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WO2025163033A1 - Composés destinés à être utilisés dans le traitement de troubles ou de maladies par modulation de l'activité du facteur de transcription gata4 - Google Patents

Composés destinés à être utilisés dans le traitement de troubles ou de maladies par modulation de l'activité du facteur de transcription gata4

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Publication number
WO2025163033A1
WO2025163033A1 PCT/EP2025/052335 EP2025052335W WO2025163033A1 WO 2025163033 A1 WO2025163033 A1 WO 2025163033A1 EP 2025052335 W EP2025052335 W EP 2025052335W WO 2025163033 A1 WO2025163033 A1 WO 2025163033A1
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WIPO (PCT)
Prior art keywords
nucleic acid
acid molecule
isolated
incrna
cells
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Pending
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PCT/EP2025/052335
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English (en)
Inventor
Giulia SPANÒ
Monika Stoll
Leon Johannes De Windt
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Universiteit Maastricht
Academisch Ziekenhuis Maastricht
Original Assignee
Universiteit Maastricht
Academisch Ziekenhuis Maastricht
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Publication of WO2025163033A1 publication Critical patent/WO2025163033A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • 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
    • 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
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0657—Cardiomyocytes; Heart cells
    • 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
    • C12N2310/11—Antisense
    • C12N2310/113—Antisense targeting other non-coding nucleic acids, e.g. antagomirs
    • 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
    • C12N2310/30—Chemical structure
    • C12N2310/33—Chemical structure of the base
    • C12N2310/333—Modified A
    • 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
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/341—Gapmers, i.e. of the type ===---===
    • 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
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells
    • 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
    • C12N2510/00—Genetically modified cells

Definitions

  • This invention pertains in general to the field of therapeutics and prophylaxis of diseases, in particular cardiopathies. It also relates to method of identifying new compounds for the diseases.
  • the proposed compounds target the interaction of the transcription factor GATA4 with downstream proteins, which ultimately allow the transcription of genes of atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), a-myosin heavy chain (a- MHC) and p-MHC, relevant for cardiomyocyte functioning.
  • ADP atrial natriuretic peptide
  • BNP B-type natriuretic peptide
  • a- MHC a-myosin heavy chain
  • p-MHC relevant for cardiomyocyte functioning.
  • GATA-4 Transcription factor GATA-4 (herewith abbreviated GATA4) is a protein that in humans is encoded by the GATA4 gene.
  • the GATA4 gene encodes for the zinc finger GATA4 transcription factor that regulates cardiogenic signaling pathways associated with embryonic cardiac development, promotes myocardial specification and promotes cardiomyocyte proliferation in neonatal hearts. In adulthood, GATA4 plays an important role in the development of cardiac hypertrophy. Interestingly, when reexpressed in sites of myocardial injury of the heart, GATA4 can stimulate cardiomyocyte proliferation and regeneration.
  • GATA4 is involved in the transcriptional regulation of genes within the respiratory epithelium of the lung, the regulation of epithelial cell differentiation in gut development, in the regulation of liver-specific gene expression and is an important regulator of gene expression within the gonads (testis and ovary).
  • This transcription factor is, thus, an interesting target to elucidate the mechanisms of cell differentiation and regeneration in several tissues.
  • the present invention is directed to the surprising finding that GATA4 is regulated by a long intergenic non-coding RNA (IncRNA) molecule.
  • IncRNA non-coding RNA
  • the inventors went to the finding and isolation of an as of yet undescribed IncRNA annotated as C8orf49 or ENST00000625198 in H. sapiens genome assembly GRCh38/hg38.
  • This transcription factor regulator is only present in primates (according to in silico investigation of the corresponding genomic region across mammalian species), which means that it could not have been derived from the assays involving GATA4 performed with non-primates.
  • GREEN human Gata4 Regulator Enhancer
  • a first aspect of the invention relates to an isolated ribonucleic acid molecule, which is a long intergenic non-coding RNA (IncRNA) molecule, comprising or consisting in a nucleic acid sequence at least 80 % identical to SEQ I D NO: 1 .
  • IncRNA intergenic non-coding RNA
  • the isolated ribonucleic acid molecule of the invention is herewith termed also long non-coding RNA (IncRNA) molecule.
  • IncRNA long non-coding RNA
  • This newly identified and isolated IncRNA molecule of the invention can be seen as a switch off-switch on tool for GATA4 expression. Hence, any compound with the ability to quench (in the sense of put out, suppress, kidnap or scavenge) it will be useful to tune GATA4 expression in a given cell scenario or condition. Due to the nucleic acid nature of the IncRNA molecule of the invention, one of the possible compounds is another nucleic acid that can be paired (e.g., base paired) with said IncRNA molecule.
  • the sequence of the IncRNA molecule of the invention, and any interference nucleic acid molecule (i.e., any nucleic acid-binding nucleic acid molecule) with a sequence that comprises or transcribes into a sequence that quenches the isolated IncRNA molecule, are embodied in the inventive concept defined by the new identified function of the isolated ribonucleic acid molecule (IncRNA molecule).
  • This interference nucleic acid molecule or nucleic acid-binding nucleic acid molecule is complementary to the IncRNA molecule at least partially.
  • a second aspect of the invention is an isolated interference nucleic acid molecule or nucleic acid-binding nucleic acid molecule, which comprises or transcribes into a sequence that quenches the isolated ribonucleic acid molecule as defined in the first aspect.
  • any mode to provide a cell with the IncRNA molecule of the invention will result in the promotion of GATA4 expression further to any endogenous expression.
  • IncRNA molecule can be done by direct administration of this nucleic acid molecule. Alternatively, it can be provided in a way that ultimately results in its expression. The same applies for the interference nucleic acid molecule/ nucleic acid-binding nucleic acid molecule of the invention.
  • nucleic acid vector comprising: (a) the sequence of the isolated ribonucleic acid molecule as defined above, or a desoxyribonucleic nucleic acid sequence that is transcribed to said ribonucleic acid; and/or (b) the interference nucleic acid molecule or nucleic acid-binding nucleic acid molecule as defined above.
  • the invention provides a host isolated mammalian cell, preferably a human cell, comprising the isolated IncRNA, and/or the interference nucleic acid molecule or nucleic acid-binding nucleic acid molecule, and/or the nucleic acid vector, all as defined above in the previous aspects.
  • the host cell of the invention is, as such, a cell that will express GATA4 promoted by the presence in it of the IncRNA (i.e. , the IncRNA molecule comprising or consisting in a nucleic acid sequence at least 80 % identical to SEQ ID NO: 1), in case it comprises the IncRNA or if it expresses it from the vector. This will assure that those cell processes that are mediated by GATA4 expression do take place.
  • the IncRNA i.e. , the IncRNA molecule comprising or consisting in a nucleic acid sequence at least 80 % identical to SEQ ID NO: 1
  • the interference nucleic acid molecule herewith also referred as a nucleic acid-binding nucleic acid molecule
  • the IncRNA endogenously transcribed will be kidnapped and the final cell phenotype will be that of a cell in which the expression of GATA4 is hindered.
  • the cells are not only carriers of the compounds of interests. They are also entities that can be used in cell therapy. [022] In order to facilitate any therapeutic effect associated to the IncRNA of the invention, or to any interference nucleic acid sequence/nucleic acid-binding nucleic acid molecule with the capability to block it, the invention also provides for pharmaceutical compositions.
  • composition comprising, together with one or more pharmaceutically acceptable excipients and/or carriers, a therapeutically effective amount of the isolated ribonucleic acid molecule as defined above, and/or a therapeutically effective amount of the isolated interference nucleic acid molecule or nucleic acid-binding nucleic acid molecule, and/or a therapeutically effective amount of the nucleic acid vector as defined above, and/or a therapeutically effective amount of the isolated host cell, all as defined above.
  • composition also encompasses a veterinary composition.
  • an isolated ribonucleic acid molecule and/or an isolated interference nucleic acid molecule or nucleic acid-binding nucleic acid molecule, and/or a nucleic acid vector, and/or an isolated host cell, and/or a pharmaceutical composition, all of them as defined in any of the previous aspects, for use in the prevention and/or treatment of a disease selected from one or more of a cardiopathy, a respiratory epithelium -related disease, a gut development disease, a liver disease, and a gonadal development disease.
  • a disease selected from one or more of a cardiopathy, a respiratory epithelium -related disease, a gut development disease, a liver disease, and a gonadal development disease.
  • This aspect can also be formulated as the use of any of an isolated ribonucleic acid molecule, and/or an isolated interference nucleic acid molecule or nucleic acidbinding nucleic acid molecule, and/or a nucleic acid vector, and/or an isolated host cell, and/or a pharmaceutical composition, all of them as defined in any of the previous aspects, for the preparation of a medicament for the prevention and/or treatment of a disease selected from one or more of a cardiopathy, a respiratory epithelium -related disease, a gut development disease, a liver disease, and a gonadal development disease.
  • a disease selected from one or more of a cardiopathy, a respiratory epithelium -related disease, a gut development disease, a liver disease, and a gonadal development disease.
  • the invention also relates to a method for the prevention and/or treatment of a disease selected from one or more of a cardiopathy, a respiratory epithelium -related disease, a gut development disease, a liver disease, and a gonadal development disease, wherein the treatment comprises administering to a subject in need thereof, a therapeutically effective amount of one or more of an isolated ribonucleic acid molecule, and/or an isolated interference nucleic acid molecule or nucleic acid-binding nucleic acid molecule, and/or a nucleic acid vector, and/or an isolated host cell, and/or a pharmaceutical composition, all of them as defined in any of the previous aspects.
  • a disease selected from one or more of a cardiopathy, a respiratory epithelium -related disease, a gut development disease, a liver disease, and a gonadal development disease
  • the treatment comprises administering to a subject in need thereof, a therapeutically effective amount of one or more of an isolated ribonucleic acid molecule, and/
  • GATA4 is a transcription factor that has been identified to be involved in several processes that ultimately alter the phenotype of the cells leading to cell hypertrophy. Thus, in some circumstances or pathological states it may be convenient to hinder, reduce or inhibit its action. But also in other pathological states the convenience of its expression is desirable due to its capacity to promote cell proliferation and specification. Therefore, the invention relates to the prevention and/or treatment of all these pathological states, originating from divergent causes, and in which the modulation of the activity of transcription factor GATA4 will suppose a benefit. “Modulation of the activity” is an expression that in this description refers to either the ability to modulate the transcription and/or translation levels of GATA4, as well as its function as transcription factor.
  • the invention relates, in another aspect, to the use of a IncRNA molecule comprising or consisting in a nucleic acid sequence at least 80 % identical to SEQ ID NO: 1 , preferably in an isolated sample comprising mammal cells, as modulator of the activity of the transcription factor GATA4.
  • the invention provides an in vitro or ex vivo method to induce cell differentiation, and/or to obtain organoids, the method comprising: a) providing a source of undifferentiated cells; b) inducing a first differentiation stage, in which the cells acquire a first phenotype or degree of specialization; c) providing to the differentiated cells of step (b) an isolated ribonucleic acid molecule comprising or consisting in a nucleic acid sequence at least 80 % identical to SEQ ID NO: 1 (i.e.
  • IncRNA of the invention culturing the cells in a culture medium, and under conditions suitable to obtain cells in a second differentiation stage, in which the cells have a higher phenotype or degree of specialization than the first phenotype in (b).
  • This sequence of steps allows to conduct an undifferentiated cell of any origin to a desired differentiation stage, in which said differentiation is expressly promoted with the enhancement of the expression and action of GATA4.
  • the invention relates, moreover, to any kind of composition, including the previously disclosed pharmaceutical composition, comprising an effective amount of the isolated ribonucleic acid molecule as defined above (i.e., comprising or consisting in a sequence 80 % identical to SEQ ID NO: 1), and/or an effective amount of the isolated interference nucleic acid molecule or nucleic acid-binding nucleic acid molecule, and/or an effective amount of the nucleic acid vector as defined above, and/or an effective amount of the isolated host cell, all as defined above.
  • These compositions comprise, in some examples and embodiments, carriers, buffers and solvents which are adequate for the nucleic acid compounds, or cells comprising it.
  • the effective amounts are those required for the intended use of the compositions.
  • compositions are dried or lyophilized, and they can be resuspended in adequate solvents.
  • These compositions may be used in any experimental setup, as well as may be for use as medicaments, preferably in the prevention and/or treatment of a disease selected from one or more of a cardiopathy, a respiratory epithelium -related disease, a gut development disease, a liver disease, and a gonadal development disease.
  • a cell culture medium preferably a cardiomyocyte cell culture medium, comprising: a) a ribonucleic acid molecule, which is a long non-coding RNA (IncRNA) molecule, said IncRNA comprising or consisting of a nucleic acid sequence with a percentage of identity of at least 80 % with SEQ ID NO: 1 , the sequence preferably comprising one or more N6-methyladenosine residue(s); or, alternatively, b) an interference nucleic acid molecule or nucleic acid-binding nucleic acid molecule, which comprises or transcribes into a sequence that quenches the IncRNA nucleic acid molecule in (a).
  • a ribonucleic acid molecule which is a long non-coding RNA (IncRNA) molecule, said IncRNA comprising or consisting of a nucleic acid sequence with a percentage of identity of at least 80 % with SEQ ID NO: 1 , the sequence preferably comprising one or more N6-methyladeno
  • FIG. 1 Human METTL16 methyltransferase is reduced in more advanced stages of human cardiomyocyte specification, (a) Schematic overview of the protocol used to differentiate hiPSCs into hiPSC-CMs. The arrows indicate the day of sample collection, (b) qRT-PCR analysis of the m6Amethyltransferase METTL16 across the four collected hiPSC-differentiated cellular stages (dO, d3, d10, d25). (c) Representative western blot analysis and (d) relative quantification of the METTL16 protein detected across the four collected hiPSC-differentiated cellular stages (dO, d3, d10, d25).
  • the data represent means ⁇ SEM from three independent experiments. P values were calculated using one-way ANOVA followed by Tukey’s multiple comparison test.
  • FIG. 2 m6A-methylated IncRNA transcripts are enriched during cardiomyocyte specification, (a) qRT-PCR with expression levels of GREEN IncRNA and its c/s-located genes GATA4 and NEIL2 across the four collected hiPSC- differentiated cellular stages (dO, d3, d10, d25). (b) Correlation matrix showing the Pearson correlation coefficient of GREEN IncRNA, GATA4 and NEIL2 transcripts expressed in the showed tissues. The data represent means ⁇ SEM from three independent experiments. P values were calculated using one-way ANOVA followed by Tukey’s multiple comparison test (*P ⁇ 0.05; **P ⁇ 0.01 ; ***P ⁇ 0.001 ; ****p ⁇ 0.0001 ; ns P > 0.05).
  • FIG. 3 IncRNA GREEN is required for GATA4 expression
  • a portion of this disclosure contains material that is subject to copyright protection (such as, but not limited to, diagrams, device photographs, or any other aspects of this submission for which copyright protection is or may be available in any jurisdiction.).
  • copyright protection such as, but not limited to, diagrams, device photographs, or any other aspects of this submission for which copyright protection is or may be available in any jurisdiction.
  • the copyright owner has no objection to the facsimile reproduction by anyone of the patent document or patent disclosure, as it appears in the Patent Office patent file or records, but otherwise reserves all copyright rights whatsoever.
  • At least a particular value means that particular value or more.
  • “at least 2" is understood to be the same as “2 or more” i.e. , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, ... , etc.
  • “at least 80 %” is understood to be the same as "80 or more” i.e., 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, ... , etc.
  • sequence identity refers to the degree of relatedness between two or more nucleic acid sequences (polynucleotide sequences), as determined by comparing the sequences.
  • sequences polynucleotide sequences
  • sequence identity may be accomplished using a mathematical algorithm; those skilled in the art will be aware of computer programs available to align two sequences and determine the percent identity between them. The skilled person will appreciate that different algorithms may yield slightly different results.
  • the “percent identity” between a query nucleic acid sequence and a subject nucleic acid sequence is the “identities” value, expressed as a percentage, that is calculated by, for example, the BLASTN algorithm when a subject nucleic acid sequence has 100% query coverage with a query nucleic acid sequence after a pair- wise BLASTN alignment is performed.
  • pairwise BLASTN alignments between a query nucleic acid sequence and a subject nucleic acid sequence are performed by using the default settings of the BLASTN algorithm available on the National Center for Biotechnology Institute's website with the filter for low complexity regions turned off.
  • a query nucleic acid sequence may be described by a nucleic acid sequence identified in one or more claims herein.
  • the “percent identity” between a query amino acid sequence and a subject amino acid sequence which is the “identities” value, expressed as a percentage, that is calculated by the BLASTP algorithm when a subject amino acid sequence has 100% query coverage with a query amino acid sequence after a pair-wise BLASTP alignment is performed, in this case by using the default settings of the BLASTN algorithm available on the National Center for Biotechnology Institute's website with the same filters indicated before for the nucleic acid sequences.
  • a gap i.e., a position in an alignment where a residue is present in one sequence but not in the other, is regarded as a position with non-identical residues and is counted as a compared position.
  • a polynucleotide having a nucleic acid sequence (subject) having at least, for example, 95% identity to a reference nucleic acid sequence of SEQ ID NO:1 (query) is intended that the nucleic acid sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five nucleotide alterations per each 100 nucleotides of the reference nucleic acid of SEQ ID NO: 1.
  • nucleotide having an nucleic acid sequence of at least 95% identical to a reference nucleic acid sequence up to 5% of the nucleotide residues in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotide residues in the reference sequence may be inserted into the reference sequence.
  • alterations of the reference sequence may occur at the 5’or 3’ positions of the reference nucleic acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
  • sequence identity between two nucleic acid sequences is preferably determined using algorithms based on global alignment, such as the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), preferably implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277); or the BLAST Global Alignment tool (Altschul et al., “Basic local alignment search tool”, 1990, J. Mol. Biol, v. 215, pages 403-410), using default settings. Local alignment also can be used when the sequences being compared are substantially the same length.
  • Needleman-Wunsch algorithm Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453
  • EMBOSS European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277
  • BLAST Global Alignment tool Altschul
  • nucleic acid such as a ribonucleic acid or an interference nucleic acid
  • nucleic acid refers to nucleic acids being present in a non-naturally occurring environment, e. g. are separated from their naturally occurring environment.
  • an isolated polypeptide according to the invention relates to a protein which is no longer in its natural environment, for example, it is being processed or handled in in vitro assays, in a recombinant host cell or in a compositions (i.e. , pharmaceutical composition).
  • compositions i.e. , pharmaceutical composition
  • a “modulator” or “agent that modulates” refers to a compound that alters the activity of a target activity, for example the activity of a target protein.
  • the modulator may be an inhibitor (antagonist) or an enhancer (agonist).
  • the modulator may alter the activity by modulation of, for example, the enzymatic activity of a target protein, by modulation the interaction of the target protein with a further factor, such as a further protein, by modulation of the activity of a regulator of the target protein, and/or by modulating expression of the target protein.
  • agonist refers to a compound or agent having the ability to initiate or enhance a biological function of a target protein or polypeptide, such as increasing the activity or expression of the target protein or polypeptide. Accordingly, "agonist” is defined in the context of the biological role of the target protein or polypeptide. While some agonists herein may specifically interact with (e.g., bind to) the target, compounds and/or agents that initiate or enhance a biological activity of the target protein or polypeptide by interacting with other members of the signal transduction pathway of which the target polypeptide is a member are in embodiments specifically included within this definition.
  • antagonists are used interchangeably, and they refer to a compound or agent having the ability to reduce or inhibit a biological function of a target protein or polypeptide, such as by reducing or inhibiting the activity or expression of the target protein or polypeptide. Accordingly, the terms “antagonist” and “inhibitor” are defined in the context of the biological role of the target protein or polypeptide. While some antagonists herein may specifically interact with (e.g., bind to) the target, compounds that inhibit a biological activity of the target protein or polypeptide by interacting with other members of the signal transduction pathway of which the target protein or polypeptide are in embodiment also included within this definition.
  • test compound refers to a molecule that may be screened for, or be identified as, modulating activity of a IncRNA molecule, such as the activity of a IncRNA molecule comprising or consisting in a nucleotide sequence at least 80 % identical to SEQ ID NO: 1 , preferably 100 % identical to SEQ ID NO: 1.
  • a molecule that modulates the interaction of a IncRNA molecule of the invention with other compounds such as a molecule that modulates the interaction with a methyltransferase, preferably an N6-methyladenosine methyltransferase, more in particular and optionally selected from m6A- methyltransferase METTL16 and/or m 6 A-methyltransferase METTL3.
  • composition refers to a composition formulated in pharmaceutically acceptable or physiologically acceptable compositions for administration to a cell or subject.
  • the compositions of the invention may be administered in combination with other agents as well, provided that the additional agents do not adversely affect the ability of the composition to deliver the intended therapy.
  • the pharmaceutical composition often comprises, in addition to a pharmaceutical active agent, one or more pharmaceutical acceptable carriers (or excipients).
  • compositions be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes; (2) parenteral administration, for example, by subcutaneous, intramuscular or intravenous injection as, for example, a sterile solution or suspension; (3) topical application, for example, as a cream, ointment or spray applied to the skin; (4) intravaginally or intrarectally, for example, as a pessary, cream or foam; or (5) aerosol, for example, as an aqueous aerosol, liposomal preparation or solid particles containing the compound.
  • oral administration for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes
  • parenteral administration for example, by subcutaneous, intramuscular or intravenous injection as, for example,
  • pharmaceutically acceptable refers to compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical (and veterinary) judgment, suitable for use in contact with the tissues of a subject (e.g. human or any other animal) without significant toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • a subject e.g. human or any other animal
  • Each carrier, excipient, etc. must also be “acceptable” in the sense of being compatible with the other ingredients of the pharmaceutical composition. It must also be suitable for use in contact with the tissue or organ of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity or other problems or complications commensurate with a reasonable benefit/risk ratio.
  • Suitable carriers, excipients, etc. can be found in standard pharmaceutical texts, and include, as a way of example preservatives, agglutinants, humectants, emollients, and antioxidants. The skilled person in the art will know the method to determine the said therapeutically effective amount and well as the possible pharmaceutically acceptable carriers or excipients.
  • nucleic acid or “polynucleotide” (used interchangeably) refers to any polymers or oligomers of (contiguous) nucleotides.
  • the nucleic acid may be DNA or RNA, or a mixture thereof, and may exist permanently or transitionally in single-stranded or double-stranded form, including homoduplex, heteroduplex, and hybrid states.
  • the present invention also contemplates any deoxyribonucleotide, ribonucleotide or peptide nucleic acid component, and any chemical variants thereof, such as methylated, hydroxymethylated or glycosylated forms of these bases, and the like.
  • the polymers or oligomers may be heterogeneous or homogenous in composition, and may be isolated from naturally occurring sources or may be artificially or synthetically produced.
  • long non-coding RNA or “long intergenic non-coding RNA” (abbrv. IncRNA) refers to a ribonucleic acid molecule transcript of about more than 200 nucleotides that are not translated into protein.
  • the arbitrary limit around 200 ribonucleotides distinguishes long ncRNAs from small non-coding RNAs, such as microRNAs (miRNAs), small interfering RNAs (siRNAs), Piwi-interacting RNAs (piRNAs), small nucleolar RNAs (snoRNAs), and other short RNAs, which are also considered non-coding RNAs.
  • these ribonucleic acid molecules may comprise modified ribonucleotides, such as methylated, hydroxymethylated or glycosylated forms of these bases, and the like.
  • modified ribonucleotides such as methylated, hydroxymethylated or glycosylated forms of these bases, and the like.
  • An example of modification is the methylation of the nitrogen base of the ribonucleotide, for example N-methylation of the nitrogen atoms of the nitrogen base.
  • sequence refers to the order of nucleotides of, or within a nucleic acid/polynucleotide. In other words, any order of nucleotides may be referred to as a sequence (nucleotide sequence).
  • a "subject" is to indicate an organism from which (cell) material may be obtained.
  • the subject may be any subject in accordance with the present invention, including, but not limited to humans, with no restriction by gender, sex or age, and/or other primates or mammals.
  • Preferably the subject is a human patient.
  • a subject may have been diagnosed with a disease, for example a cardiovascular disease.
  • nucleic acid construct As used herein, the terms “construct”, “nucleic acid construct”, “nucleic acid vector”, “vector”, and “expression vector” may be used interchangeably and are defined as man-made nucleic acid molecules resulting from the use of recombinant DNA technology. These constructs and vectors therefore do not include naturally occurring nucleic acid molecules although a nucleic acid construct may comprise (parts of) naturally occurring nucleic acid molecules.
  • interference nucleic acid molecule refers to any RNA or DNA or combination thereof that by nitrogen base complementarity is capable to quench or kidnap another nucleic acid molecule (e.g., the IncRNA of the invention), and this way to prevent the later to perform its function.
  • interference nucleic acid molecules include interference RNA (iRNA) or the mostly synthetic antisense oligonucleotides (ASO). Most of them promote, at certain extent, that the quenched nucleic acid molecule is degraded by an endonuclease (e.g., RNase H).
  • iRNA generically spoken, operates sequence specifically and post-transcriptionally by activating ribonucleases which, along with other enzymes and complexes, coordinately degrade the RNA after the original RNA target has been cut into smaller pieces.
  • iRNA molecules examples include small-hairpin RNA (shRNA), microRNA (miRNAs), and siRNAs.
  • shRNA small-hairpin RNA
  • miRNAs microRNA
  • siRNAs siRNAs.
  • ASO bind to their target nucleic acid via Watson-Crick base pairing, and inhibit or alter gene expression via steric hindrance, splicing alterations, initiation of target degradation, or other events.
  • a nucleic acid sequence is understood as “complementary” to another nucleic acid sequence, in this case to a IncRNA molecule, when due to the nitrogen base complementarity these two sequences are paired at least under physiological conditions, i.e. at a temperature about 34-38 ° C, and at the physiological pH of a particular tissue environment.
  • the sequences are also considered complementary if under astringent conditions they are maintained hybridized to each other. Although it depends on the lenght of the sequences that hybridize, generally astringency conditions can be selected to be 5 ° C lower than the value of the melting temperature (Tm) corresponding to the specific sequence and its complement under certain conditions of pH and ionic strength.
  • severe astringency conditions may use hybridization or washes of 1 to 4 ° C lower than Tm; moderately stringent conditions can utilize hybridization and washings from 11 to 20 0 C lower than the Tm.
  • the salt concentration is less than 1.% M of Na ions, and typically between 0.01 and 1.0 M of concentration of Na ions (or other salts) at a pH of 7.0 at 8.3 and a temperature of at least 60 ° C for sequences with a number of nucleotides from 500 nt and on.
  • astringent conditions applied to “complementarity” between nucleic acid sequences may also be applied to determine if a nucleic acid sequence, for example a nucleic acid-binding nucleic acid molecule or interference nucleic acid molecule, has the property to hybridize with a target (e.g., with the IncRNA) and quench it to interfere in its function.
  • a target e.g., with the IncRNA
  • a cell differentiation stage refers to the phenotype of a cell, preferably of a mammalian cell, given in a particular moment in relation to an undifferentiated cell from which it derives from. Differentiation makes a cell specialized. As the cells differentiate, they develop different characteristics and structures within the cell, which then can carry out a specific function. This is what is meant when a cell is specialized. When a cell is specialized is also call that is a mature cell, meanwhile in the path from undifferentiated to a differentiated stage, several intermediate maturity (or immaturity) stages are observed.
  • the cells derive from stem cells that first acquire a cell differentiation stage called mesoderm, and then they progressively evolve to more differentiated states that give rise to specialized cells with differing phenotypes, such as endothelial cell or a beating cardiomyocyte, for example.
  • any compound, method, use, or composition described herein can be implemented with respect to any other compound, method, use or composition described herein.
  • Embodiments discussed in the context of compounds, methods, use and/or compositions of the invention may be employed with respect to any other compound, method, use or composition described herein.
  • an embodiment pertaining to one compound, method, use or composition may be applied to other compounds, methods, uses and compositions of the invention as well.
  • references in the description to methods of treatment refer to the compounds, pharmaceutical compositions, and medicaments of the present invention for use in a method for treatment of the human (or animal) body by therapy.
  • the present invention is directed to the surprising finding that the transcription factor GATA4 is regulated, in primate mammals, by a long non-coding RNA (IncRNA) molecule, which positively cisregulates GATA4 transcription and/or expression (i.e. , translation).
  • IncRNA non-coding RNA
  • the invention referred in a first aspect relates to an isolated ribonucleic acid molecule, which is a long non-coding RNA (IncRNA) molecule, comprising or consisting in an nucleic acid sequence at least 80 % identical to SEQ ID NO: 1 , preferably 100 % identical to SEQ ID NO: 1.
  • the isolated IncRNA molecule comprises one or more methylated residues, preferably one or more N6- methyladenosine residue(s), more preferably one N6-methyladenosine residue.
  • the one or more residues are in a sequence defined by the consensus sequence [TTCAGATGA]
  • the isolated IncRNA molecule comprises one methylated residue in a sequence defined by the consensus sequence [TTCAGATGA], preferably one N6-methyladenosine residue at the first, or second, or third adenosine in this sequence defined by the consensus sequence [TTCAGATGA]
  • the isolated IncRNA molecule comprises two methylated residues in a sequence defined by the consensus sequence [TTCAGATGA], preferably two N6-methyladenosine residue at the first and second, or at the first and third, or at the second and third adenosine residues in this sequence defined by the consensus sequence [TTCAGATGA]
  • the isolated IncRNA molecule comprises three methylated residues in a sequence defined by the consensus sequence [TTCAGATGA], preferably three N6- methyladenosine residues.
  • the isolated IncRNA comprises or consists in a nucleic acid sequence at least 90 % identical, preferably at least 91 % identical, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % identical to SEQ ID NO: 1.
  • the isolated IncRNA molecule comprises or consists in an nucleic acid sequence 100 % identical to SEQ ID NO: 1.
  • IncRNA of the invention When along this description is referred to this isolated IncRNA of the invention, it is to be understood as the molecule comprising a sequence or consisting in a sequence from at least 80 % up to 100 % identical to SEQ ID NO: 1.
  • Interference nucleic acid molecules also referred, synonymously, as nucleic acid- bindinq nucleic acid molecules
  • the invention also relates to any interference nucleic acid molecule which comprises or transcribes into a sequence that quenches the isolated ribonucleic acid molecule as defined in the first aspect.
  • This interference nucleic acid molecule can also be referred here as a nucleic acid-binding nucleic acid molecule, both terms used interchangeably.
  • the interference nucleic acid molecule, or nucleic acidbinding nucleic acid molecule is selected from one or more of an antisense nucleic acid oligonucleotide (ASO), an small interfering RNA (siRNA), a small hairpin RNA (shRNA), and a microRNA (miRNA).
  • Aptamers or chemical antibodies are other type of single-stranded DNA or RNA oligonucleotides that bind proteins and small molecules with high affinity and specificity by recognizing tertiary or quaternary structures as antibodies.
  • the interference nucleic acid molecule, or nucleic acidbinding nucleic acid molecule is selected from one or more of an antisense nucleic acid oligonucleotide (ASO), an small interfering RNA (siRNA), a small hairpin RNA (shRNA), and a microRNA (miRNA), preferably an antisense nucleic acid oligonucleotide (ASO).
  • ASO antisense nucleic acid oligonucleotide
  • siRNA small interfering RNA
  • shRNA small hairpin RNA
  • miRNA microRNA
  • the interference nucleic acid molecule, or nucleic acid-binding nucleic acid molecule is an antisense nucleic acid oligonucleotide (ASO).
  • ASO antisense nucleic acid oligonucleotide
  • said ASO comprises from 15 to 20 nucleotides, preferably is 16, 17 or 18 nucleotides long, and it binds by complementary base-pairing to fragments all along the sequence of SEQ I D NO: 1 .
  • the interference nucleic acid molecule or nucleic acid-binding nucleic acid molecule is an antisense nucleic acid oligonucleotide (ASO), preferably comprising or consisting in a sequence at least 80 % identical to SEQ ID NO: 2 (also referred in this description as Gapmer GREEN).
  • ASO antisense nucleic acid oligonucleotide
  • the ASO is a nucleic acid molecule that comprises RNA and DNA fragments, and which sequence is at least 80 % identical to SEQ ID NO: 2 (Gapmer GREEN). More preferably is at least 82 %, at least 85 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % identical to SEQ ID NO: 2 (Gapmer GREEN). In a more preferred embodiment, the ASO is 100 % identical to SEQ ID NO: 2 (Gapmer GREEN).
  • the sequence of the isolated IncRNA of the invention is, in a preferred embodiment, comprised in a nucleic acid vector.
  • nucleic acid vector comprising: (a) the sequence of the isolated ribonucleic acid molecule as defined above, or a desoxyribonucleic nucleic acid sequence that is transcribed to said ribonucleic acid; and/or (b) the interference nucleic acid molecule or nucleic acid-binding nucleic acid molecule as defined above.
  • the vectors are, indeed, polynucleotide constructs that comprise, within their sequence, one or more of the enumerated sequences related with the IncRNA of the invention, as well as other regulatory sequences or stabilizing sequences that aid to maintain the sequence of interest inside cells to modulate this way the expression of GATA4.
  • Particular regulatory sequences or stabilizing sequences are selected from one or more of a promoter, an enhancer and/or a silencer nucleic acid sequence, a 5’ untranslated region (5’-UTR) sequence and/or a 3’ untranslated region (3’-UTR) nucleic acid sequence, 3’ and/or 5’ inverted terminal repeats, an intron(s) sequence, a polyadenylation signal nucleic acid sequence, a nucleic acid sequence that encodes for a gene operably linked to the promoter and that confers nucleotide sequence or fragment of the promoter.
  • the vector comprises the sequence of the isolated ribonucleic acid molecule as defined above, or a desoxyribonucleic nucleic acid sequence that is transcribed to said ribonucleic acid as defined in the first aspect; and a sequence that codifies for the GATA4 transcription factor, wherein both sequences are operatively linked to a promoter and, preferably in a consecutive order.
  • the vectors are expression vectors, preferably viral vectors, in particular selected from one or more of a retrovirus nucleic acid, an adenovirus nucleic acid, an adeno-associated virus (AAV) nucleic acid and a lentivirus nucleic acid.
  • the vector is an adeno-associated virus nucleic acid, in particular from a serotype selected from the group consisting of AAV2, AAV6, AAV9 or a combination with AAV8.
  • the serotype of the adeno-associated virus is AAV6.
  • the vectors, or directly the isolated IncRNA of the invention can be integrated into host cells as defined above.
  • all cells that express GATA4 are useful host cells.
  • Preferred examples of cells are selected from cardiomyocytes, pneumocytes, enterocytes, hepatocytes and cells that constitute the gonads. More preferred host cells are cardiomyocytes.
  • the cells can be at any stage of specialization observed in the tissues where they reside.
  • progenitor cells of a cell type are used in preferred embodiments. These progenitor cells have a phenotype of maturity/specification lower than a full specialized cell and are, for example, selected from immature cells derived from induced pluripotent stem cells or from cells differentiated from embryonic stem cells.
  • embryonic stem cells in particular human embryonic stem cells, are referred to in this description, they do not result from the destruction of any human embryo.
  • preferred examples of host cells are selected from cells at any stage of maturation/specialization, from the mesoderm stage to full differentiated state.
  • cardiac cells they can be cells with a cardiac mesoderm phenotype, with a cardiac progenitor phenotype, and/or with a full differentiated and specialized phenotype, such as a beating cardiomyocyte.
  • Cardiac mesoderm phenotype is usually characterized by the expression of one or more of EOMES and BRACHYURY.
  • T-box transcription factor T also known as Brachyury protein, is encoded for in humans by the TBXT gene, and is it has a conserved role in defining the midline of a bilaterian organism and thus the establishment of the anterior-posterior axis; it also defines the mesoderm during gastrulation and thus it is used as a mesoendoderm marker.
  • Eomesodermin also known as T-box brain protein 2 (Tbr2) is a protein that in humans is encoded by the EOMES gene.
  • the host cells are cardiac cells with a cardiac mesoderm phenotype and express one or more of the EOMES and BRACHYURY proteins, preferably both.
  • Cardiac progenitor phenotype and full differentiated beating cardiomyocytes are usually characterized by the expression of one or more of TNNT2 and MYH6 proteins.
  • Cardiac muscle troponin T (cTnT) is a protein that in humans is encoded by the TNNT2 gene.
  • Cardiac TnT is the tropomyosin-binding subunit of the troponin complex, which is located on the thin filament of striated muscles and regulates muscle contraction in response to alterations in intracellular calcium ion concentration.
  • Myosin heavy chain, a isoform (MHC-a) is a protein that in humans is encoded by the MYH6 gene.
  • MHC-a isoform is abundantly expressed in both cardiac atria and cardiac ventricles during embryonic development. Following birth, cardiac ventricles predominantly express another isoform, the MHC-p isoform, and cardiac atria predominantly express the MHC-a isoform.
  • the host cells are cardiac cells with a cardiac progenitor phenotype, and/or with a full differentiated and specialized phenotype, and they express one or more of TNNT2 and MYH6 proteins, preferably both.
  • the host cells are beating cardiomyocytes that express one or more of TNNT2 and MYH6 proteins, preferably both.
  • the host cells of the invention or directly any vector or nucleic acid sequence comprising the IncRNA molecule of the invention, or any interference nucleic acid molecule or nucleic acid-binding nucleic acid molecule as defined above, are useful active ingredients for compositions, such as for pharmaceutical compositions.
  • the invention also relates to pharmaceutical compositions comprising, together with one or more pharmaceutically acceptable excipients and/or carriers, a therapeutically effective amount of the isolated ribonucleic acid molecule, and/or a therapeutically effective amount of the isolated interference nucleic acid molecule or nucleic acid-binding nucleic acid molecule, and/or a therapeutically effective amount of the nucleic acid vector as defined above, and/or a therapeutically effective amount of the isolated host cell, all as defined above in the previous aspects or embodiments.
  • liposomes comprising cationic lipids are used. These cationic lipids in the liposome complex with the negatively charged nucleic acid molecules to allow them to overcome the electrostatic repulsion of the cell membrane.
  • cationic lipids used in liposomes, LNP or NLC are selected from DOSPA (2,3-dioleoyloxy-N- [2(sperminecarboxamido)ethyl]-N,N- dimethyl-1-propaniminium trifluoroacetate) and DOPE (1 ,2-Dioleoyl-sn- glycerophosphoethanolamine).
  • DOSPA 2,3-dioleoyloxy-N- [2(sperminecarboxamido)ethyl]-N,N- dimethyl-1-propaniminium trifluoroacetate
  • DOPE 1,2-Dioleoyl-sn- glycerophosphoethanolamine
  • Lipofectamine is an example of these cationic lipid carriers. It consists of a 3:1 mixture of DOSPA and DOPE. Lipofectamine's cationic lipid molecules are formulated with a neutral co-lipid (helper lipid).
  • the DNA-containing liposomes (positively charged on their surface) can fuse with the negatively charged plasma membrane of living cells, due to the neutral co-lipid mediating fusion of the liposome with the cell membrane, allowing nucleic acid cargo molecules to cross into the cytoplasm for replication or expression.
  • the skilled person in the art will recognize the rationale behind this operational and will understand that other cationic and neutral lipid combinations are possible.
  • the IncRNA molecule of the invention or any nucleic acid molecule as herewith disclosed and related with the same can also be used as ingredients in cell culture media.
  • a cell culture medium preferably a cardiomyocyte cell culture medium, comprising: a) a ribonucleic acid molecule, which is a long non-coding RNA (IncRNA) molecule, said IncRNA comprising or consisting of a nucleic acid sequence with a percentage of identity of at least 80 % with SEQ ID NO: 1 , preferably 100 %, the sequence preferably comprising one or more N6-methyladenosine residue(s); or, alternatively, b) an interference nucleic acid molecule or nucleic acid-binding nucleic acid molecule, which comprises or transcribes into a sequence that quenches the IncRNA nucleic acid molecule in (a).
  • a ribonucleic acid molecule which is a long non-coding RNA (IncRNA) molecule, said IncRNA comprising or consisting of a nucleic acid sequence with a percentage of identity of at least 80 % with SEQ ID NO: 1 , preferably 100 %, the sequence preferably
  • IncRNA Due to the new discovered regulatory role of the isolated IncRNA on GATA4, it derives its use in therapy. Thus, it is herewith disclosed for use as a medicament any of an isolated ribonucleic acid molecule, and/or an isolated interference nucleic acid molecule or nucleic acid-binding nucleic acid molecule, and/or a nucleic acid vector, and/or an isolated host cell, and/or a pharmaceutical composition, all of them as defined in any of the previous aspects, all of them as defined in any of the previous aspects and preferred embodiments.
  • GATA4 is known to be involved in processes that lead to the proliferation and differentiation/specification of a cell, and therefore in processes favoring regeneration of damaged tissues.
  • a first therapeutic approach takes advantage of the new identified role of the IncRNA as enhancer of the expression of GATA4, and relates to said IncRNA molecule for use in the prevention and/or treatment of a disease which benefits from GATA4 expression, wherein said IncRNA molecule comprises or consists in a sequence at least 80 % identical to SEQ ID NO:1 , preferably 100 % identical to SEQ ID NO: 1.
  • any nucleic acid vector comprising or transcribing for this isolated IncRNA molecule, and/or any an isolated host cell, and/or a pharmaceutical composition that in some form comprise or provide the IncRNA molecule of the invention in a subject with need thereof.
  • GATA4 is also known to be involved in processes leading to pathology as a result of its role in the response to cell stress (e.g., cell hypertrophy).
  • the IncRNA molecule of the invention is, thus, itself a target to modulate the expression of the transcription factor. Therefore, in a second therapeutic approach, compounds that can hinder the action of the IncRNA are for use in the prevention and/or treatment of a disease mediated by the expression of the transcription factor GATA4.
  • the expression “disease mediated by the expression of the transcription factor GATA4”, encompasses those pathologies in which an aberrant or higher expression of the transcription factor in relation to an acknowledged health state is observed.
  • an isolated interference nucleic acid molecule or nucleic acidbinding nucleic acid molecule as previously disclosed, and which comprises or transcribes into a sequence that quenches the IncRNA molecule of the invention, is for use in the prevention and/or treatment of a disease mediated by the expression of the transcription factor GATA4. Also are so any nucleic acid vector, and/or an isolated host cell, and/or a pharmaceutical composition, all of them comprising or providing for this interference nucleic acid molecule.
  • the IncRNA molecule, and/or the isolated interference nucleic acid molecule or nucleic acid-binding nucleic acid molecule, and/or the nucleic acid vector, and/or the isolated host cell, and/or the pharmaceutical composition, all of them as defined in any of the previous aspects and embodiments are for use in the prevention and/or treatment of a disease selected from one or more of a cardiopathy, a respiratory epithelium -related disease, a gut development disease, a liver disease, and a gonadal development disease.
  • GATA4 and IncRNA GREEN SEQ ID NO: 1 expression in different mesodermal-derived tissues (e.g., heart and gonads) and endodermal-derived tissues (e.g., stomach, liver and pancreas).
  • GATA4 is involved in cell differentiation and specification, as well as in cell proliferation and regeneration. GATA4 has also been associated with hypertrophy of cells when overexpressed in these mesodermal- derived tissues, and endodermal-derived tissues. Due to the surprising finding that a IncRNA in primates comprising or consisting in SEQ ID NO: 1 regulates the expression of GATA4, any disease or condition resulting in a damaged mesoderm or endoderm tissue, can be therapeutically approached by increasing GATA4 expression through the administering of the said IncRNA. On the other side, in case of a disease or condition involving hypertrophy of the cells in these tissues linked to the expression of GATA4, the isolated IncRNA of the invention is a key target to be blocked and so, to block the pathological consequences associated to GATA4 expression.
  • the IncRNA molecule, and/or the isolated interference nucleic acid molecule or nucleic acid-binding nucleic acid molecule, and/or the nucleic acid vector, and/or the isolated host cell, and/or the pharmaceutical composition are for use in a cardiopathy selected from one or more of a hypertrophic cardiomyopathy, preferably ventricular hypertrophy and/or left ventricular remodeling; a cardiovascular disease, preferably selected from ischemic disease, hypertension, heart failure, and valvular disease; malignant arrhythmia; myocardial infarction; and a myocardial congenital heart disease.
  • the IncRNA molecule comprising or consisting in a nucleotide sequence at least 80 % identical to SEQ I D NO: 1 , preferably 100 % identical to SEQ ID NO: 1 , is for use in the treatment of a myocardial infarction.
  • This therapeutic application results from the regenerative role of GATA4 observed in sites of ischemic injury of the heart after a myocardial infarction, where GATA4 has been seen to stimulate cardiomyocyte proliferation and regeneration.
  • this embodiment illustrates the applicability of the isolated IncRNA molecule of the invention, or any nucleic acid molecule providing it, in regenerative medicine.
  • any isolated interference nucleic acid molecule i.e., nucleic acid-binding nucleic acid molecule
  • nucleic acid-binding nucleic acid molecule which comprises or transcribes into a sequence that quenches the IncRNA molecule as previously disclosed, is for use in the prevention and/or treatment of a hypertrophic cardiomyopathy, preferably ventricular hypertrophy and/or left ventricular remodeling.
  • the said interference or nucleic acid-binding nucleic acid molecule sequence will be able to quench said IncRNA, and this way to impair or block the expression and activity of GATA4, which has been seen overexpressed in relation to healthy conditions under stress cell conditions that ultimately are, in part, the cause of the hypertrophic cardiomyopathy.
  • the interference nucleic acid molecule i.e., the nucleic acid-binding nucleic acid molecule
  • the interference nucleic acid molecule is selected from an antisense nucleic acid oligonucleotide (ASO), an siRNA, a shRNA, a miRNA, and an aptamer; preferably it is an ASO.
  • Antisense oligonucleotides are single-stranded oligonucleotides (10-20 nts) that have been specially chemically modified, which can bind to RNA expressed by target genes through base complementary pairing and affect protein synthesis at the level of posttranscriptional processing or protein translation
  • a preferred ASO is a single-stranded, oligonucleotide that comprises a DNA sequence (DNA portion) flanked by two locked nucleic acid (LNA) sequences.
  • LNA locked nucleic acid
  • This kind of ASO is also known as LNA GapmeRs.
  • LNA GapmeRs LNA GapmeRs that bind the IncRNA of the invention.
  • LNA GapmeR tested in the examples is a possible one, but others can be designed which are oligonucleotides complementary to any fragment of SEQ ID NO: 1 (i.e. , or to any sequence from 80 % to 100 % identical to SEQ ID NO: 1).
  • the whole or part of the sequence of the GapmeR is complementary to the target RNA, in this description to the IncRNA molecule that comprises or consists in a nucleotide sequence at least 80 % identical to SEQ ID NO: 1 , preferably 100 % identical to SEQ ID NO: 1.
  • the locked nucleic acids sequences comprise ribonucleotides that comprise a methylene bridge bond linking the 2' oxygen to the 4' carbon of the ribonucleotide pentose ring.
  • the bridge bond fixes the pentose ring in the 3'-endo conformation.
  • the ASO is a nucleic acid molecule that comprises or consists in a nucleic acid sequence at least 80% identical to SEQ ID NO: 2 (Gapmer GREEN). More preferably is at least 82 %, at least 85 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % identical to SEQ ID NO: 2 (Gapmer GREEN). In a more preferred embodiment, the ASO is 100 % identical to SEQ ID NO: 2 (Gapmer GREEN).
  • the IncRNA molecule, and/or the isolated interference nucleic acid molecule or nucleic acid-binding nucleic acid molecule are for use in the treatment of a mesodermal or endodermal tissue disease, selected from one or more of a cardiopathy, a respiratory epithelium -related disease, a gut development disease, a liver disease, and a gonadal development disease, wherein the treatment comprises the administering of a therapeutically effective amount of the IncRNA molecule and the administering of a therapeutically effective amount of the isolated interference nucleic acid molecule or nucleic acid-binding nucleic acid molecule, both as previously defined.
  • a mesodermal or endodermal tissue disease selected from one or more of a cardiopathy, a respiratory epithelium -related disease, a gut development disease, a liver disease, and a gonadal development disease
  • the treatment comprises the administering of a therapeutically effective amount of the IncRNA molecule and the administering of a therapeutically effective amount of the isolated interference nucleic acid
  • the simultaneous, concomitant or intermittent administering of these two types of molecules aims to regulate or tune at convenience the expression of GATA4. Therefore, in the context of a disease with hypertrophic cells, such as hypertrophic cardiomyopathy, the administering of adjusted amounts and doses and adequate formulations (e.g., multi-layer pharmaceutical formulations) of both types of molecules, can first promote reduction of hypertrophy due to the quenching of the IncRNA (endogenous and/or administered), and later promote the regeneration due to the action of administered IncRNA of the invention.
  • adjusted amounts and doses and adequate formulations e.g., multi-layer pharmaceutical formulations
  • the IncRNA molecule and the isolated interference nucleic acid molecule or nucleic acid-binding nucleic acid molecule can be administered as active principles in a pharmaceutical composition.
  • An example is a multi-layered pharmaceutical formulation (tablets, pills), optionally with a controlled-release of the active principles, in which first an ASO (i.e. , a nucleic acid-binding nucleic acid molecule) is released to quench the endogenous IncRNA that regulates GATA4 expression, and then in a second step an adjusted amount of IncRNA is released from a more inner layer of the multi-layered pharmaceutical formulation, which will help in any differentiation or regeneration of the tissue.
  • ASO i.e. , a nucleic acid-binding nucleic acid molecule
  • a method for identifying an agent that modulates the activity of a IncRNA molecule comprising or consisting in a nucleotide sequence with a percentage of identity of at least 80 % with SEQ ID NO: 1 , preferably with a percentage of identity of 100 % with SEQ ID NO: 1 , preferably an agent that binds to and modulates the activity of a IncRNA of the first aspect, wherein the method comprises:
  • the method comprises introducing the candidate agent in a cell, which cell comprises the IncRNA molecule of the invention, and/or that comprises the transcription factor GATA4.
  • the cells preferably mammalian primate cells, are isolated cells at any differentiation and specialization stage observed in the tissues wherein they commonly reside.
  • the cells are selected in some embodiments from cells with a mesoderm phenotype (i.e., mesoderm stage of differentiation) to a full differentiated/specialized phenotype (i.e., full cell differentiation stage to obtain a specialized cell).
  • cardiac cells they can be cells with a cardiac mesoderm phenotype, with a cardiac progenitor phenotype, and/or with a full differentiated and specialized phenotype, such as a beating cardiomyocyte.
  • the step of detecting a change in the activity of this IncRNA comprises one or more of: analysis or determination of cell differentiation stage, cell specification, and cell proliferation.
  • the candidate agent is selected from one or more of an ASO, siRNA, a shRNA, a miRNA, and an aptamer.
  • the methylation profile of the isolated IncRNA molecule of the invention may have a role in its activity, it is also herewith provided for an in vitro screening method for identifying an agent that modulates the interaction of a IncRNA molecule, comprising or consisting in a nucleotide sequence at least 80 % identical to SEQ ID NO: 1 , preferably 100 % identical to SEQ ID NO: 1 , with a methyltransferase, preferably with an N6-methyladenosine methyltransferase, more in particular and optionally selected from the m 6 A-methyltransferase METTL16 and/or the m 6 A-methyltransferase subunit METTL3, wherein the method comprises:
  • step (e) comparing the amounts obtained in step (d) to a control to determine the candidate agent’s modulatory activity.
  • the culture media comprises the compounds required to get a particular cell phenotype or specialization.
  • the undifferentiated cells are induced pluripotent stem cells
  • the first cell differentiation stage is a mesoderm stage, preferably attained with a culture media that comprises one or more of CHIR99021 , BMP4, Activin A, and bFGF
  • the second cell differentiation stage is a beating cardiomyocyte.
  • the method of the invention is a method for the obtaining of organoids from the cells of interest, which are cultured in the conditions to obtain these kind of three-dimensional structures.
  • organoids that comprise beating cardiomyocytes are attained.
  • the invention also encompasses a method to induce cell differentiation, preferably cardiomyocyte differentiation in vitro or ex vivo, the method comprising introducing a methylation modification, preferably a methylation modification in an adenosine residue, in a IncRNA molecule comprising or consisting in a sequence at least 80 % identical to SEQ ID NO: 1 , preferably 100 % identical to SEQ ID NO: 1 , wherein the method comprises:
  • step (d) providing the conditions that allow interaction of the IncRNA with the methyltransferase to obtain a cell, preferably a cardiomyocyte, at a more advanced stage of specification in relation to the stage in step (a).
  • METTL16 expression levels in each cellular stage were analyzed. METTL16 expression slightly increased when cells exited the pluripotent stage (dO), entered the mesoderm stage (d3) and transited to cardiac progenitors (d10) and reduced once cultures differentiated into beating CMs (d25; (Fig.1 b-d). METTL16 gene expression analysis was also analyzed at single cell resolution over these stages by scRNA-seq using the 10x Genomics droplet platform followed by the suggested data analysis pipeline compromising of Cell Ranger and Seurat (Satija, R., Farrell, J.
  • METTL16 transcripts were higher expressed in the pluripotent stage (dO) and cardiac mesoderm stage (d3) and gradually reduced towards stages with more advanced cardiomyocyte differentiation (data not shown).
  • dO pluripotent stage
  • d3 cardiac mesoderm stage
  • d3 cardiac mesoderm stage
  • Fig.l e analogous biphasic pattern with higher concentration in myogenic precursor cells
  • transcripts that mapped to the sex chromosomes were excluded, since hiPSC clones from one male and one female donor were used. Overall, over 7,900 transcripts were mapped which including IncRNAs, miRNAs and miscRNAs for each sequenced cellular stage, with 82% of the mapped transcripts were IncRNAs, 6% was represented by miRNAs, 7% of transcripts were scaRNAs, scRNAs, miscRNAs, snoRNAs, snRNAs and 5% represented other miscellaneous ncRNAs.
  • m6A peaks (P value ⁇ 0.05) in each biological replicate and at every cellular stage were consistently identified, and found that about 23% of the sequenced ncRNA transcripts were m6A-methylated. Since the overwhelming majority (-94%) of the m6A-methylated ncRNA fraction was represented by IncRNAs, the analysis was focused exclusively on IncRNA transcripts and a total number of 2,883; 3,037; 112 3,100 and 2,606 m6A peaks on dO, d3, d10 and d25, were respectively identified. Next, the distribution of m6A modifications on IncRNA transcripts was analyzed and compared it to the distribution on protein-coding mRNA transcripts.
  • MeRIP-seq of hiPSCs from a pluripotent stage towards spontaneously beating cardiomyocytes revealed a predominant m6A methylation pattern in the IncRNA fraction on non-canonical motifs that follows specific topological patterns that differ from m6A modified protein-coding transcripts.
  • m6A-methylated IncRNA transcripts are enriched during cardiomyocyte specification.
  • IncRNA H19 a regulator of cardiac differentiation (Han, Y. et al. Downregulation of long non-coding RNA H19 promotes P19CL6 cells proliferation and inhibits apoptosis during late-stage cardiac differentiation via miR-19b-modulated Sox6. Cell & Bioscience 6, 1-11 (2016)).
  • analysis also detected the significant hypermethylation and upregulation of IncRNA MIR22HG, which can act as a tumor-suppressor by regulating Wnt/p-catenin, epithelial-mesenchymal transition (EMT), Notch, and STAT3 pathways (Zhang, L., Li, C. & Su, X. Emerging impact of the long noncoding RNA MIR22HG on proliferation and apoptosis in multiple human cancers. Journal of Experimental & Clinical Cancer Research 39, 271 (2020)).
  • C8orf49 encodes two alternative long intergenic transcripts of 1 ,968 nt and 1 ,414 nt, respectively, and is located on chromosome 8p23.1 positioned in the same transcriptional orientation between the genes encoding GATA4 (GATA Binding Protein 4) and NEIL2 (Nei Like DNA Glycosylase 2).
  • LncRNA transcripts can regulate transcription of target genes located in their vicinity (cis-acting) or distantly located (trans-acting). Based on the evidence that cisregulating IncRNAs are often located in the proximity of genes encoding for transcriptional regulators, this transcript was named by the inventors Gata4 REgulator ENhancer or IncRNA GREEN. Indeed, GATA4 and IncRNA GREEN show similar transcription patterns, both starting from the cardiac mesodermal stage and increasing even more towards cardiomyocyte maturation. In contrast, NEIL2 transcripts significantly decreased across these stages (Fig.2a). Second, the expression correlation between the three genes was evaluated by calculating the Pearson correlation coefficient based on their expression in different human tissues.
  • GATA4 is required for early mesoderm and endoderm development
  • GATA4, IncRNA GREEN and NEIL2 Transcript per Million (TPMs) values were extrapoled from three selected mesodermal-derived tissues (heart and gonads) and three endodermal-derived tissues (stomach, liver and pancreas) from GTEx( Human genomics.
  • GTEx Genotype-Tissue Expression
  • IncRNA GREEN is required for GATA4 expression, mesodermal commitment and cardiomyocyte differentiation.
  • LncRNAs are key regulators of signaling pathways that coordinate proper cardiogenesis, showing a dynamic spatiotemporal expression over different developmental stages. Therefore, the inventors focused on differentially methylated and expressed IncRNA transcripts during different stages of hiPSCs differentiating into cardiomyocytes. In this temporal analysis, we found the largest fraction of significantly differentially methylated and expressed IncRNA transcripts (24 IncRNA transcripts in total) at the cardiac progenitor stage compared to the cardiac mesodermal stage. Interestingly, 17 out of 24 IncRNA transcripts were significantly upregulated and m6A hypermethylated.
  • H19 an established regulator of cardiac development, was among this group of IncRNAs, further corroborating our hypothesis that m6A modification has a key role in coordinating the early cardiomyocyte specification, by regulating the stability and function of IncRNAs in early cardiogenesis.
  • GATA4 gene encodes for the zing finger GATA4 TF that regulates cardiogenic signaling pathways associated with embryonic cardiac development and promotes myocardial specification.
  • GATA4 interacts with a network of transcription factors (TFs), including Nkx2.5, HAND2, MEF2, TBX5, and SRF, to generate complexes that promote the transcription of cardiogenic gene programs.
  • TFs transcription factors
  • GATA4-binding sites have been identified in a large number of cardiac-specific promoters and enhancers, including the atrial natriuretic factor (ANF) promoter, the cardiac troponin C (cTnC) enhancer, the a-myosin heavy chain (a-MHC) promoter, and the myosin light chain I (MLCI) promoter.
  • AMF atrial natriuretic factor
  • cTnC cardiac troponin C
  • a-MHC a-myosin heavy chain
  • MLCI myosin light chain I
  • GATA4 can stimulate cardiomyocyte proliferation and regeneration (Yu, W. et al. GATA4 regulates Fgf16 to promote heart repair after injury. Development 143, 936-949 (2016). Malek Mohammadi, M. et al. The transcription factor GATA4 promotes myocardial regeneration in neonatal mice. EMBO Mol Med 9, 265-279 (2017). Medlej, A., Mohammad Soltani, B., Javad Mowla, S., Hosseini, S. & Baharvand, H. A novel miRNA located in the GATA4 gene regulates the expression of IGF-1 R and AKT1/2 genes and controls cell proliferation.
  • GATA4 mutations both in human and mouse, lead to severe congenital heart defects, demonstrating the importance of this TF in human cardiogenesis.
  • Garg, V. et al. GATA4 mutations cause human congenital heart defects and reveal an interaction with TBX5. Nature 424, 443-447 (2003). Rajagopal, S.K. et al. Spectrum of heart disease associated with murine and human GATA4 mutation. J Mol Cell Cardiol 43, 677-685 (2007)).
  • GATA4 is required for maintaining normal cardiac function, and, in response to pathological stress, is reactivated causing cardiac hypertrophic remodeling (Suzuki, Y.J.
  • GATA4 In contrast, in the left ventricle, upon aortic banding, GATA4 does not change its transcriptional levels, suggesting that GATA4 is regulated via posttranslational modification (Park, A.-M. et al. Pulmonary hypertension-induced GATA4 activation in the right ventricle. Hypertension 56, 1145-1151 (2010)). Although GATA4 is largely functionally studied in the context of the heart, remarkably little is known about its transcriptional regulation. It was speculated that IncRNA GREEN cis-regulated GATA4 transcription. Other IncRNAs have been reported to cis-regulate central transcription factors in development. LncRNA Handsdown, for instance, negatively regulates its cislocated gene Hand2 in mouse during cardiac development.
  • IncRNA GREEN expression positively correlated with GATA4 expression. This result was confirmed in vitro; IncRNA GREEN expression was detected at cardiac mesodermal stages, and significantly increased during cardiomyocyte differentiation similar to GATA4. More importantly, downregulation IncRNA GREEN strongly affected GATA4 levels, confirming that it positively cis- regulates GATA4 transcription. Furthermore, IncRNA GREEN silencing significantly influenced the transcription of GATA4-regulated genes.
  • hiPSCs were differentiated into hiPSC-CMs by mesodermal induction, followed by inhibition of the WNT-signaling pathway, as previously described (see Tiburcy M, et al. Generation of Engineered Human Myocardium in a Multi-well Format. STAR Protoc. 2020;1 (1): 100032; and Chen VC, et al. Development of a scalable suspension culture for cardiac differentiation from human pluripotent stem cells. Stem Cell Res. 2015;15(2):365-375). Briefly, we started the differentiation protocol on 80-90% confluent hiPSCs, maintained in Matrigel-coated plates.
  • BSFM basal serum-free medium
  • Mesodermal induction was carried out supplementing the BSFM with 1 pM CHIR99021 (Stemgent), 5ng/ml Recombinant human BMP4 (R&D Systems), 9ng/ml Recombinant Human/Mouse/rat Activin A (R&D Systems) and 5ng/ml human FGF-2 (Miltenyi Biotec) for 3 days. Subsequently, cells were cultured for other 7 days in BSFM containing 5pM IWP-4 (Stemgent). HiPSC- CMs were maintained in BSFM for 25 days.
  • cells underwent a single round of metabolic selection between days 13-17, using RPMI 1640 medium without glucose, without HEPES (Thermo Fisher), supplemented with 1 % 100X penicillin/streptomycin (Invitrogen), 2.2mM 50% sodium lactate (Sigma) and 0.1mM p-mercaptoethanol (Invitrogen).
  • the Pluripotent Stem Cell 4-Marker Immunocytochemistry Kit (ThermoFisher Scientific, A24881) was used, according to manufacturer instructions.
  • hiPSCs were and seeded on Matrigel-coated 12- well chamber, removable (Ibidi, #81201). Cells were maintained in in E8TM Medium. When the cells reached 70% confluency, they were fixed with 4% PFA/PBS for 15 minutes at room temperature. Upon three washing steps in PBS, cells were permeabilized with the permeabilization solution for 15 min at room temperature and treated with the blocking solution for 30 min at room temperature.
  • hiPSC-CM spontaneous contractions were measured and quantified according to the published procedure (Grune T, et al. The “MYOCYTER” - Convert cellular and cardiac contractions into numbers with Imaged. Scientific Reports. 2019;9(1): 15112). Briefly, spontaneously contracting differentiated CMs (d25) were recorded through a commercially available tablet connected via a camera adapter to an optical microscope, using a 5x objective. Videos, originally saved as mp4, were converted in Fiji Imaged with the FFMPEG import function into AVI format files and analyzed in Imaged via Myocyter v1.3 macro. The beating frequency, the overall peak contraction time measured at 10% threshold, and the overall contraction time of differentiated CMs (d25) were measured.
  • ScRNA-seq data was acquired from a dataset generated on our lab.
  • cells from 5 different hiPSC-differentiated cellular stages were collected (dO, d3, d10, d25 and from engineered heart myocardium) from two cell clones (BXS0116, TC1133).
  • Cells were dissociated with Accutase and Trypsin and prepared following the Chromium Single Cell 3’ Gene Expression Solution v2 and sequenced on the NextSeq2000 according to manufacturer’s instructions. Reads were afterwards counted and mapped to the human reference genome (GrCh38/Ensembl98) the 10x Genomics Cell Ranger 5.0.0 pipeline with default parameters.
  • Data Analysis was performed using the ‘Seurat’-package (v4.02) and integrated to account for gender differences, Gene expression was assessed according to the dimensional reduction and clustering using the first 20 principal components.
  • RNA was denatured at 95°C for 3 min, spotted directly onto a positively charged Nylon membrane (Roche), and UV cross-linked at 1 ,200 microjoules [x100] using CL-1000 ultraviolet crosslinker (UVP).
  • UVP CL-1000 ultraviolet crosslinker
  • the membrane was blocked in 5% nonfat milk shaking for 1 h at room temperature and incubated with primary anti-m6A antibody (1 :1000, Cat. 202003, Synaptic Systems) at 4°C overnight. The day after, the membrane was incubated for 1 h at room temperature in secondary anti-rabbit IgG- HRP conjugated antibody (1 :2000, DAKO), before imaging using Extreme Sensitivity Chemiluminescence Substrate (PerkinElmer).
  • RNA was extracted using Trizol reagent as described above. The RNA quality and concentration were measured by Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit (Agilent). In this study, samples from both clones were considered biological replicates at each cellular time-point (n 2 per each group).
  • RNA fraction was fragmented into ⁇ 100-nt-long fragments using divalent cations under elevated temperature. Then the cleaved RNA fragments were incubated for 2h at 4 °C with m6A-specific antibody (Cat. 202003, Synaptic Systems) in IP buffer (50mM Tris-HCI, 750mM NaCI and 0.5% Igepal CA-630) supplemented with BSA (0.5pg pl-1).
  • m6A-specific antibody Cat. 202003, Synaptic Systems
  • ncRNAs only IncRNA, miRNA, scaRNA, scRNA, miscRNA, snoRNA, snRNA and sRNA transcripts. Because in this study we employed hiPSCs clones from both female and male donors, sexual chromosomes were excluded from our bioinformatics analysis to avoid clonespecific results.
  • Pearson correlation analysis was applied to study the linear correlation of GREEN IncRNA, GATA4 and NEIL2 transcript expression in human tissues (heart, pancreas, stomach, liver, ovaries and testis). TPMs values- (i.e., transcripts per million) of each target transcript in each selected tissue were acquired from GTEx (Human genomics. The Genotype-Tissue Expression (GTEx) pilot analysis: multitissue gene regulation in humans. Science. 2015;348(6235):648-660), and used as input for our analysis. Pearson correlation analysis was performed using the correlation package (v0.6.0) (Makowski D, et al.. Methods and algorithms for correlation analysis in R. Journal of Open Source Software. 2020;5(51):2306).
  • EvoACTG database http://evoactg.uni-muenster.de/ was used to evaluate the chamber specificity of the human GREEN IncRNA, GATA4 and NEIL2 transcripts in the heart (Gandhi S, et al. Evolutionarily conserved transcriptional landscape of the heart defining the chamber specific physiology. Genomics. 2021 ;113(6):3782-3792).
  • Cytoplasmic and nuclear RNA fractions were isolated from hiPSC-CMs (d25) according to the published procedure (Senichkin VV, et al. Simple and Efficient Protocol for Subcellular Fractionation of Normal and Apoptotic Cells. Cells. 2021 ;10(4):852). In brief, adherent cells were washed and harvested in BSFM on d25 of the differentiation protocol. Cells were centrifuged at 4°C at 500g for 4 min, and gently washed in phosphate-buffered saline (PBS).
  • PBS phosphate-buffered saline
  • the endogenous knockdown of IncRNA GREEN was achieved using an LNA “GapmeR” specifically targeting the two human GREEN transcripts.
  • the LNA “GapmeR” was purchased at Qiagen Inc (Hilden, Germany). Briefly, cells were transfected on d3 of the differentiation protocol with 125nM LNA “GapmeR” using Lipofectamine RNAiMAX, according to the manufacturer’s protocol. After 24h the transfection mix was replaced with fresh medium. Finally, 48h post-transfection, cells were harvested and processed for gene expression analysis. Untreated cells were used as control.

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Abstract

L'invention concerne une nouvelle molécule d'acide ribonucléique isolée, qui est une longue molécule d'ARN non codant (ARNlnc), et qui régule l'activité du facteur de transcription GATA4. L'invention porte notamment sur l'utilisation de cette molécule à des fins thérapeutiques, ainsi que de toute molécule d'acide nucléique d'interférence pouvant l'éteindre. L'invention concerne également des procédés particuliers pour induire la différenciation cellulaire et l'obtention d'organoïdes.
PCT/EP2025/052335 2024-01-30 2025-01-30 Composés destinés à être utilisés dans le traitement de troubles ou de maladies par modulation de l'activité du facteur de transcription gata4 Pending WO2025163033A1 (fr)

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US20070042381A1 (en) * 2002-12-05 2007-02-22 Rosetta Genomics Bioinformatically detectable group of novel regulatory viral and viral associated oligonucleotides and uses thereof
US20110178283A1 (en) * 2006-03-03 2011-07-21 International Business Machines Corporation Ribonucleic acid interference molecules and binding sites derived by analyzing intergenic and intronic regions of genomes
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