyog A, Csont T, Burley DS, Baxter GF, Ferdinandy P (2010) Role of cGMP-PKG signaling in the protection of neonatal rat cardiac myocytes subjected to simulated ischemia / reoxygenation. Basic research in cardiology 105:643-650 doi: 10.1007 / s00395-010-0097-0

[0334] Hausenloy DJ, Garcia-Dorado D, Botker HE, Davidson SM, Downey J, Engel FB, Jennings R, Lecour S, Leor J, Madonna R, Ovize M, Perrino C, Prunier F, Schulz R, Sluijter JPG, Van Laake LW, Vinten-Johansen J, Yellon DM, Ytrehus K, Heusch G, Ferdinandy P (2017) Novel targets and future strategies for acute cardioprotection: Position Paper of the European Society of Cardiology Working Group on Cellular Biology of the Heart. Cardiovascular research 113:564-585 doi: 10.1093 / cvr / cvx049

[0335] Hausser, J., Syed, A.P., Selevsek, N., van Nimwegen, E., Jaskiewicz, L., Aebersold, R., Zavolan M. (2013). Timescales and bottlenecks in miRNA-dependent gene regulation. Mol Sy st Biol. 9:711.

[0336] Huang CK, Kafert-Kasting S, Thum T (2020) Preclinical and Clinical Development of Noncoding RNA Therapeutics for Cardiovascular Disease. Circulation research 126:663-678 doi:10.1161 / circresaha.119.315856

[0337] Jia P, Wu X, Dai Y, Teng J, Fang Y, Hu J, Zou J, Liang M, Ding X (2017) MicroRNA-21 Is Required for Local and Remote Ischemic Preconditioning in Multiple Organ Protection Against Sepsis. Critical care medicine 45:e703-e710 doi: 10.1097 / ccm.0000000000002363

[0338] Jia Z, Lian W, Shi H, Cao C, Han S, Wang K, Li M, Zhang X (2017) Ischemic Postconditioning Protects Against Intestinal Ischemia / Reperfusion Injury via the HIF-la / rniR-21 Axis. Scientific reports 7:16190 doi:10.1038 / s41598-017-16366-6

[0339] Kozomara Ana et al., miRBase: from microRNA sequences to function, Nucleic Acids Research, Volume 47, Issue DI, 08 January 2019, Pages D155-D162, https: / / doi.org / 10.1093 / nar / gkyl l41

[0340] Kozomara, Ana and Griffiths-Jones, Sam miRBase: annotating high confidence microRNAs using deep sequencing data, Nucleic Acids Research, Volume 42, Issue DI, 1 January 2014, Pages D68-D73, https: / / doi.org / 10.1093 / nar / gktl l81

[0341] Kim HW, Mallick F, Durrani S, Ashraf M, Jiang S, Haider KH (2012) Concomitant activation of miR- 107 / PDCD10 and hypoxamir-210 / Casp8ap2 and their role in cytoprotection during ischemic preconditioning of stem cells. Antioxidants & redox signaling 17: 1053-1065 doi: 10.1089 / ars.2012.4518

[0342] Koudstaal S, Jansen of Lorkeers S, Gho JM, van Hout GP, Jansen MS, GriindemanPF, Pasterkamp G, Doevendans PA, Hoefer IE, Chamuleau SA (2014) Myocardial infarction and functional outcome assessment in pigs. Journal of visualized experiments : JoVE doi: 10.3791 / 51269

[0343] Lecour S, Botker HE, Condorelli G, Davidson SM, Garcia-Dorado D, Engel FB, Ferdinandy P, Heusch G, Madonna R, Ovize M, Ruiz-Meana M, Schulz R, Sluijter JP, Van Laake LW, Yellon DM, Hausenloy DJ (2014) ESC working group cellular biology of the heart: position paper: improving the preclinical assessment of novel cardioprotective therapies. Cardiovascular research 104:399-411 doi: 10.1093 / cvr / cvu225

[0344] Lee TJ, Yuan X, Kerr K, Yoo JY, Kim DH, Kaur B, Eltzschig HK. Strategies to Modulate MicroRNA Functions for the Treatment of Cancer or Organ Injury. Pharmacol Rev. 2020 Jul;72(3):639-667. doi: 10.1124 / pr.119.019026. PMID: 32554488; PMCID: PMC7300323.

[0345] Lennox KA, Behlke MA. (2011) Chemical modification and design of anti-miRNA oligonucleotides. Gene Ther. 2011 Dec;18(12):l l l l-20. doi: 10.1038 / gt.2011.100. Epub 2011 Jul 14. PMID: 21753793. Li, Y-m, Sun, J-g, Hu, L-h, Ma, X-c, Zhou, G, Huang, X-z. (2019). Propofol-mediated cardioprotection dependent of microRNA-451 / HMGBl against myocardial ischemia-reperfusion injury. J Cell Physiol. 2019; 234: 23289- 23301. https: / / doi.org / 10.1002 / jcp.28897

[0346] Li, W., Dong, M., Chu, L., Feng, L., & Sun, X. (2019). MicroRNA-451 relieves inflammation in cerebral ischemia-reperfusion via the Toll-like receptor 4 / MyD88 / NF-KB signaling pathway. Molecular Medicine Reports, 20, 3043-3054. https: / / doi.org / 10.3892 / mmr.2019.10587

[0347] Lim GB (2019) MicroRNA-directed cardiac repair after myocardial infarction in pigs. Nat Rev Cardiol 16:454- 455 doi: 10.1038 / s41569-019-0216-z

[0348] Lim LP, Lau NC, Garrett-Engele P, Grimson A, Schelter JM, Castle J, Bartel DP, Linsley PS, Johnson JM (2005) Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs. Nature 433:769-773 doi:10.1038 / nature03315

[0349] Lima JF et al. (2018) Anti-miRNA oligonucleotides: A comprehensive guide for design. RNA Biol. 2018 Mar 4; 15(3):338-352. doi: 10.1080 / 15476286.2018.1445959. Epub 2018 Mar 23. PMID: 29570036; PMCID: PMC5927725.

[0350] Liu L, Chen Y, Shu J, Tang CE, Jiang Y, Luo F (2020) Identification of microRNAs enriched in exosomes in human pericardial fluid of patients with atrial fibrillation based on bioinformatic analysis. J Thorac Dis 12:5617-5627 doi:10.21037 / jtd-20-2066

[0351] Liu L, Luo F, Lei K (2021) Exosomes Containing LINC00636 Inhibit MAPK1 through the miR-450a-2-3p Overexpression in Human Pericardial Fluid and Improve Cardiac Fibrosis in Patients with Atrial Fibrillation. Mediators of Inflammation 2021:9960241 doi: 10.1155 / 2021 / 9960241

[0352] Louch WE, Sheehan KA, Wolska BM (2011) Methods in cardiomyocyte isolation, culture, and gene transfer. Journal of molecular and cellular cardiology 51:288-298 doi: 10.1016 / j.yjmcc.2011.06.012

[0353] Luo M, Weng Y, Tang J, Hu M, Liu Q, Jiang F, Yang D, Liu C, Zhan X, Song P, Bai H, Li B, Shi Q (2012) Micro RNA-450a-3p represses cell proliferation and regulates embryo development by regulating Bubl expression in mouse. PLoS One 7:e47914 doi:10.1371 / journal.pone.0047914

[0354] Lv XW, He ZF, Zhu PP, Qin QY, Han YX, Xu TT (2021) miR-451-3p alleviates myocardial ischemia / reperfusion injury by inhibiting MAPlLC3B-mediated autophagy. Inflamm Res 70:1089-1100 doi:10.1007 / s00011-021- 01508-4

[0355] Makkos A, Szantai A, Paloczi J, Pipis J, Kiss B, Poggi P, Ferdinandy P, Chatgilialoglu A, Gorbe A (2019) A Comorbidity Model of Myocardial Ischemia / Reperfusion Injury and Hypercholesterolemia in Rat Cardiac Myocyte Cultures. Frontiers in physiology 10:1564 doi: 10.3389 / fphys.2019.01564

[0356] Moghaddam AS, Afshari JT, Esmaeili SA, Saburi E, Joneidi Z, Momtazi-Borojeni AA (2019) Cardioprotective microRNAs: Lessons from stem cell-derived exosomal microRNAs to treat cardiovascular disease. Atherosclerosis 285:1-9 doi: 10.1016 / j. atherosclerosis.2019.03.016

[0357] Ong SB, Katwadi K, Kwek XY, Ismail NI, Chinda K, Ong SG, Hausenloy DJ (2018) Non-coding RNAs as therapeutic targets for preventing myocardial ischemia-reperfusion injury. Expert opinion on therapeutic targets 22:247-261 doi:10.1080 / 14728222.2018.1439015

[0358] Onodi Z, Visnovitz T, Kiss B, Hambalko S, Koncz A, Agg B, Varadi B, Toth V, Nagy RN, Gergely TG, Gergo D, Makkos A, Pelyhe C, Varga N, Ree D, Apati A, Leszek P, Kovacs T, Nagy N, Ferdinandy P, Buzas El, Gorbe A, Giricz Z, Varga ZV (2022) Systematic transcriptomic and phenotypic characterization of human and murine cardiac myocyte cell lines and primary cardiomyocytes reveals serious limitations and low resemblances to adult cardiac phenotype. Journal of molecular and cellular cardiology 165: 19-30 doi: 10.1016 / j .yjmcc.20 1.1 .007

[0359] Paloczi J, Szantai A, Kobolak J, Bock I, Ruivo E, Kiss B, Gaspar R, Pipis J, Ocsovszki I, Tancos Z, Feher A, Dinnyes A, Onodi Z, Madonna R, Ferdinandy P, Gorbe A (2020) Systematic analysis of different pluripotent stem cell-derived cardiac myocytes as potential testing model for cardiocytoprotection. Vascular pharmacology 133-134: 106781 doi: 10.1016 / j.vph.2020.106781

[0360] Perrino C, Barabasi AL, Condorelli G, Davidson SM, De Windt L, Dimmeler S, Engel FB, Hausenloy DJ, Hill JA, Van Laake LW, Lecour S, Leor J, Madonna R, Mayr M, Prunier F, Sluijter JPG, Schulz R, Thum T, Ytrehus K, Ferdinandy P (2017) Epigenomic and transcriptomic approaches in the post-genomic era: path to novel targets for diagnosis and therapy of the ischaemic heart? Position Paper of the European Society of Cardiology Working Group on Cellular Biology of the Heart. Cardiovascular research 113:725-736 doi:10.1093 / cvr / cvx070

[0361] Rane S, He M, Sayed D, Vashistha H, Malhotra A, Sadoshima J, Vatner DE, Vatner SF, Abdellatif M (2009) Downregulation of miR-199a derepresses hypoxia-inducible factor- 1 alpha and Sirtuin 1 and recapitulates hypoxia preconditioning in cardiac myocytes. Circulation research 104:879-886 doi:10.1161 / circresaha.l08.193102

[0362] Roberts TC, Langer R, Wood MJA (2020) Advances in oligonucleotide drug delivery. Nature Reviews Drug Discovery 19:673-694 doi:10.1038 / s41573-020-0075-7

[0363] Schreckenberg R, Klein J, Kutsche HS, Schulz R, Gbmbri K, Bencsik P, Benczik B, Agg B, Saghy E, Ferdinandy P, Schluter KD (2020) Ischaemic post-conditioning in rats: Responder and non-responder differ in transcriptome of mitochondrial proteins. Journal of cellular and molecular medicine 24:5528-5541 doi:10.1111 / jcmm.l5209

[0364] Scrimgeour NR, Wrobel A, Pinho MJ, Hoydal MA (2020) microRNA-451a prevents activation of matrix metalloproteinases 2 and 9 in human cardiomyocytes during pathological stress stimulation. American journal of physiology. Cell physiology 318:C94-clO2 doi: 10.1152 / ajpcell.00204.2019

[0365] Shah AM, Giacca M. Small non-coding RNA therapeutics for cardiovascular disease. Eur Heart J. 2022 Nov 14;43(43):4548-4561. doi: 10.1093 / eurheartj / ehac463. PMID: 36106499; PMCID: PMC9659475. Sluijter JP, Condorelli G, Davidson SM, Engel FB, Ferdinandy P, Hausenloy DJ, Lecour S, Madonna R, Ovize M, Ruiz- Meana M, Schulz R, Van Laake LW (2014) Novel therapeutic strategies for cardioprotection. Pharmacol Ther 144:60-70 doi:10.1016 / j.pharmthera.2014.05.005

[0366] Taubel J, Hauke W, Rump S, Viereck J, Batkai S, Poetzsch J, Rode L, Weigt H, Genschel C, Lorch U, Theek C, Levin AA, Bauersachs J, Solomon SD, Thum T (2021) Novel antisense therapy targeting microRNA-132 in patients with heart failure: results of a first-in-human Phase lb randomized, double-blind, placebo -controlled study. European heart journal 42:178-188 doi: 10.1093 / eurheartj / ehaa898 van Rooij E, Sutherland LB, Thatcher JE, DiMaio JM, Naseem RH, Marshall WS, Hill JA, Olson EN (2008) Dysregulation of microRNAs after myocardial infarction reveals a role of miR-29 in cardiac fibrosis. Proceedings of the National Academy of Sciences of the United States of America 105: 13027-13032 doi:10.1073 / pnas.0805038105 Varga ZV, Giricz Z, Bencsik P, Madonna R, Gyongyosi M, Schulz R, Mayr M, Thum T, Puskas LG, Ferdinandy P (2015) Functional Genomics of Cardioprotection by Ischemic Conditioning and the Influence of Comorbid Conditions: Implications in Target Identification. Current drug targets 16:904-911 doi: 10. 174 / 1389450116666150427154203 Varga ZV, Kupai K, Szucs G, Gaspar R, Paloczi J, Farago N, Zvara A, Puskas LG, Razga Z, Tiszlavicz L, Bencsik

[0367] P, Gorbe A, Csonka C, Ferdinandy P, Csont T (2013) MicroRNA-25 -dependent up-regulation of NADPH oxidase 4 (N0X4) mediates hypercholesterolemia-induced oxidative / nitrative stress and subsequent dysfunction in the heart. Journal of molecular and cellular cardiology 62: 111-121 doi: 10.1016 / j .yjmcc.2013.05.009 Varga ZV, Zvara A, Farago N, Kocsis GF, Pipicz M, Gaspar R, Bencsik P, Gorbe A, Csonka C, Puskas LG, Thum

[0368] T, Csont T, Ferdinandy P (2014) MicroRNAs associated with ischemia-reperfusion injury and cardioprotection by ischemic pre- and postconditioning: protectomiRs. American journal of physiology. Heart and circulatory physiology 307:H216-227 doi: 10.1152 / ajpheart.00812.2013

[0369] Voigt N, Pearman CM, Dobrev D, Dibb KM (2015) Methods for isolating atrial cells from large mammals and humans. Journal of molecular and cellular cardiology 86:187-198 doi: 10.1016 / j.yjmcc.2015.07.006

[0370] Wang X, Zhu H, Zhang X, Liu Y, Chen J, Medvedovic M, Li H, Weiss MJ, Ren X, Fan GC (2012) Loss of the miR-144 / 451 cluster impairs ischaemic preconditioning -mediated cardioprotection by targeting Rac-1. Cardiovascular research 94:379-390 doi: 10.1093 / cvr / cvs096

Claims

CLAIMS1. A miRNA compound for use in a treatment to protect cells, tissues and / or organs of a subject against consequences of ischemic injury and / or reperfusion injury in a patient predisposed to or affected by ischemia, wherein said miRNA compound is a miRNA agonist of a miRNA species miR-450a, said miRNA agonist having the same target specificity as miR-450a or overlapping target specificity as miR-450a.

2. The miRNA compound for use according to claim 1, wherein said miRNA agonist comprises an oligonucleotide moiety, wherein optionally said oligonucleotide moiety is a nucleic acid analogue, wherein preferably one or more nucleotides of said oligonucleotide moiety is / are chemically modified nucleotides, and / or said miRNA agonist comprises one or two tag(s) at either or both end of the oligonucleotide moiety.

3. The miRNA compound for use according to claim 2, wherein said miRNA compound comprises a a double stranded oligonucleotide or a single stranded oligonucleotide.

4. The miRNA compound for use according to any of claims 1 to 3, wherein said miRNA compound shows a cell protective effect in a cardiomyocyte cell viability assay.

5. The miRNA compound for use according to any of claims 1 to 4, wherein said miRNA agonist comprises a nucleotide sequence identical to the seed region of the corresponding miRNA species or is different at most in 1, 2, 3, 4 or 5 nucleotide(s) from said sequence and / or said miRNA agonist comprises a 17 to 27 nucleotides long nucleic acid segment the sequence of which is identical to the sequence of said miRNA species miR-450a, or is different at most in 1, 2, 3, 4, 5, 6 or 7 nucleotide(s) from said sequence, respectively, with the proviso that chemically modified nucleotides of natural nucleotides or base analogs of natural bases are not considered as being different in terms of sequences.

6. The miRNA compound for use according to any of claims 1 to 5, wherein the treatment is to protect cells, tissues and / or organs against ischemic and reperfusion injury, preferably against acute ischemic and reperfusion injury.

7. The miRNA compound for use according to claim 6, wherein the treatment is to protect cells, tissues and / or organs against short term and / or direct consequences of acute ischemic and reperfusion injury.

8. The miRNA compound for use according to any of claims 1 to 7, wherein the treatment is to protect tissues and / or organs, preferably heart tissues or the heart, against acute ischemic and reperfusion injury in the heart of said patient.

9. The miRNA compound for use according to any of claims 1 to 8, wherein- said miRNA compound is administered to the patient within 24 hours after the ischemic attack in said tissue and / or organ or simultaneously with reperfusion therein, and / or- said miRNA compound is administered to the patient less than 24 hours before surgery or intervention.

10. The miRNA compound for use according to any of claims 1 to 9, wherein said miRNA compound is used in combination with a miRNA agonist of a miRNA species miR-451.

11. A combination of miRNA compounds for use in a treatment as defined in any of claims 1 to 9, wherein said combination comprises a miRNA compound according to claim 1 and a miRNA agonist of a miRNA species miR-451, wherein the miRNA agonist of miRNA species miR-451 is defined as the same way as the miRNA agonist of miRNA species miR-450a defined in any of claims 1 to 10, mutatis mutandis.

12. The miRNA compound for use in combination according to claim 10 or the combination for use according to claim 11, wherein said miRNA agonist of a miRNA species miR-451 comprises a nucleic acid sequence identical with the seed region of said miRNA species and / or which comprises a 17 to 27 nucleotides long nucleic acid segment, the sequence of which is identical to the sequence of said miRNA species or is different at most in 1, 2, 3, 4, 5 or 6 nucleotide(s) therefrom, preferably while maintaining the agonizing effect, preferably as defined in claim 4.

13. A pharmaceutical composition for use in a treatment to protect cells, tissues and / or organs against short term and / or direct consequences of acute ischemic and reperfusion injury in a patient predisposed to or affected by ischemia, in the heart of a patient predisposed to or attacked by acute ischemia, said composition comprising a miRNA compound for use according to any of claims 1 to 9 or a combination for use as defined in claim 11 or 12, and pharmaceutically acceptable excipient and / or carrier.

14. A pharmaceutical composition for use in a treatment to protect cells, tissues and / or organs against short term and / or direct consequences of acute ischemic and reperfusion injury in a patient predisposed to or affected by ischemia, in the heart of a patient predisposed to or attacked by acute ischemia, said composition comprising a further cardioprotective miRNA compound.

Images