WO2025163033A1

WO2025163033A1 - Compounds for use in the treatment of disorders or diseases through modulation of transcription factor gata4 activity

Info

Publication number: WO2025163033A1
Application number: PCT/EP2025/052335
Authority: WO
Inventors: Giulia SPANÒ; Monika Stoll; Leon Johannes De Windt
Original assignee: Universiteit Maastricht; Academisch Ziekenhuis Maastricht
Current assignee: Universiteit Maastricht; Academisch Ziekenhuis Maastricht
Priority date: 2024-01-30
Filing date: 2025-01-30
Publication date: 2025-08-07
Anticipated expiration: 2026-07-30

Abstract

The invention relates to a new isolated ribonucleic acid molecule, which is a long non-coding RNA (lncRNA) molecule, and that regulates the activity of the GATA4 transcription factor. The invention discloses the use in therapy of this molecule, as well as of any interference nucleic acid molecule able to quenched it. Particular methods to induce cell differentiation and the obtention of organoids are also disclosed.

Description

Title: Compounds for use in the treatment of disorders or diseases through modulation of transcription factor GATA4 activity.

FIELD OF THE INVENTION

[001] 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.

BACKGROUND OF THE INVENTION

[002] The background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[003] There are diseases and disorders, which origin or key aspect relates with the capacity of the cells of one or more tissue types to correctly develop (i.e., mainly differentiate and proliferate) for performing their function. Under this umbrella many developmental diseases are counted. On the other hand, other diseases derive from the alteration of cell size (i.e., hypertrophies) and/or alteration of cell number that ultimately affect the functioning of the organ or tissue in which those cells reside.

[004] Examples of these two kinds of etiologies leading to pathology can be seen in most of the organs. In the particular case of cardiac tissue, for example, the cardiogenic heart defect results from an improper development during embryogenesis, and pathological ventricular remodeling is a pathology that appears with the presence of hypertrophic cardiomyocytes configuring the layers of the ventricles as a compensation mechanism after a myocardial infarct. Different therapeutical approaches are required considering the different etiologies of disease in a tissue.

[005] As disclosed in the international patent application with the publication number WO201 8/055235, some of the new research lines proposed for the treatment of myocardial infarction rely on the administration of therapeutic agents which have the ability to induce regeneration. The authors in this international publication propose isoxazole-amide compounds that target the networks of cardiac transcription factors that control cardiac gene expression and play, this way, a central role in transcriptional regulation during cardiogenesis and in the adaptive pathophysiological processes in the adult heart. 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.

[006] 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. Apart from cardiac specification, 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).

[007] This transcription factor is, thus, an interesting target to elucidate the mechanisms of cell differentiation and regeneration in several tissues.

[008] In light of this, new products, compositions, methods and uses for in the treatment of disorders or diseases through modulation of transcription factor GATA4 activity, such as cardiopathies, would be highly desirable but are not yet readily available. In particular, there is still a clear need in the art for reliable, efficient, and reproducible products, compositions, methods and uses that allow to be used in the treatment of these disorders. Accordingly, the technical problem underlying the present invention can been seen in the provision of such products, compositions, methods and uses for complying with any of the aforementioned needs, or at least providing the public with a useful choice. The technical problem is solved by the embodiments characterized in the claims and herein below.

SUMMARY OF THE INVENTION

[009] As embodied and broadly described herein, the present invention is directed to the surprising finding that GATA4 is regulated by a long intergenic non-coding RNA (IncRNA) molecule. Within the frame of an independent research to analyze the non- coding RNA (ncRNA) methylome, and its role in the specification of cardiomyocytes, 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. With now an identified function, the inventors termed this new isolated InRNA as “human Gata4 Regulator Enhancer (GREEN)” (SEQ ID NO: 1), as it was confirmed that it regulated the transcription and expression of the cardiogenic transcription factor GATA4, located in the vicinity (cisacting IncRNA).

[010] Thus, 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 .

[011] For the purpose of easy wording, the isolated ribonucleic acid molecule of the invention is herewith termed also long non-coding RNA (IncRNA) molecule.

[012] The Examples below, stemmed on a model of cardiomyocyte differentiation from induced pluripotent stem cells, substantiate that this IncRNA molecule promotes the expression of GATA4. This transcription factor is of interest for cell differentiation and specification, as well as for cell proliferation and regeneration in case of tissue damage. Besides, the data provide evidence that in case of hypertrophy of the cells 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.

[013] 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.

[014] 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.

[015] Therefore, 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.

[016] On the other hand, 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.

[017] The provision of the 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.

[018] Therefore, another aspect of the invention is a 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.

[019] Yet in another aspect, 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.

[020] 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. On the other hand, if what is contained in the host cell is the interference nucleic acid molecule (herewith also referred as a nucleic acid-binding nucleic acid molecule), or a vector that comprises it, 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.

[021] The skilled person in the art will understand that 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.

[023] Thus, another aspect of the invention is pharmaceutical 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.

[024] In the sense of the invention a pharmaceutical composition also encompasses a veterinary composition.

[025] Directly derivable from the capability to modulate the expression of the transcription factor GATA4, which is known to be involved either in the hypertrophy of cells, or in the promotion of cell proliferation and differentiation, it results the application in therapy of the IncRNA of the first aspect of invention, or of any interference nucleic acid molecule that quenches it.

[026] Thus, it is also herewith disclosed as another aspect of the invention, 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 as a medicament.

[027] Yet in another aspect of the invention, it relates to 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.

[028] 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. The invention, thus, 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.

[029] As previously indicated, 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.

[030] 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. [031] Yet in another aspect, 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); and d) 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).

[032] 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.

[033] Herewith disclosed are also induced differentiated cells, and organoids obtained or obtainable by the in vitro or ex vivo method to induce cell differentiation, and/or to obtain organoids, as disclosed in the previous aspect.

[034] 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. Moreover, in some embodiments, the 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. [035] For the cell manipulation with any of an IncRNA molecule comprising or consisting in a nucleic acid sequence at least 80 % identical to SEQ ID NO: 1 , or an interference nucleic acid molecule or nucleic acid-binding nucleic acid molecule, the later which comprises or transcribes into a sequence that quenches the IncRNA, the provision of them in a cell culture medium is advantageous.

[036] Thus, herewith disclosed is also 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).

BRIEF DESCRIPTION OF THE DRAWINGS

[037] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:

[038] Figure 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). GAPDH served as loading control, (e) RNA dot blot analysis of m6A modification expression 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.]

[039] Figure 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).

[040] Figure 3: IncRNA GREEN is required for GATA4 expression, (a) Scheme describing the workflow of the experiment. The arrows on the bottom part indicate the days of GapmeR transfection (Day 3) and day of sample collection Day 5). (b) qRT- PCR analyses of endogenous GREEN IncRNA, its c/s-located genes GATA4 and NEIL2 and MALAT1 IncRNA 48h post-transfection on cardiac mesoderm cells (d3) treated with GapmeR, and compared to control cells, (c) qRT-PCR analyses of a subset of genes including mesoderm-specific genes, atrial-specific genes, ion channel specific genes and cardiomyocyte specific genes (top); and a subset of selected GATA4-driven genes (bottom) in GapmeR transfected cells and their Control, (d) schematic representation of the proposed model.

[041] The data represent means ± SEM from three independent experiments. P values were calculated using unpaired f-test. Results were considered statistically significant when the P value was < 0.05 (*P < 0.05; **P < 0.01 ; ***P < 0.001 ; ****p < 0.0001 ; # P > 0.05).

DESCRIPTION

Definitions

[042] 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.). 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.

[043] Various terms relating to the methods, compositions, uses and other aspects of the present invention are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art to which the invention pertains, unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definition provided herein. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein. [044] For purposes of the present invention, the following terms are defined below. [045] As used herein, the singular form terms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a long non coding RNA” includes a combination of two or more long no coding RNAs, and the like; or “a cell” includes a combination of two or more cells, and the like.

[046] As used herein, “about” and “approximately", when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1 %, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed invention. Unless otherwise clear from context, all numerical values provided herein include numerical values modified by the term “about.” [047] As used herein, “and/or” refers to a situation wherein one or more of the stated cases may occur, alone or in combination with at least one of the stated cases, up to with all of the stated cases.

[048] As used herein, "at least" a particular value means that particular value or more. For example, "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. Also for example, “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.

[049] As used herein “identity” or “sequence identity” refers to the degree of relatedness between two or more nucleic acid sequences (polynucleotide sequences), as determined by comparing the sequences. The comparison of sequences and determination of 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.

[050] Thus, 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. Such 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. Importantly, a query nucleic acid sequence may be described by a nucleic acid sequence identified in one or more claims herein. The same applies for 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.

[051] Therefore, the term "identity" refers to the percentage of residues that are identical in the two sequences when the sequences are optimally aligned. If, in the optimal alignment, a position in a first sequence is occupied by the same amino acid residue as the corresponding position in the second sequence, the sequences exhibit identity with respect to that position. The percentage of identity determines the number of identical residues over a defined length in a given alignment. Thus, the level of identity between two sequences or ("percent sequence identity") is measured as a ratio of the number of identical positions shared by the sequences with respect to the number of positions compared (i.e. , percent sequence identity = (number of identical positions/total number of positions compared) x 100). 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.

[052] As an illustration, by 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. In other words, to obtain a polynucleotide 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. These 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. A number of mathematical algorithms for rapidly obtaining the optimal alignment and calculating identity between two or more sequences are known and incorporated into a number of available software programs. For purposes of the present invention, the 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.

[053] As used herein, the term “isolated” when referring to a polynucleotide (nucleic acid, such as a ribonucleic acid or an interference nucleic acid), refers to nucleic acids being present in a non-naturally occurring environment, e. g. are separated from their naturally occurring environment. For example, 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). The term also refers to such nucleic acid being artificially or synthetically produced. Within the context of the present invention it will be clear for the skilled person if a reference to a nucleic acid, or polynucleotide includes reference to an “isolated” nucleic acid, or polynucleotide. [054] As used herein, 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.

[055] As used herein, “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.

[056] As used herein, "antagonist" and/or "inhibitor" 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.

[057] As used herein a “test compound”, “candidate agent” or “agent,” 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. It also refers to 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 m⁶A-methyltransferase METTL3.

[058] As used herein, the term “pharmaceutical 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). The pharmaceutical 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.

[059] The term "pharmaceutically acceptable" as used herein pertains 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. 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.

[060] As used herein the term “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.

[061] As used herein, the term “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. InRNAs have been detected involved in the regulation of transcriptional programs affecting many (patho)physiological processes. As previously indicated, these ribonucleic acid molecules may comprise 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.

[062] As used herein, “sequence”, or “(poly)nucleotide 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).

[063] As used herein, 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.

[064] 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. [065] As used herein, the term “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. It can also be referred herewith as “nucleic acid-binding nucleic acid molecule”. Examples of 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. Examples of iRNA molecules include small-hairpin RNA (shRNA), microRNA (miRNAs), and siRNAs. On the other hand, 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.

[066] As used herein, 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. However, 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. In stringent conditions 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. These 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. For the determining of this complementarity, as well as the ability to quench a nucleic acid molecule, there are conventional techniques which are broadly known and routine for the skilled person in the art.

[067] As used herein, “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. In the particular case of cardiac cells, 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.

[068] As used herein, “conventional techniques” or “methods known to the skilled person” refer to a situation wherein the methods of carrying out the conventional techniques used in methods of the invention will be evident to the skilled worker. The practice of conventional techniques in molecular biology, biochemistry, cell culture, genomics, sequencing, medical treatment, pharmacology, immunology and related fields are well-known to those of skill in the art and are discussed, in various handbooks and literature references.

[069] As used herein, “comprising” or “to comprise” is construed as being inclusive and open ended, and not exclusive. Specifically, the term and variations thereof mean the specified features, steps or components are included. These terms are not to be interpreted to exclude the presence of other features, steps or components. It also encompasses the more limiting “to consist of”. Detailed description

[070] The invention is defined herein, and in particular in the accompanying claims. Subject-matter which is not encompassed by the scope of the claims does not form part of the present claimed invention.

[071] It is contemplated that 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. Thus, 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.

[072] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[073] Any 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.

[074] As embodied and broadly described herein, 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).

[075] This has prompted the manipulation of cells, namely cardiomyocyte precursor or progenitor cells, to elucidate the role of the newly identified tandem lncRNA-GATA4 in health and pathological states.

Isolated ribonucleic acid molecule

[076] As previously indicated, 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. [077] In a particular embodiment of the first aspect, the isolated IncRNA molecule comprises one or more methylated residues, preferably one or more N6- methyladenosine residue(s), more preferably one N6-methyladenosine residue. In an embodiment the one or more residues are in a sequence defined by the consensus sequence [TTCAGATGA], In an embodiment, 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], In another embodiment, 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], In another embodiment, the isolated IncRNA molecule comprises three methylated residues in a sequence defined by the consensus sequence [TTCAGATGA], preferably three N6- methyladenosine residues.

[078] In another particular embodiment of the first aspect, 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. In a preferred embodiment, the isolated IncRNA molecule comprises or consists in an nucleic acid sequence 100 % identical to SEQ ID NO: 1.

[079] 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)

[080] 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. [081] In some embodiments, 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.

[082] In some embodiments, 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).

[083] In a preferred embodiment, the interference nucleic acid molecule, or nucleic acid-binding nucleic acid molecule, is an antisense nucleic acid oligonucleotide (ASO). In also another preferred embodiment 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 .

[084] In a preferred embodiment, 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).

[085] In a more preferred embodiment 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).

[086] These types of interfering nucleic acids are known by the skilled person. Any sequence with the capability to interact, mainly by complementarity of the nitrogen base pairing, with the sequence of the IncRNA of the invention, is encompassed as candidate to be comprised in a longer polynucleotide sequence used as vector according to the invention, or as an isolated compound provided by other means, such as encapsulated in micro or nanoparticles, or in liposomes.

Vectors/nucleic acid constructs and cells

[087] The sequence of the isolated IncRNA of the invention is, in a preferred embodiment, comprised in a nucleic acid vector.

[088] Another aspect of the invention is, thus, a 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.

[089] 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.

[090] 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.

[091] In another particular embodiment, 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. [092] In a preferred embodiment, 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. [093] In some preferred embodiments, 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. In an embodiment, the serotype of the adeno-associated virus is AAV6.

[094] As previously indicated, the vectors, or directly the isolated IncRNA of the invention can be integrated into host cells as defined above. In principle 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.

[095] The cells can be at any stage of specialization observed in the tissues where they reside. Thus, as host cells, also 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. When 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.

[096] Thus, preferred examples of host cells are selected from cells at any stage of maturation/specialization, from the mesoderm stage to full differentiated state.

[097] In the particular case of 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.

[098] 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 (EOMES) also known as T-box brain protein 2 (Tbr2) is a protein that in humans is encoded by the EOMES gene. In early in development, Eomesodermin/Tbr2 controls early differentiation of the cardiac mesoderm. [099] Thus, in a particular embodiment, the host cells are cardiac cells with a cardiac mesoderm phenotype and express one or more of the EOMES and BRACHYURY proteins, preferably both.

[100] 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.

[101] Thus, in a particular embodiment, 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. In another embodiment, the host cells are beating cardiomyocytes that express one or more of TNNT2 and MYH6 proteins, preferably both.

[102] The detection of all these proteins (i.e. , markers) in the hosts cells can be done by conventional techniques widely known by the skilled person, and they include for example techniques employing specific antibodies or fragments thereof.

[103] In the same way, the detection of a beating cardiomyocyte can be visualized through the microscope.

Compositions and uses

[104] 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.

[105] Therefore, 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.

[106] In some embodiments of the pharmaceutical composition, the nucleic acid of the invention, either the isolated IncRNA or the isolated interference nucleic acid molecule capable to quench it, or any of nucleic acid sequence that transcribe to any of them, is encapsulated in a delivery vehicle, for example in a liposome or in a micro/nanoparticle, such as in lipid nanoparticles (LNP) or nanostructured lipid carriers (NLC). The skilled person in the art will know the technologies behind the preparation of pharmaceutical compositions that comprise nucleic acids or cells.

[107] For the delivery of nucleic acids in the cells, in particular the IncRNA molecules of the invention or any nucleic acid that transcribes to it, 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. Examples of 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). 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.

[108] The skilled person will understand that these liposomes, nano or microparticles as previously disclosed in detail are also useful for the encapsulation of any interference nucleic acid molecule or nucleic acid-binding nucleic acid molecule with a sequence that comprises or transcribes into a sequence that quenches the isolated IncRNA molecule. [109] Besides the pharmaceutical compositions, 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.

[110] Thus, it is also an object of the present invention 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).

[111] The invention relates also to the use, preferably in an isolated sample comprising mammal cells, more preferably primate cells, of a IncRNA molecule comprising or consisting in a nucleic acid sequence at least 80 % identical to SEQ ID NO: 1 , preferably 100 % identical to SEQ ID NO: 1 , as modulator of the transcription factor GATA4 activity. It also relates to the use, preferably in an isolated sample comprising mammal cells, more preferably comprising primate cells, of an interference nucleic acid molecule or nucleic acid-binding nucleic acid molecule which comprises or transcribes into a sequence that quenches the said IncRNA molecule. In the first use, the modulator acts as an agonist of the GATA4 activity. In the second application, the nucleic acid molecule is an inhibitor or antagonist of the GATA4 activity, since it quenches the IncRNA molecule. The use as disclosed is, in an example, useful for in vitro and/or ex vivo experimental assays, in which the activity of GATA4 is desired to be modulated (increased or decreased) for any reason.

IncRNA for use in therapy

[112] 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.

[113] Two therapeutic approaches are possible derived from the new identified regulatory role of the isolated IncRNA molecule.

[114] 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. For use in this prevention and/or treatment are also 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.

[115] On the other hand, 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.

[116] 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.

[117] Therefore, 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. [118] In a preferred embodiment, 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.

[119] As will be further illustrated in the Examples, there is a strong linear correlation between 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). The data (in vitro) in the examples performed with cardiac mesoderm, and the results that are derived therefrom, make plausible that any of the IncRNA molecule, and/or the isolated interference nucleic acid molecule or nucleic acid-binding nucleic acid molecule, all of them as defined in any of the previous aspects and embodiments, may be used in the prevention and/or treatment of a disease in any of the mesodermal-derived tissues, and endodermal-derived tissues.

[120] As previously disclosed, 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.

[121] In a more preferred embodiment, 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 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.

[122] In also a more preferred embodiment, 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.

[123] Thus, this embodiment illustrates the applicability of the isolated IncRNA molecule of the invention, or any nucleic acid molecule providing it, in regenerative medicine.

[124] In the alternative, and as another embodiment, any isolated interference nucleic acid molecule (i.e., 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.

[125] In a preferred embodiment, and when so required due to the etiology of the disease, the interference nucleic acid molecule (i.e., the nucleic acid-binding 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.

[126] 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

[127] As will be illustrated in the Examples, a preferred ASO is a single-stranded, oligonucleotide that comprises a DNA sequence (DNA portion) flanked by two locked nucleic acid (LNA) sequences. This kind of ASO is also known as LNA GapmeRs. [128] ASO, and among them LNA GapmeRs that bind the IncRNA of the invention (SEQ ID NO: 1), are designed following conventional techniques, and they encompass all those that can bind to any fragment of SEQ ID NO:1 , preferably blocking the function of the IncRNA of the invention, and ultimately the function of the transcription factor GATA4.

[129] The 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).

[130] 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. Once the GapmeR and the target are paired, the duplex of the DNA sequence and the RNA catalyzes the RNase H-dependent degradation of the RNA. The presence in the ASO of the LNA flanking sequences are known to increase the affinity for the target. 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.

[131] In a preferred embodiment, 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).

[132] In also another embodiment, the IncRNA molecule, and/or the isolated interference nucleic acid molecule or nucleic acid-binding nucleic acid molecule, as defined in any of the aspects and embodiments, 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.

[133] As the skilled person in the art will understand, 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.

[134] 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.

Screening methods

[135] Herewith identified for the first time the role of a IncRNA molecule with a sequence at least 80 % identical to SEQ ID NO:1 , preferably 100 % identical to SEQ ID NO: 1 , it derives from the same the convenience of modulating its activity or role in the cell to, ultimately, switch-on or switch-off the activity of the transcription factor GATA4.

[136] Thus, herewith disclosed is also 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:

(a) contacting the IncRNA with a candidate agent; and (b) detecting a change in the activity of the IncRNA compared to a control to determine the candidate agent’s modulatory activity.

[137] In a particular embodiment of the screening method for identifying an agent (i.e., candidate agent) that modulates the activity of a IncRNA molecule of the invention, 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. Thus, 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). In the particular case of 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.

[138] In another embodiment of the in vitro screening method for identifying an agent that modulates the activity of a IncRNA molecule as defined above, 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.

[139] In also another embodiment of the screening method, the candidate agent is selected from one or more of an ASO, siRNA, a shRNA, a miRNA, and an aptamer.

[140] Derived from the knowledge that 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 m⁶A-methyltransferase METTL16 and/or the m⁶A-methyltransferase subunit METTL3, wherein the method comprises:

(a) providing a mammalian cell or an assay-container that comprises the IncRNA, and optionally the transcription factor GATA4, and the methyltransferase;

(b) adding a candidate agent;

(c) provide conditions that allow interaction of the candidate agent with the IncRNA molecule and/or the methyltransferase; (d) determining the amount of IncRNA molecule that is methylated;

(e) comparing the amounts obtained in step (d) to a control to determine the candidate agent’s modulatory activity.

[141] The skilled person in the art is aware of the conventional techniques for the analysis of the methylation of nucleic acid molecules, some of which are disclosed in the Examples section.

Methods to induce cell differentiation

[142] Given that the newly identified IncRNA molecule of the invention intervenes in the network of transcription factors that ultimately promote cell differentiation, it derives its use in a method to obtain differentiated cells, including organoids.

[143] The invention relates to a method to induce cell differentiation, in vitro, or ex vivo, the method comprising: a) providing a source of undifferentiated cells, preferably of mammalian cells; b) inducing a first cell differentiation stage, in which the cells acquire a first phenotype or degree of maturity or specialization; c) providing to the differentiated cells of step (b) a IncRNA molecule comprising or consisting in a nucleic acid sequence at least 80 % identical to SEQ ID NO: 1 , preferably 100 % identical to SEQ ID NO: 1 ; and d) culturing the cells in a culture medium, and under conditions suitable to obtain cells in a second cell differentiation stage, in which the cells have a higher phenotype or degree of maturity or specialization than the first phenotype in (b).

[144] For the induction of cell differentiation from undifferentiated cells, several culture media known by the skilled person may be used. In some embodiments, for the induction of the first cell differentiation stage in the method, the cells are cultured in a culture medium that comprises one or more of modulators of the Wnt pathway, the Fgf/MAPK pathway, the BMP pathway, and the Tgfp/Nodal pathway, which pathways and modulators are common general knowledge for the skilled person. In some embodiments, the culture for the induction of a first differentiation stage comprises one or more of CHIR99021 (a inhibitor of GSK-3), Bone morphogenic protein 4-BMP4, Activin A, and one or more fibroblast growth factors (FGF), preferably basic FGF (bFGF). [145] The culturing is performed for a period of time that is suitable to achieve the desired first cell differentiation stage, usually for a period of 2 to 5 days, such as 3 days.

[146] In the same way and in another embodiment of the method to induce cell differentiation, to get the cells in a second differentiation stage and after the provision in the culture medium of the IncRNA molecule of the invention, the culture media comprises the compounds required to get a particular cell phenotype or specialization.

[147] The conditions are thus defined by the compounds in the culture media, the skilled person will recognize, and they include the commonly applied temperature and relative humidity in cell culturing.

[148] In some embodiments when the cells are cardiac cells, 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; and the second cell differentiation stage is a beating cardiomyocyte.

[149] The method of the invention, and as the skill person will recognize, 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.

[150] In the particular case of starting from undifferentiated cells that are allowed to differentiate to cardiac cells through the path of several stages of differentiation, organoids that comprise beating cardiomyocytes are attained.

[151] Also herewith disclosed is another method to induce cell differentiation, preferably in a mammalian cell, in this case taking advantage of the possibility to introduce methylation modifications at specific nucleotide residues of the IncRNA molecule newly identified.

[152] Thus, 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:

(a) providing a source of cells, preferably cardiomyocytes at a particular stage of differentiation; (b) providing the conditions under which IncRNA and optionally the transcription factor GATA4 is transcribed in the cells, preferably in the cardiomyocytes, and/or providing to the cells, preferably cardiomyocytes, the IncRNA molecule and optionally the transcription factor GATA4;

(c) providing a methyltransferase or the conditions to allow the cells to express an endogenous methyltransferase; and

(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).

[153] The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art (including the contents of the references cited herein), readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein.

[154] All references cited herein, including journal articles or abstracts, published or corresponding patent applications, patents, or any other references, are entirely incorporated by reference herein, including all data, tables, figures, and text presented in the cited references. Additionally, the entire contents of the references cited within the references cited herein are also entirely incorporated by references.

[155] It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance presented herein, in combination with the knowledge of one of ordinary skill in the art.

[156] It will be understood that all details, embodiments, and preferences discussed with respect to one aspect of embodiment of the invention is likewise applicable to any other aspect or embodiment of the invention and that there is therefore not need to detail all such details, embodiments, and preferences for all aspect separately.

[157] Having now generally described the invention, the same will be more readily understood through reference to the following examples which is provided by way of illustration and is not intended to be limiting of the present invention. Further aspects and embodiments will be apparent to those skilled in the art.

EXAMPLES

Example

[158] Isolation of IncRNA molecule of SEQ ID NO: 1 in the context of a study of cardiac non coding RNOME (ncRNOME)

[159] The materials and methods accompanying the following results are listed at the ens of this section.

[160] Human METTL16 methyltransferase is reduced in more advanced stages of human cardiomyocyte specification.

[161] Two different healthy hiPSC clones from a non-Hispanic Caucasian female donor and a male Hispanic Caucasian donor were differentiated to account for biological variance and susceptibility to the differentiation protocol. Small molecule, biphasic Wnt signaling modulation was used to initiate mesodermal specification and cardiac differentiation (Fig.1a). The sample collection times were chosen according to their cell specific informative stage during differentiation and included pluripotent hiPSCs (day 0: dO), cardiac mesoderm (day 3: d3), cardiac progenitors (day 10: d10) and differentiated cardiomyocytes (CMs; day 25: d25).

[162] Since m6A methylation of ncRNAs mainly depends on METTL16 activity, 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. A., Gennert, D., Schier, A.F. & Regev, A. Spatial reconstruction of single-cell gene expression data. Nature Biotechnology 33, 495-502 (2015). Hao, Y. et al. Integrated analysis of multimodal single-cell data. Cell 184, 3573-404 3587.e3529 (2021)). A LIMAP was used to produce a low-dimensional embedding of the single cell data while preserving global structure. After data integration, cells from both hiPSC clones showed similar expression profiles and clustered according to pre-determined time points, rather than individual biological- or batch effects. 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). In line, global m6A cellular levels followed an analogous biphasic pattern with higher concentration in myogenic precursor cells (Fig.l e). Taken together, the data indicate that METTL16 expression and m6A cellular abundance are higher in earlier stages of human cardiomyocyte lineage specification compared to more advanced stages of cardiomyocyte differentiation.

[163] The m6A IncRNA methylome in hiPSCs differentiating into beating cardiomyocytes.

[164] To explore the m6A modified ncRNOME over stages of pluripotency towards cardiomyocyte differentiation, the two hiPSC clones as biological replicates were subjected to m6A RNA immunoprecipitation followed by high-throughput sequencing (MeRIP-seq) at informative stages of pluripotency (dO), cardiac mesoderm stage (d3), cardiac progenitors (d10) and differentiated cardiomyocytes (d25). A principal component analysis (PCA) of the input and immunoprecipitated (IP) samples was performed, which showed that the generated m6A data were sufficiently repeatable and of high-quality for each biological replicate (data not shown).

[165] To avoid clone-specific artifacts, 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. Interestingly, from the analysis, a single m6A-peak on the majority of the m6A- methylated IncRNA transcripts was observed, which is in line with previously reported m6A-methylation of IncRNAs (Xie, S.-J. et al. Characterization of Long Non-coding RNAs Modified by m(6)A RNA Methylation in Skeletal Myogenesis. Frontiers in cell and developmental biology 9, 762669-762669 (2021). Wang, S. et al. Comprehensive Analysis of Long Noncoding RNA Modified by m(6)A Methylation in Oxidative and Glycolytic Skeletal Muscles. Int J Mol Sci 23 (2022)) and these m6A peaks were highly enriched in exons, with a higher density on the first and the last exons and a lower density on internal exons. In contrast, in m6A-methylated mRNAs more than one m6A- peak per transcript was found in the majority of transcripts, with only 44% of m6A- methylated mRNA transcripts were methylated in a single position. Moreover, in protein-coding transcripts, m6A peaks were primarily enriched in 3' UTRs and if methylation occurred in exons, a higher density on the internal exons was observed. Finally, a non-canonical consensus motif was identified where IncRNA m6A peaks occurred (i.e. , the sequence [TTCAGATGA]).

[166] Taken together, 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.

[167] m6A-methylated IncRNA transcripts are enriched during cardiomyocyte specification.

[168] Next, the relationship between transcriptomic and epitranscriptomic changes was analyzed. Each hiPSC-differentiated cellular stage was compared with its predecessor (d3 versus dO, d10 versus d3 and d25 versus d10), m6A modifications were correlated with transcript abundance of the IncRNAs and the correlations as four- quadrant scatter plots were visualized (Data not shown). Interestingly, the highest number of differentially m6A-methylated and differentially-expressed IncRNA transcripts were obtained when comparing stages d10 and d3, which corresponds to the stage of cardiomyocyte lineage specification. Specifically, among the quadrant with hypermethylated and upregulated IncRNA transcripts, which occupied the main portion of the plot, we found IncRNA H19, previously demonstrated as 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)).

Likewise, 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)). Interestingly, MIR22HG IncRNA has been recently described as a regulator of skeletal muscle differentiation and proliferation by producing miR-22-3p that targets Histone deacetylase 4 (HDAC4), therefore promoting the activity of MEF2C transcription factors and its downstream gene program (Li, R. et al. Long noncoding RNA Mir22hg-derived miR-22-3p promotes skeletal muscle differentiation and regeneration by inhibiting HDAC4. Molecular Therapy - Nucleic Acids 24, 200-211 (2021)). However, the involvement of IncRNA MIR22HG in cardiac muscle differentiation remains unknown. Additionally, IncRNA HELPPAR (HELLP syndrome associated IncRNA), previously studied in pregnancy-associated diseases, also appeared in our analysis. I ntriguingly, apart from IncRNA H19, all the other differentially m6A-methylated and differentially-expressed IncRNAs in the study, such as TIBILA and KC6 transcripts, remain functionally undescribed in human cardiac specification or have not been described yet. These findings indicate that exploring m6A profiles in human cardiac lineage specification revealed new regulatory ncRNAs that function in cardiovascular development.

[169] Discovery of IncRNA Gata4 REgulator ENhancer (GREEN, or SEQ ID NO: 1).

[170] Further inspection of the Hyper-up quadrant revealed an as of yet undescribed IncRNA annotated as C8orf49 or ENST00000625198 in H. sapiens genome assembly GRCh38/hg38. Within the sequence the presence of the identified m6A motif was confirmed. 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). In silico investigation of the corresponding genomic region across mammalian species highlighted positional synteny and high sequence conservation only in primate species and, surprisingly, the complete absence of IncRNA C8orf49 orthologs in other mammalian species such as rodents or even large animal species (Table 1 , below).

[171] Table 1. hGREEN sequence homology in Primates

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. Because the transcription factor 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. The Genotype-Tissue Expression (GTEx) pilot analysis: multitissue gene regulation in humans. Science 348, 648-660 (2015)). There was a strong linear correlation between GATA4 and IncRNA GREEN expression, while NEIL2 expression is only moderately correlated to either GATA4 or IncRNA GREEN expression (Fig.2b). Moreover, chamber-specific expression of these genes was evaluated in the heart using the EvoACTG database (Gandhi, S. et al. Evolutionarily conserved transcriptional landscape of the heart defining the chamber specific physiology. Genomics 113, 3782-3792 (2021)) and it was observed an overlap in the right atrium, left atrium and left ventricle (Data not shown). On a subcellular level, IncRNA GREEN localized predominantly in the nuclear fraction, consistently with its putative transcriptional regulatory role (Data not shown). In silico analysis of the secondary structure of IncRNA GREEN based on the Minimum Free Energy (MFE) prediction resulted in negative free energy (-639.79 kcal/mol) indicating a stably folded structure (data not shown). In conclusion, our results corroborate the hypothesis that m6A modification regulates the expression of IncRNA transcripts with putative function in early human cardiac lineage specification and discovered IncRNA GREEN (SEQ ID NO: 1) with cis-regulatory function on the cardiogenic transcription factor GATA4, which appears to be a de novo invention in primates.

[172] IncRNA GREEN is required for GATA4 expression, mesodermal commitment and cardiomyocyte differentiation.

[173] To explore the biological function of IncRNA GREEN and test whether it acts as a transcriptional regulator of GATA4, endogenous IncRNA GREEN was silenced in cardiac mesoderm (d3) with a “GapmeR” antisense oligonucleotide specifically designed to target the IncRNA (SEQ ID NO: 2). 48h post-transfection no cellular toxicity was observed and a robust and specific silencing of IncRNA GREEN was found. Interestingly, an equally robust downregulation of GATA4 expression was observed. NEIL2 expression was also affected but to a much lesser extent, confirming the hypothesis that IncRNA GREEN acts in cis as a transcriptional regulator of the neighboring gene GATA4 (Fig.3b). In line, the effect of IncRNA GREEN silencing was evaluated directly on the expression levels of validated downstream target genes of the transcription factor GATA4 as shown by GATA4 chromatin occupancy in their enhancer or promoter regions during heart development. It was observed that nearly all GATA4 target genes were substantially affected, including the cardiomyocyte sarcomeric components ACTN1 and DES, as well as the cardiogenic transcriptional regulators NKX2.5, TBX5, GATA6 and HAND2 (Fig.3c). Finally, to directly assess the contribution of IncRNA GREEN to mesodermal commitment and cardiomyocyte lineage specification, the expression of prototypical mesodermal markers EOMES, BRACHYURY and MESP1 was measured and it was observed a strong repression, indicating that mesodermal commitment was impaired by the absence of IncRNA GREEN (Fig.3c). Additionally, a very robust downregulation of the pacemaker markers HCN4 and KCNJ3 was observed, as well as a profound downregulation of the cardiomyocyte-specific marker genes TNNT2, MYH6. I ntriguingly, a significant upregulation of the atrial-specific cardiomyocyte marker NR2F2 was evident, suggesting that ventricular cardiomyocyte lineage specification was more specifically affected by silencing IncRNA GREEN (Fig.3c). Conclusively, the data show that m6A modification stabilizes the expression of the primate-specific IncRNA GREEN which is essential for proper human cardiomyocyte specification through cis-regulation of the canonical cardiogenic transcription factor GATA4.

[174] DISCUSSION

In this study, we demonstrated the importance of m6A modification in regulating a novel IncRNA that we termed “Gata4 REgulator Enhancer” or “GREEN” (SEQ ID NO: 1) for its cis-regulation of GATA4, which is required for human cardiomyocyte lineage specification. Recent evidence reported that IncRNAs can be m6A-methylated through human METTL16 enzyme, which was described as an essential modulator of embryonic development. In fact, METTL16 regulates MAT2A mRNA expression, which eventually affects SAM levels, and therefore the methylation capacity of the cells Pendleton, K.E. et al. The U6 snRNA m6A Methyltransferase METTL16 regulates SAM Synthetase Intron Retention. Cell 169, 824-835. e814 (2017)). Due to this function, METTL16 knockout in vivo resulted in murine embryonic lethality, as other authors have reported. Our results show that transcription and translation of METTL16 are active during hiPSC differentiation into cardiomyocytes, supporting the importance of METTL16 in cardiac development. We systematically profiled the IncRNA methylome in four hiPSC stages towards cardiomyocyte differentiation and demonstrated that IncRNA transcripts are the majority of ncRNAs that are m6A-methylated. Secondly, our m6A distribution analysis illustrated important topological differences between IncRNAs and mRNAs, with the vast majority of IncRNA transcripts single m6A methylated and with methylation peaks that were mainly located in exons and enriched within the first and the last exons. In contrast, most of protein-coding mRNA transcripts showed more than one single m6A modification, methylation peaks were mainly located in the in 3' UTRs, and methylation peaks were enriched in internal exons. Overall, these features reveal that coding and non-coding transcripts undergo differential m6A methylation likely with distinct downstream molecular ramifications.

[175] 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. Of note, 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.

[176] GATA4 gene encodes for the zing finger GATA4 TF that regulates cardiogenic signaling pathways associated with embryonic cardiac development and promotes myocardial specification. On a molecular level, 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. 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. Interestingly, forced expression of Gata4, Mef2c, and Tbx5 in mouse fibroblasts induced their reprogramming into functional cardiomyocytes (leda, M. et al. Direct Reprogramming of Fibroblasts into Functional Cardiomyocytes by Defined Factors. Cell 142, 375-386 (2010)). In addition, when re-expressed in sites of the injured heart, 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. J Cell Biochem 121 , 3438-3450 (2020)). Lack of GATA4 expression in mice is embryonically lethal at E8.5 due to the failure in the formation of the heart tube and ventral morphogenesis Molkentin, J.D., Lin, Q., Duncan, S.A. & Olson, E.N. Requirement of the transcription factor GATA4 for heart tube formation and ventral morphogenesis. Genes & development 11 , 1061-1072 (1997). Kuo, C.T. et al. GATA4 transcription factor is required for ventral morphogenesis and heart tube formation. Genes & development 11 , 1048-1060 (1997)). Moreover, 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)). In the adult heart, GATA4 is required for maintaining normal cardiac function, and, in response to pathological stress, is reactivated causing cardiac hypertrophic remodeling (Suzuki, Y.J. Cell signaling pathways for the regulation of GATA4 transcription factor: Implications for cell growth and apoptosis. Cell Signal 23, 1094- 1099 (2011). Nemer, G. & Nemer, M. Chapter 9.2 - GATA4 in Heart Development and Disease, in Heart Development and Regeneration, (eds. N. Rosenthal & R.P. Harvey) 599- 616 (Academic Press, Boston; 2010). Dirkx, E., da Costa Martins, P.A. & De Windt, L.J. Regulation of fetal gene expression in heart failure. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease 1832, 2414-2424 (2013). Bisping, E. et al. Gata4 is required for maintenance of postnatal cardiac function and protection from pressure overload-induced heart failure. Proceedings of the National Academy of Sciences 103, 14471-14476 (2006)). Park et al. suggested the presence of GATA4 regulatory regions, containing a CCAAT box, 250 bp from its transcriptional start site, but they claimed different GATA4 regulatory mechanisms in the right and left ventricles. In rat hypoxic pulmonary hypertension models, the DNA binding proteins CBF/NF-Y and Annexinl compete to bind the CCAAT box, respectively, to activate or repress GATA4 expression in the right ventricle. 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. Similarly, murine IncRNA Evxlas cis-regulates the transcription of its neighbor gene EVX1 to facilitate the mesendodermal differentiation of embryonic stem cells. In our study, computational analysis indicated that 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. Altogether, these results strongly indicate and demonstrate that IncRNA GREEN intervenes in the delicate gene regulatory network governing human cardiomyocyte specification by controlling GATA4 locus activation. Our finding covers the knowledge gap concerning the regulatory network underlying GATA4 actions between rodent models and humans, explaining why, so far, our community has struggled with fully understanding GATA4 transcriptional modulation.

[177] MATERIALS AND METHODS

[178] Human induced pluripotent stem cell (hiPSCs) maintenance

[179] Two human iPSCs lines were used in this study. Respectively, wildtype human ATCC-BXS0116 iPSCs were derived from a healthy non-Hispanic Caucasian female donor and purchased from ATCC (ATCC® ACS-1030™); wildtype human TC1133 iPSCs were derived from a Hispanic Caucasian male donor and kindly provided by Prof. Dr. WH. Zimmermann, Institute of Pharmacology (Gottingen, Germany), and used for MeRIP-Seq. The remaining experiments were performed using the ATCC^-1- BXS0116 iPSC line. Both lines were cultured and maintained in Essential 8™ Medium (Gibco™) in Matrigel-coated plates (Corning®). 80% confluent cells were passaged with Versene Solution 1X (Gibco™) and maintained in E8™ Medium supplemented with 5pM ROCK inhibitor (Stemolecule™ Y27632) on the first day after passaging.

[180] Directed hiPSCs differentiation into Cardiomyocytes

[181] 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. Cells were cultured in basal serum-free medium (BSFM), consisting of RPMI 1640 Medium, GlutaMAX™ Supplement (Gibco™) supplemented with 1% 100X sodium pyruvate (Invitrogen), 2% 50X B-27® Supplement (Gibco™), 200pM L-ascorbic acid 2 phosphate sesquimagnesium salt hydrate (Sigma). 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. Additionally, to enrich the hiPSC-CMs yield, 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).

[182] Total RNA isolation, reverse transcription, and quantitative real-time PCR

[183] Total RNA was isolated from each hiPSC-differentiated cellular stage (dO, d3, d10 and d25) using Trizol reagent and the Direct-zol™ RNA MiniPrep kit (both from Zymo Research) according to the manufacturer’s instructions. The RNA quality and concentration were assessed with NanoDrop 2000 (Thermo Fisher). 1 pg of total RNA was retrotranscribed into cDNA using M-MLV reverse transcriptase (Promega), RNasin Plus RNase inhibitor (Promega), dNTPs (Promega), oligo(dT) primers, and random hexamers (both from IDT). Differences in gene expression were analyzed via quantitative real-time PCR (qRT-PCR) on a BioRad iCycler (BioRad), using SYBR Green Supermix (BioRad). The fold change values were determined using the 2'^AACt method, normalizing the values to the human housekeeping gene L7. All experiments were performed in biological triplicates. Primer sequences used in the study are listed in Table 2 below:

Table 3. Other sequences mentioned in this description

[184] Western Blot analysis

[185] Cells were lysed in RIPA buffer (Sigma) supplemented with PhosSTOP/ Protease inhibitor cocktail (Roche Applied Science), incubated 20 min in ice, sonicated 30s on/20s off for 15 cycles, and centrifuged at 4°C at 15000g for 10 min. The protein concentrations were quantified by a BCA kit (Thermo-Fisher). Prior to SDS-PAGE separation and migration to PVDF membrane, proteins were denatured in 1X Leammli buffer, including 2% p-mercaptoethanol, for 5 min at 95 °C. Membranes were incubated at 4°C overnight with the following primary antibodies: METTL16 (1 :1000, Abeam - #ab186012), GAPDH (1 : 10000, Millipore - #MAB374), Histone H3 (1 :1000, Cell Signaling Technology - #9715s). The following secondary HRP conjugated antibodies were applied for 1 hour at room temperature: polyclonal swine anti-rabbit IgG-HRP (1 :2000, DAKO), polyclonal rabbit anti-goat IgG-HRP (1 :2000, DAKO - #P0399). After antibody incubations, membranes were washed three times in 0,05% PBS-Tween 20 and imaged using Extreme Sensitivity Chemiluminescence Substrate (PerkinElmer) and the LAS-3000 documentation system (FujiFilm, Life Science). Protein quantification was performed with Imaged, normalizing for loading control.

[186] Immunofluorescence staining

[187] To stain hiPSCs, 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 E8™ 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. Cells were then stained over night at 4°C for anti-human OCT4, Anti-Human SOX2, anti-human SSEA4 and anti-human TRA-1-60 primary antibodies diluted in blocking solution. Subsequently, cells were rinsed three times and incubated for 1 h at room temperature with Alexa Fluor® 555 goat anti-mouse lgG2a and Alexa Fluor® 488 donkey anti rat. Finally, cells were washed again three times and nuclei were stained with NucBlue™ Fixed Cell (DAPI) for 5 min. The grid of the 12-well chamber was removed, and the cells were prepared for visualization with Vectashield mounting medium (VWR). All images were acquired using Leica DMI3000 B inverted fluorescence microscope with 20x objectives. Imaged Fiji was used to analyze the acquired images.

[188] Measurement and quantification of hiPSC-CM contractility

[189] 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.

[190] scRNA-seq and data analysis

[191] ScRNA-seq data was acquired from a dataset generated on our lab. In brief, 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.

[192] m⁶A dot-blot assay

[193] Total 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). 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).

[194] MeRIP-Seq and data analysis

[195] Total 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).

[196] To determine the methylation status of ncRNAs we performed m6A RNA immunoprecipitation followed by high-throughput sequencing (MeRIP-Seq) on total RNA (rRNA removal). MeRIP-Seq was performed by LC Bio-Technology CO, according to published procedure with some modifications (see Dominissini D, et al. Transcriptome-wide mapping of N(6)-methyladenosine by m(6)A-seq based on immunocapturing and massively parallel sequencing. Nat Protoc. 2013;8(1):176-189; and Meyer KD, et al. Comprehensive analysis of mRNA methylation reveals enrichment in 3' UTRs and near stop codons. Cell. 2012;149(7):1635-1646). Briefly, more than 25pg of total RNA was used and depleted of ribosomal RNA using Epicentre Ribo-Zero Gold Kit (Illumina) according to the manufacturer's instructions. Following purification, the 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). The mixture was then incubated with protein-A beads and eluted in elution buffer (1 x |p buffer and 6.7mM m6A), followed by ethanol precipitation. Eluted m6A-containing fragments (IP) and untreated input control fragments were converted to final cDNA library in accordance with a strand-specific library preparation by dllTP method. The average insert size for the paired-end libraries was ~100±50bp. Paired-end 2x150bp sequencing was performed on an Illumina Novaseq 6000 platform following the vendor's recommended protocol.

[197] MeRIP-seq and RNA-seq data analysis was carried out. Cutadapt and in-house Perl scripts were used to trim raw reads and remove adaptor contamination, low quality and undetermined bases (Martin M. Cutadapt removes adapter sequences from high- throughput sequencing reads. 2011. 2011 ;17(1):3). FastQC software was used for quality control checks (FastQC. ln:2015). Reads were mapped to H. sapiens genome (Version: v101) by Bowtie with default parameters (Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nature Methods. 2012;9(4):357-359). Mapped reads of IP and input libraries were used for subsequent peak analysis via exomePeak R and the P value < 0.05 was considered to be a peak (Meng J., et al. A protocol for RNA methylation differential analysis with MeRIP-Seq data and exomePeak R/Bioconductor package. Methods (San Diego, Calif). 2014;69(3):274-281). Called peaks were annotated by intersection with gene architecture using ChlPseeker (Yu G, Wang L-G, He Q-Y. ChlPseeker: an R/Bioconductor package for ChIP peak annotation, comparison and visualization. Bioinformatics. 2015;31 (14):2382-2383). Finally, transcript expression levels were calculated as FPKM (FPKM= [total_exon_fragments/mapped_reads(millions)xexon_length(kB)]) using StringTie (Pertea M, et al. StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. Nature Biotechnology. 2015;33(3):290-295.). The differentially expressed transcripts were selected with Iog2 fold change = |1 | and P value < 0.05 by edgeR, and filtered per transcript biotype (Robinson MD, et al. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics. 2010;26(1):139-140.). In this analysis we considered as 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.

[198] Motif analysis was performed using the findMotif pipeline within the Homer software (v4.11). As input the unique differential expressed IncRNAs and default parameters were used to generate motifs. [199] Pearson correlation analysis in human tissues

[200] 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).

[201] GREEN IncRNA secondary structure prediction

[202] To predict GREEN IncRNA secondary structure we used the RNAFold webserver (htp://rna.tbi.univie.ac.at/) using GREEN genomic sequence as input (Gruber AR, et al. The Vienna RNA websuite. Nucleic Acids Res. 2008;36(Web Server issue):W70-74).

[203] GREEN IncRNA, GATA4 and NEIL2 cardiac chamber-specificity

[204] 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).

[205] Subcellular RNA fractionation

[206] 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). Upon centrifugation, cells were resuspended in cold hypotonic buffer (20mM Tris-HCI pH 7.4, 10mM KCI, 2mM MgCI2, 1mM EGTA, 0.5mM DTT, 0.5mM PMSF) and incubated on ice for 3 min. Cellular membranes were disrupted in 0.1% NP-40 followed by 3 min incubation on ice. Subcellular fractions were separated by centrifugation at 4°C at 1000g for 5 min: nuclei (pellet) and cytoplasm (supernatant). [207] The nuclear fraction (pellet) was rinsed with isotonic buffer (20mM Tris-HCI pH 7.4, 150mM KCI, 2mM MgCI2, 1mM EGTA, 0.5mM DTT, 0.5mM PMSF) containing NP- 40, incubated in ice, and centrifuged at 4 °C at 1000g for 3 min. The cytosolic fraction (supernatant) was subjected to high-speed centrifugation to remove pellet debris. Finally, Trizol reagent was added to the fractions and RNA was extracted as described above.

[208] Cellular transfection

[209] 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.

[210] Statistics and reproducibility

[211] Results shown for images or blots were repeated independently at least once with similar results. Data are presented as the mean ± standard error of the mean (SEM). Statistical methods for bioinformatics analyses are described above. All other statistical analyses were performed with GraphPad Prism 8 and included ordinary oneway ANOVA followed by Tukey’s multiple comparison tests when group differences were detected at 5% significance level, and unpaired t-test when comparing two experimental groups. Results were considered statistically significant when the P value was < 0.05 (*P < 0.05; **P < 0.01 ; ***P < 0.001 ; ****P < 0.0001 ; ns P > 0.05).

[212] Having now fully described this invention, it will be appreciated by those skilled in the art that the same can be performed within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation.

[213] Reference to known method steps, conventional methods steps, known methods or conventional methods is not in any way an admission that any aspect, description, or embodiment of the present invention is disclosed, taught, or suggested in the relevant art.

Claims

1. An isolated ribonucleic acid molecule comprising or consisting in a nucleic acid sequence at least 80 % identical to SEQ ID NO: 1.

2. The isolated ribonucleic acid molecule according to claim 1 , which comprises one or more N6-methyladenosine residue(s).

3. 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 any one of claims 1-2.

4. The isolated interference nucleic acid molecule, or nucleic acid-binding nucleic acid molecule, according to claim 3, which is selected from one or more of an antisense nucleic acid oligonucleotide (ASO), a small interfering RNA (siRNA), a small hairpin RNA (shRNA), and a microRNA (miRNA).

5. The isolated interference nucleic acid molecule, or nucleic acid-binding nucleic acid molecule, according to any one of claims 3-4, which is an antisense nucleic acid oligonucleotide (ASO), preferably comprising or consisting in a sequence at least 80 % identical to SEQ ID NO: 2.

6. The isolated interference nucleic acid molecule according to claim 5, which is an antisense nucleic acid oligonucleotide (ASO) comprising or consisting in a sequence at least 82 % identical to SEQ ID NO: 2, preferably at least 85 %, at least 90 %, at least 95 %, or 100 % identical to SEQ ID NO: 2.

7. A nucleic acid vector comprising: (a) the sequence of the isolated ribonucleic acid molecule as defined in any one of claims 1-2, 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 in any one of claims 3-6.

8. A host isolated mammalian cell, preferably a human cell, comprising the isolated ribonucleic acid molecule as defined in any one of claims 1-2, and/or the interference nucleic acid molecule or nucleic acid-binding nucleic acid molecule as defined in any one of claims 3-6, and/or the nucleic acid vector as defined in claim 7.

9. A pharmaceutical 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 in any one of claims 1-2, or a therapeutically effective amount of an isolated interference nucleic acid molecule or nucleic acid-binding nucleic acid molecule as defined in any one of claims 3-6, and/or a therapeutically effective amount of the nucleic acid vector as defined in claim 7, and/or a therapeutically effective amount of the isolated host cell as defined in claim 8.

10. An isolated ribonucleic acid molecule as defined in any one of claims 1-2, and/or an isolated interference nucleic acid molecule or nucleic acid-binding nucleic acid molecule as defined in any one of claims 3-6, and/or a nucleic acid vector as defined in claim 7, and/or an isolated host cell as defined in claim 8, and/or a pharmaceutical composition as defined in claim 9, for use as a medicament.

11. The isolated ribonucleic acid 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, for use according to claim 10, which is 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.

12. The isolated ribonucleic acid 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, for use according to claim 11 , wherein the disease is a cardiomyopathy selected from one or more of 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.

13. The isolated ribonucleic acid 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, for use according to claim 12, wherein the cardiomyopathy is hypertrophic cardiomyopathy, preferably ventricular hypertrophy and/or left ventricular remodeling.

14. The isolated ribonucleic acid 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, for use according to claim 12, wherein the cardiomyopathy is a myocardial infarction.

15. Use of an isolated ribonucleic acid molecule comprising or consisting in an nucleic acid sequence at least 80 % identical to SEQ ID NO: 1 , preferably in an isolated sample comprising mammal cells, preferably primate cells, as modulator of the activity of the transcription factor GATA4.

16. 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 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 with SEQ ID NO: 1 ; and (d) 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 phenotype of specialization higher than the first phenotype in (b).

//end