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WO2025003718A1 - Composés pour le traitement d'une lésion ischémique - Google Patents

Composés pour le traitement d'une lésion ischémique Download PDF

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Publication number
WO2025003718A1
WO2025003718A1 PCT/HU2024/050049 HU2024050049W WO2025003718A1 WO 2025003718 A1 WO2025003718 A1 WO 2025003718A1 HU 2024050049 W HU2024050049 W HU 2024050049W WO 2025003718 A1 WO2025003718 A1 WO 2025003718A1
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Prior art keywords
mirna
mir
ischemia
ischemic
compound
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Inventor
Péter FERDINANDY
Anikó GÖRBE
András MAKKOS
Bence ÁGG
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PHARMAHUNGARY 2000 KFT
Tamogatott Kutatocsoportok Irodaja
Semmelweis Egyetem
Original Assignee
PHARMAHUNGARY 2000 KFT
Tamogatott Kutatocsoportok Irodaja
Semmelweis Egyetem
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Publication of WO2025003718A1 publication Critical patent/WO2025003718A1/fr
Anticipated expiration legal-status Critical
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • C12N2310/141—MicroRNAs, miRNAs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • the 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.
  • Ischemic conditioning stimuli have been shown to induce cardioprotective signaling in experimental animal models of acute ischemia/reperfusion (I/R) injury.
  • I/R acute ischemia/reperfusion
  • 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].
  • MI myocardial infarction
  • 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].
  • 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].
  • 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].
  • 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.
  • said miRNA compound is a miRNA agonist of miR-125b* (used in itself or in combination with other miRNA compounds).
  • said miRNA compound is a miRNA antagonist of miR-487b (used in itself or in combination with other miRNA compounds).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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. reviewed the preclinical/clinical trials about miRNA therapeutics. However, they did not disclose any effect of miR-450a.
  • 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.
  • 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.
  • 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.
  • said subject is a vertebrate animal, preferably a mammal, highly preferably a human.
  • the subject is an animal having a cardiovascular system.
  • the subject is a patient.
  • 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.
  • said miRNA compound comprises a a double stranded oligonucleotide or a single stranded oligonucleotide.
  • 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.
  • 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.
  • 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.
  • 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.
  • the treatment is to protect cells, tissues and/or organs against ischemic and reperfusion injury.
  • ischemic and reperfusion injury is acute ischemic and reperfusion injury.
  • the treatment is to protect cells, tissues and/or organs against short term and/or direct consequences of acute ischemic and reperfusion injury.
  • 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.
  • 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.
  • 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.
  • 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.
  • said miRNA compound is administered to the patient less than one day before surgery or intervention.
  • 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.
  • the miRNA compound of the invention is used in combination with one or more further cardioprotective miRNA compound(s).
  • the tissue is a cardiovascular tissue, preferably a heart tissue, and/or the organ is an organ of the cardiovascular system, preferably the heart.
  • the miRNA compound of the invention is used in combination with a miRNA agonist of a miRNA species miR-451, as defined herein.
  • 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.
  • 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.
  • 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.
  • 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.
  • said miRNA compound is administered to the patient less than 24 hours before surgery or intervention.
  • 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.
  • 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.
  • 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 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.
  • 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
  • said miRNA compound is a miRNA agonist of
  • acute myocardial infarction is treated or prevented.
  • 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.
  • 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,
  • 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.
  • 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 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 as
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • PCI percutaneous coronary intervention
  • 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 surgery of the brain or nervous system
  • tumors from the heart, brain, lung, kidney, liver, bowels peripheral vessel diseases.
  • the miRNA compounds are applied typically for preventive purposes.
  • 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.
  • the patient predisposed to ischemia is a patient predisposed to acute ischemia and/or acute reperfusion injury.
  • 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.
  • 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).
  • 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.
  • 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
  • the miRNA compound is a miRNA agonist of miR-450a, preferably miRNA agonist of miR-450a and a miRNA agonist of miR-451.
  • 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.
  • the miRNA agonist is a miRNA mimic of said miRNA species.
  • 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).
  • the pharmaceutical composition comprises multiple compounds.
  • the invention relates to a pharmaceutical kit comprising multiple compounds.
  • 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-ische
  • miRNA micro-RNA
  • the miRNA species are termed as defined above dependent on their up -or down-regulation in the samples.
  • the calculated ratios of the expression levels are obtained by a) assessing the expression level of miRNA species
  • RNA ribonucleic acid
  • MiRNAs play an essential role in the posttranscriptional regulation of gene expression by repression or activation of translation/transcription ("RNA interference").
  • miRNA sequences 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],
  • 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.
  • 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.
  • MiRNA compounds may comprise natural nucleotides or nucleotide analogs.
  • 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).
  • 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.
  • a miRNA mimic may comprise one or more modified or artificial base(s), sugar unit(s) or internucleotide linkage(s)
  • a publishedmiRNA 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.
  • 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.
  • 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.
  • the miRNA compound has a nucleic acid moiety comprising a nucleotide sequence identical with the seed region of the corresponding miRNA species.
  • 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.
  • 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.
  • 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.
  • 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.
  • Acute ischemia of the brain is "stroke".
  • 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.
  • Ischemia may be cardiac ischemia, e.g. ischemia of the myocardium.
  • TIA transient ischemic attack
  • 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).
  • 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
  • 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.
  • PMI postoperative myocardial infarction
  • 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).
  • 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).
  • 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.
  • 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.
  • 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.
  • cell death occurs, at least pre-eminently, via a necrotic and/or apoptotic mechanism and/or via autophagy.
  • acute reperfusion injury is insensitive to anti-inflammatory treatment.
  • 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.
  • AMI 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.
  • IHD Ischemic heart disease
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • administering 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.
  • 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.
  • PCI Percutaneous Coronary Intervention
  • angioplasty involves the insertion of a balloontype device into a patient’s artery to widen and open the blocked artery.
  • 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.
  • microRNAs are 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.
  • 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.
  • 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.
  • 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.
  • sI/R simulated ischemia-reperfusion
  • 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.
  • SI/R simulated ischemia-reperfusion injury
  • Fig. 9 Effect on cell viability of miR-450a-3p after simulated ischemia-reperfusion (sI/R) in human AC 16 cardiomyocytes.
  • 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.
  • 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.
  • 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.
  • AMI acute myocardial infarction
  • 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.
  • 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.
  • 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.
  • the inventors identified potential cardioprotective microRNAs (protectomiRs) in a porcine model of reperfused acute myocardial infarction (AMI) and cardioprotection and validated their cardiocytoprotective effect.
  • AMI 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.
  • 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].
  • 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.
  • miRNA-29b, - 133a, and -146b as miRNAs are associated with postconditioning [Baars et al., 2014].
  • 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].
  • 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.
  • 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.
  • 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].
  • 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].
  • the present inventors performed cross-species pig-rat microRNA sequence similarity matching and tested mimic or antagomiR protectomiRs with 100% homology to pig miRNAs.
  • 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.
  • the sequence of rat miR-450a-3p 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.
  • 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.
  • 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.
  • the miRNA agonist of a miRNA species miR-450a is a useful drug candidate and is further developed as therapeutic for cardioprotection.
  • 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.
  • nucleic acids 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.
  • 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.
  • TNA threose nucleic acids
  • LNA locked nucleic acid
  • HNA beta-hexitol nucleic acids
  • 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.
  • 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.
  • Phosphodiester bond 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.
  • PS phosphorothioate
  • Internucleotide modifications may be combined with sugar modifications to improve potency and resistance to the oligo- nucleotide sequence.
  • 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.
  • 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.
  • bases are for example isoguanine, isocytosine and 2,6-diaminopurine for adenine.
  • base replacements can be considered as mutations, in particular if binding properties are changed.
  • Typical single base replacements are e.g.
  • Cytosine yC, xC, isocytosine
  • Guanin isoguanine, xG.
  • nucleic analogs in the form of oligonucleotides is contemplated in the present invention.
  • 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.
  • miRNA mimic preparation is a routine task and such technology (available from e.g. ThermoScientific, Dharmacon) may in itself be suitable.
  • a nucleic analog may be part of a larger molecule considered as a miRNA compound.
  • it may comprise flanking tags and sequences for stabilization, targeting or reporting.
  • flanking tags and sequences may be removed in the patient’s body e.g. by nucleases or may be pharmaceutically tolerable.
  • both double-stranded and single stranded variants may work.
  • a stranded variants i.e. an RNA analogue is applied.
  • lipid-based delivery e.g. by liposomes is one of the further most accepted approach for targeting miRNA to the site of the injury.
  • lipids e.g. cationic lipid and other, stabilizing molecules like PEG or a further helper lipid
  • 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].
  • 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]
  • Polymers like polyethylene imine (PEI) has been known as a means for delivering nucleic acid based medicinal agents.
  • PEI polyethylene imine
  • Various alternatives have been developed like copolymers.
  • Another potential option is Poly(lactic-co-glycolic acid) (PLGA) which has been proposed for oligonucleotide delivery.
  • PLGA Poly(lactic-co-glycolic acid)
  • antagomiR-92 encapsulated in PLGA microspheres was shown to improve cardiac function and angiogenesis after MI in pigs [Bellera N et al., 2014].
  • 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].
  • 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.
  • such therapies include administration of a miRNA of the invention to a patient in need of such treatment.
  • a formulation e.g. a formulation as disclosed herein is carried out depending on the target tissue.
  • a formulation e.g. a formulation as disclosed herein is administered intravenously (iv.) to the patient.
  • 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].
  • 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:
  • Sham operated control (Sham): balloon catheter was placed into the coronary artery but remained deflated.
  • Isch Ischemia-reperfusion group
  • Ischemic preconditioning group IPreC: 3 * 5 min myocardial ischemia was applied before the 90 min LAD occlusion.
  • 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).
  • 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.
  • Table 2A miRNA HT-qPCR data table
  • Table 2B miRNA HT-qPCR data table (cont.)
  • 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.
  • 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.
  • the present inventors selected the protectomiR candidate miRNAs associated with all three cardioprotective maneuver (IPreC, IPostC and RIPerC), the triple overlap.
  • IPreC cardioprotective maneuver
  • IPostC IPostC
  • RIPerC RIPerC
  • 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.
  • 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.
  • 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].
  • 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.
  • the transfected cells were subjected to a simulated ischemia-reperfusion (sI/R) protocol as previously described [Makkos et al., 2019; Paloczi etal., 2020].
  • sI/R simulated ischemia-reperfusion
  • 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.
  • 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.
  • 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.
  • microRNAs differentially expressed by one or multiple ischemic conditionings versus Isch are presented on a Venn diagram (Fig. 2).
  • 29 microRNAs were up- and 28 microRNAs were down-regulated.
  • 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.
  • 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.
  • 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.
  • 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.
  • MiRNAs modulated by three conditionings versus Ischemia were considered to have the highest probability to play a role in cardioprotection.
  • 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.
  • 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).
  • rat homologs of 12 out of 14 selected miRNAs 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).
  • 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.
  • ssc-miR pig
  • rat mi-miR
  • 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 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).
  • microRNAs may have cardioprotective effect in in vitro rat models of ischemia-reperfusion injury (I/R) [Varga et al., 2014].
  • I/R ischemia-reperfusion injury
  • 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.
  • AMI acute myocardial infarction
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • rno-miR-450a-3p mimic Horizon Discovery, Cat# C-320808-00-0005
  • Dharmacon miRIDIAN mimic negative control miRNA Horizon Discovery, Cat# CN-001000-01-05
  • the transfected cells were then subjected to simulated (I/R) as described before [Onodi et al., 2022].
  • I/R simulated
  • 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.
  • cell viability was measured with CellTiter-Glo Luminescent Viability Assay (Promega, Cat# G7571) according to the manufacturer’s protocol.
  • 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.
  • Group 2 simulated ischemia + vehicle
  • Group 3 normoxia + negative control miRNA
  • Group 4 simulated ischemia + negative control miRNA
  • Group 5 simulated ischemia + rno-miR-450a-3p
  • 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.
  • 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.
  • 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.
  • 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, Anthonyy P, Heusch G (2016) 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
  • 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
  • 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
  • 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.

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Abstract

La présente invention concerne des composés de microARN et une composition pharmaceutique les comprenant destinés à être utilisés dans une prophylaxie et/ou un traitement de cellules, de tissus et/ou d'organes pour les protéger contre une lésion d'ischémie-reperfusion chez un patient présentant une prédisposition pour, ou affecté par, une ischémie, l'utilisation des composés de miARN et des acides nucléiques codant pour ceux-ci dans le diagnostic et dans la préparation de compositions pharmaceutiques et des méthodes de traitement d'un patient nécessitant une protection des cellules ou des tissus contre des conséquences d'une lésion d'ischémie-reperfusion aiguë.
PCT/HU2024/050049 2023-06-30 2024-06-29 Composés pour le traitement d'une lésion ischémique Pending WO2025003718A1 (fr)

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Title
ANDRAS MAKKOS ET AL.: "Systematic review and network analysis of microRNAs involved in cardioprotection against myocardial ischemia/reperfusion injury and infarction: Involvement of redox signalling", FREE RADICAL BIOLOGY AND MEDICINE, vol. 172, 2021, pages 237 - 251, XP086727041, DOI: 10.1016/j.freeradbiomed.2021.04.034 *
FENG GUANGHANG, LIU JIE, LU ZITAO, LI YAOKUN, DENG MING, LIU GUANGBIN, SUN BAOLI, GUO YONGQING, ZOU XIAN, LIU DEWU: "miR-450-5p and miR-202-5p Synergistically Regulate Follicle Development in Black Goat", INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, MOLECULAR DIVERSITY PRESERVATION INTERNATIONAL (MDPI), BASEL, CH, vol. 24, no. 1, Basel, CH , pages 401, XP093257668, ISSN: 1422-0067, DOI: 10.3390/ijms24010401 *
HAOYU ZHENG, ZHOU YU, HAIRONG WANG, HONGXUE LIU & XIAOQIN CHEN: "MiR-125b-5p ameliorates hypoxia/reoxygenation-induc:ed endothelial cell dysfunction and attenuates reduced uterine perfusion pressure-induced hypertension in pregnant rats via targeting BMF", HYPERTENSION IN PREGNANCY, MARCEL DEKKER, NEW YORK, NY, US, vol. 41, no. 2, 16 February 2022 (2022-02-16), US , pages 79 - 88, XP009559998, ISSN: 1064-1955, DOI: 10.1080/10641955.2022.2036753 *
LV, X. ET AL.: "miR -451-3p alleviates myocardial ischemia/reperfusion injury by inhibiting MAP1LC3B-mediated autophagy", INFLAMMATION RESEARCH, vol. 70, 11 October 2021 (2021-10-11), pages 1089 - 1100, XP037611007, DOI: 10.1007/s00011-021-01508-4 *
WANG WEI, ZHENG HAO: "Myocardial Infarction: The Protective Role of MiRNAs in Myocardium Pathology", FRONTIERS IN CARDIOVASCULAR MEDICINE, FRONTIERS MEDIA S.A., vol. 8, XP093257670, ISSN: 2297-055X, DOI: 10.3389/fcvm.2021.631817 *

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