WO2011080261A1

WO2011080261A1 - Method for improved cardiomyogenic differentiation of pluripotent cells

Info

Publication number: WO2011080261A1
Application number: PCT/EP2010/070786
Authority: WO
Inventors: Daniel Aberdam; Matthieu Rouleau
Original assignee: INSERM (Institut National de la Santé et de la Recherche Médicale)
Priority date: 2009-12-28
Filing date: 2010-12-28
Publication date: 2011-07-07

Abstract

The invention relates to a method for obtaining a population of cardiomyocyte-lineage cells wherein said method comprises a step of culturing pluripotent cells, in particular human pluripotent cells in the presence of an inhibitor of ΔNp63 expression and/or activity.

Description

METHOD FOR IMPROVED CARDIOMYOGENIC DIFFERENTIATION

OF PLURIPOTENT CELLS

FIELD OF THE INVENTION:

The invention relates to a method for obtaining a population of cardiomyocyte- lineage cells wherein said method comprises a step of culturing pluripotent cells, in particular human pluripotent cells, in the presence of an inhibitor of ΔΝρ63 expression and/or activity. BACKGROUND OF THE INVENTION:

For several years, technologies in the field of regenerative medicine have focused on stem cells, as these cells have the capacity to differentiate into specialized cell types. Indeed, certain tissues or organs, such as heart tissue cannot regenerate alone or, at least, cannot regenerate efficiently, due to their very limited capacity of self-renewal.

Regenerative medicine involves transplanting cells of interest with the goal of repairing and regenerating a target tissue and/or target organ. Therefore, intensive efforts have been devoted to the development of methods for cardiac repair based on cell transplantation and more particularly to the development of methods for obtaining cardiomyocytes namely from pluripotent or multipotent stem cells such as embryonic or adult stem cells.

Therefore, in order to increase cardiomyogenic induction, different factors were used during the differentiation process of both mouse and human pluripotent cells such as embryonic stem cells. These agents include growth factors/hormones (e.g. transforming growth factor-β, vascular endothelial growth factor and bone morphogenetic factor-2) and chemical reagents (e.g. nitric oxide and 5-azacytidine). However, although preliminaries studies are encouraging, further studies are required, particularly with human pluripotent cells, to optimize conditions for maximal yield of cardiomyocytes. Indeed, enrichment strategies of pluripotent cells-derived cardiomyocytes prior to transplantation are highly desirable (Zhang et al. 2008).

For instance, international patent application WO 2006/066320 describes a method for enhancing cardiomyocyte differentiation of human embryonic stem cells comprising culturing said cells in the presence of ascorbic acid or a derivative or functional equivalent thereof. p63 is a transcription factor that plays an important role in skin epidermal development and differentiation. The p63 gene encodes for two major protein isoforms, one containing an amino -terminal trans-activation domain (TAp63) and one lacking this domain (ΔΝρ63). Both the TAp63 and ΔΝρ63 transcripts are also alternatively spliced at the 3' end producing proteins with unique C-termini that are designated as α, β and γ isoforms. Recent research has suggested that ΔΝρ63 is the predominant isoform expressed and active in keratinocytes. ΔΝρ63 was thus shown to control epidermal commitment (Medawar et al. 2008). Moreover, international patent application WO 2008/120201 discloses that ΔΝρ63 is useful for differentiating ectodermal progenitor cells into keratinocytes.

Therefore, there is a strong need for a method for the cardiomyogenic differentiation of pluripotent cells in order to obtain cardiomyocyte- lineage cells with a high-efficiency for use in the treatment of a cardiac pathology and/or cardiac reconstruction or regeneration. SUMMARY OF THE INVENTION:

The invention relates to a method for obtaining a population of cardiomyocyte- lineage cells wherein said method comprises a step of culturing pluripotent cells in the presence of an inhibitor of ΔΝρ63 expression and/or activity. The invention also relates to the use of an inhibitor of ΔΝρ63 expression and/or activity for increasing the cardiomyogenic differentiation of pluripotent cells.

The invention also relates to a population of cardiomyocyte- lineage cells obtainable by a method as defined above as well as pharmaceutical composition thereof.

The invention further relates to a method of screening for compounds having a cardioprotective or cardiotoxic effect wherein said method comprises the steps of:

a) culturing a population of cardiomyocyte-lineage cells obtainable by a method of the invention in the presence of a test compound;

b) comparing the survival of the cells of step a) to that of a population of cardiomyocyte-lineage cells as defined above cultured in the absence of said test compound; wherein a survival of the cells cultured in the presence of said test compound is higher to the survival of the cells cultured in the absence of said test compound is indicative of a cardioprotective effect of said test compound. DETAILED DESCRIPTION OF THE INVENTION:

The inventors have demonstrated that an inhibitor of ΔΝρ63 expression significantly improves the cardiomyogenic differentiation of pluripotent cells. Definitions:

Throughout the specification, several terms are employed and are defined in the following paragraphs.

As used herein, the term "cardiomyocyte- lineage cells" refers generally to both cardiomyocyte precursor cells and mature cardiomyocytes. Reference to cardiomyocyte- lineage cells, precursors, or cardiomyocytes in this disclosure can be taken to apply equally to cells at any stage of cardiomyocyte ontogeny without restriction, as defined above, unless otherwise specified. The main phenotypic markers of cardiomyocyte-lineage cells include cardiomyogenic markers such as GATA-4, Nkx2.5, Tbx5, Mlc2v, cardiac troponin-T (cTnT), ventricular myosin, desmin, atrial natriuretic peptide and sarcomeric alpha (a) actinin.

As used herein, the term "cardiomyocytes" refers to fully differentiated, post-mitotic cells of the cardiomyogenic lineage. Moreover, cardiomyocytes have specific morphologic, structural and functional properties since such cells are namely beating cells.

As used herein, the term "marker" refers to a protein, glycoprotein or other molecule expressed on the surface of a cell or inside a cell, and which is useful for identifying the cell {e.g., identify the type of cell). A marker can generally be detected by conventional methods. Specific non-limiting examples of methods that may be used for the detection of a cell surface marker are immunohistochemistry (IHC), fluorescence activated cell sorting (FACS) and enzymatic analysis.

As used herein, the terms "ΔΝρ63" or "DeltaNp63" are used interchangeably and have their general meaning in the art. They refer to a specific N-terminal isoform of p63 in which the transactivation domain is deleted. Indeed, the TP63 gene, a member of the TP53 gene family, encodes several isoforms with (TAp63) or without (ΔΝρ63) a typical transactivation domain in N-terminal by using two different promoters. Furthermore, alternative splicing within the 3' end results in several C-terminus isoforms called α, β, γ for ΔΝρ63. ΔΝρ63 suppresses transcriptional activity of p53 and/or TAp63 in a dominant-negative manner. The naturally occurring human ANp63protein has a nucleotidic sequence shown in Genbank Accession number NM 001114980.1. An "inhibitor of gene expression" refers to a natural or synthetic compound that has the biological effect of inhibiting or significantly reducing the expression of a gene. An "inhibitor of ΔΝρ63 expression" thus refers to a natural or synthetic compound that has the biological effect of inhibiting or significantly reducing the expression of the gene encoding for the ΔΝρ63 isoform. Preferably, to be considered as efficient inhibitor, a compound must reduce the expression of the ΔΝρ63 protein by at least 60%.

The term "inhibitor of ΔΝρ63 activity" as used herein refers to any compound, natural or synthetic, which results in a decreased activation of the ΔΝρ63 signaling pathway, which is the series of molecular signals generated as a consequence of ΔΝρ63 binding site in gene promoters of interest such p53.

Such a compound may be identified by screening compounds by using a test based on the use of an embryonic stem (ES) cell line in which the green fluorescent EGFP gene has been inserted into one allel of the ΔΝρ63 -specific exons. ES cells will be committed to epidermal differentiation and thus will become green. Therefore any compound that will inhibit ΔΝρ63 expression will significantly reduce EGFP production as detected by UV microscopy and FACS analysis.

In the context of the present invention, inhibitors of ΔΝρ63 activity are selective for ΔΝρ63 as compared with TAp63. By "selective" it is meant that the affinity of the inhibitor for ΔΝρ63 is at least 10-fold, preferably 25-fold, more preferably 100-fold, still preferably 200-fold higher than the affinity for the TAp63. Selectivity of an inhibitor of ΔΝρ63 activity may be assayed for instance by determining by screening the compounds on a home made microarray containing about 10 known p63-target genes (such as p21, MDM2, PERP, BMP7, GATA-3, ITGB4, LAMA3, c-ΕΒΡδ, IKKcc) and 20 non relevant genes (such genes are genes for example involved in the hematopoietic signaling pathways like GATA-1, CD34 and mesenchymal cells like CD73). The affinity of an inhibitor for ΔΝρ63 (or TAp63) may be quantified by measuring the activity of ΔΝρ63 (or TAp63) in the presence a range of concentrations of said inhibitor in order to establish a dose-response curve.

The term "small organic molecule" refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macro molecules (e. g., proteins and nucleic acids). Preferred small organic molecules range in size up to about 5 kDa, more preferably up to 2 kDa, and most preferably up to about 1 kDa.

In the context of the invention, the terms "treating" or "treatment", as used herein, refer to a method that is aimed at delaying or preventing the onset of a pathology, such as reversing, alleviating, inhibiting, slowing down or stopping the progression, aggravation or deterioration of the symptoms of a pathology, or bringing about improvements of the symptoms of a pathology, and/or curing a pathology.

As used herein, the term "subject" denotes a mammal, such as a rodent, a feline, a canine and a primate. Preferably, a subject according to the invention is a human.

Methods for obtaining a population of cardiomyocyte-lineage cells

from plunpotent cells

The invention provides a method for obtaining a population of cardiomyocyte-lineage cells wherein said method comprises a step of culturing pluripotent cells in the presence of an inhibitor of ΔΝρ63 expression and/or activity.

In one embodiment, the cardiomyocyte-lineage cells are cardiomyocytes. The term "pluripotent cells" as used herein refers to undifferentiated cells which are capable of differentiating into cells of all three embryonic germs layers (i.e. endoderm, ectoderm and mesoderm) Typically, pluripotent cells may express the following markers OCT-4, SOX2, Nanog, SSEA-3 and 4, TRA 1/81, see International Stem Cell Initiative recommendations, 2007.

In one embodiment, the pluripotent cells are human pluripotent cells.

In another embodiment, the pluripotent cells are non-human pluripotent cells, such as mouse cells.

In one embodiment, the pluripotent cells are stem cells.

Typically, said stem cells are embryonic stem cells.

In a preferred embodiment, the pluripotent cells are human embryonic stem cells (hES cells). According to an embodiment of the invention, hES cells may be selected from any hES cell lines. Examples of hES cell lines include but are not limited to, SA-01, VUB-01, HI (Thomson JA et al. 1998), and H9 (Amit M et al. 2000).

Alternatively, hES cells may be obtained according a method not involving embryo destruction as described in Chung et al. 2008 or in Revazova et al. 2008.

In one embodiment, the pluripotent cells are non-human embryonic stem cells, such a mouse stem cells.

In one embodiment, the pluripotent cells are induced pluripotent stem cells (iPS). The terms "induced pluripotent stem cells" or "iPS" refer to a type of pluripotent stem cell artificially derived from a non-pluripotent cell (e.g. an adult somatic cell). Human induced pluripotent stem cells are identical to human embryonic stem cells in the ability to form any adult cell, but are not derived from an embryo. Typically, a human induced pluripotent stem cell may be obtained through the induced expression of Oct3/4, Sox2, Klf , and c-Myc genes in any adult somatic cell (e.g. fibroblast). For example, human induced pluripotent stem cells may be obtained according to the protocol as described by Takahashi K. et al. 2007, by Yu et al. 2007 or else by any other protocol in which one or the other agents used for reprogramming cells in these original protocols are replaced by any gene or protein acting on or transferred to the somatic cells at the origin of the iPS lines. Basically, adult somatic cells are transfected with viral vectors, such as retroviruses, which comprises Oct3/4, Sox2, Klf4, and c-Myc genes.

Alternatively, hES cells or human iPS cells may be selected from master cell banks that may be constituted in a therapeutic purpose. In a preferred manner, hES cells or human iPS may be selected to avoid or limit immune rejection in a large segment of the human population. Typically hES cells or human iPS cells are HLA-homozygous for genes encoding major histocompatibility antigens A, B and DR, meaning that they have a simple genetic profile in the HLA repertory. The cells could serve to create a stem cell bank as a renewable source of cells that may be suitable for preparing human heart substitutes for use in cell therapy of pathologies associated with heart damage (e.g. heart failure, myocardial infarction and cardiac ischemia...).

Pluripotent cells may be cultured with an inhibitor of ΔΝρ63 expression and/or activity in any culture medium capable of promoting the growth and the differentiation of pluripotent cells into cardiomyocyte-lineage cells.

In one embodiment, pluripotent cells are cultured in a cardiomyogenic differentiation culture medium comprising factors inducing the differentiation of pluripotent cells into cardiomyocyte-lineage cells. Different cardiomyogenic differentiation culture media are known in the art and are described for example in Leschik et al. 2008, Mery et al, 2005, Czyz et al. 2001 and Braam et al. 2009.

In a preferred embodiment, the cardiomyogenic differentiation culture medium comprises the cardiogenic morphogen bone morphogenetic protein 2 (BMP2) and SU5402 (a FGF receptor inhibitor) as described in Leschik et al., 2008 and also in the international patent application WO 2009/112496. In another embodiment, pluripotent cells are cultured with an inhibitor of ΔΝρ63 expression and/or activity in a culture medium allowing spontaneous cardiomyogenic differentiation.

Preferred culture media formulations that will promote the growth and the differentiation of pluripotent cells into cardiomyocyte- lineage cells include chemically defined media (CDM). As used herein, the term "chemically defined media" (CDM) refers to a nutritive solution for culturing cells which contains only specified components, preferably components of known chemical structure. A chemically defined medium is a serum- free and feeder-free medium. As used herein, "serum-free" refers to a culture medium containing no added serum. As used herein, "feeder-free" refers to culture medium containing no added feeder cells. The term feeder-free encompasses, inter alia, situations where cardiomyocyte- lineage cells are passaged from a culture with feeders into a culture medium without added feeders even if some of the feeders from the first culture are present in the second culture. Thus, a chemically defined medium is devoided of components derived from non-human animals, such as Foetal Bovine Serum (FBS), Bovine Serum Albumin (BSA) and animal feeder cells such mouse feeder cells.

Suitable CDM include humanised Johansson and Wiles CDM. Such CDM, described in 21 is supplemented with insulin, transferrin and defined lipids to which was added polyvinyl alcohol (PVA) as substitute for Bovine Serum Albumin (BSA). Thus, "CDM-PVA" refers to a chemically defined medium comprising polyvinyl alcohol (PVA) instead of bovine or human serum albumin. Thus, an appropriate CDM according to the invention may consist of 50 % IMDM (Invitrogen, Cergy, France) and 50% F12 NUT MIX (Invitrogen), supplemented with 7 μg/ml of insulin (Roche, Sandhofer, Germany), 15 μg/ml of transferrin (Roche), 450 μΜ of monothioglycerol (Sigma- Aldrich, St Quentin, France) and 1 mg /ml of Polyvinyl Alcohol (PVA; Sigma).

The step of culturing pluripotent cells with an inhibitor of ΔΝρ63 expression and/or activity shall be carried out for the necessary time required for the production of cardiomyocyte-lineage cells. Typically, the culture of pluripotent cells with an inhibitor of ΔΝρ63 expression and/or activity shall be carried out for at least 5 days, preferably at least 7 days, even more preferably at least 10 days. If necessary, the culture medium may be renewed, partly or totally, at regular intervals. Typically, the culture medium may be replaced with fresh culture medium of the invention every other day for 10 days. In one aspect, an inhibitor of ΔΝρ63 expression is used.

According to this aspect, said inhibitor of ΔΝρ63 expression is selected from the group consisting of antisense R A or DNA molecules, small inhibitory R As (siR As), short hairpin RNAs (shRNAs) and ribozymes.

Inhibitors of ΔΝρ63 expression for use in the invention may be based on anti-sense oligonucleotide constructs. Anti-sense oligonucleotides, including anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of ΔΝρ63 mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of ΔΝρ63, and hence ΔΝρ63 activity, in a cell. For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence encoding ΔΝρ63 can be synthesized, e.g., by conventional phosphodiester techniques and administered e.g., by intravenous injection or infusion. Methods for using antisense techniques for specifically inhibiting gene expression of genes whose sequence is known are well known in the art (e.g. see Tuschl, T. et al. (1999); Hannon, GJ. (2002); U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732).

Small inhibitory RNAs (siRNAs) can also function as inhibitors of ΔΝρ63 gene expression for use in the invention. ΔΝρ63 gene expression can be reduced by contacting a subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that ΔΝρ63 gene expression is specifically inhibited (i.e. RNA interference or RNAi). Methods for selecting an appropriate dsRNA or dsRNA-encoding vector are well known in the art for genes whose sequence is known (e.g. see U.S. Pat. Nos. 6,573,099 and 6,506,559; and International Patent Publication Nos. WO 01/36646, WO 99/32619, and WO 01/68836).

In a preferred embodiment, the sequence of the mouse si-ANp63 RNA is represented by SEQ ID NO: 1.

In another preferred embodiment, the sequence of the human sh-ANp63 RNA is represented by SEQ ID NO: 2.

Ribozymes can also function as inhibitors of ΔΝρ63 gene expression for use in the invention. Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleo lytic cleavage. Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleo lytic cleavage of ΔΝρ63 mRNA sequences are thereby useful within the scope of the invention. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences, GUA, GUU, and GUC. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable. The suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using, e.g., ribonuclease protection assays.

Both antisense oligonucleotides and ribozymes useful as inhibitors of ΔΝρ63 gene expression can be prepared by known methods. These include techniques for chemical synthesis such as, e.g., solid phase phosphoramadite chemical synthesis. Alternatively, anti- sense RNA molecules can be generated by in vitro or in vivo transcription of DNA sequences encoding the RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Various modifications to the oligonucleotides of the invention can be introduced as a means of increasing intracellular stability and half-life. Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2'-0-methyl rather than phosphodiesterase linkages within the oligonucleotide backbone.

Antisense oligonucleotides siRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide siRNA or ribozyme nucleic acid to the cells and preferably cells expressing ΔΝρ63. Preferably, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide siRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and R A virus such as a retrovirus. One can readily employ other vectors not named but known to the art.

Preferred viral vectors are based on non-cytopathic eukaryotic viruses in which nonessential genes have been replaced with the gene of interest. Non-cytopathic viruses include retroviruses (e.g., lentivirus), the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA. Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell lined with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with viral particles) are provided in Kriegler, 1990 and in Murry, 1991).

Preferred viruses for certain applications are the adeno-viruses and adeno-associated viruses, which are double-stranded DNA viruses that have already been approved for human use in gene therapy. The adeno-associated virus can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species. It further has advantages such as, heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hemopoietic cells; and lack of superinfection inhibition thus allowing multiple series of transductions. Reportedly, the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection. In addition, wild-type adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event. The adeno- associated virus can also function in an extrachromosomal fashion.

Other vectors include plasmid vectors. Plasmid vectors have been extensively described in the art and are well known to those of skill in the art (e.g. see Sambrook et al., 1989). In the last few years, plasmid vectors have been used as DNA vaccines for delivering antigen-encoding genes to cells in vivo. They are particularly advantageous for this because they do not have the same safety concerns as with many of the viral vectors. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operatively encoded within the plasmid. Some commonly used plasmids include pBR322, pUC18, pUC19, pRC/CMV, SV40, and pBlueScript. Other plasmids are well known to those of ordinary skill in the art. Additionally, plasmids may be custom designed using restriction enzymes and ligation reactions to remove and add specific fragments of DNA. Plasmids may be delivered by a variety of parenteral, mucosal and topical routes. For example, the DNA plasmid can be injected by intramuscular, intradermal, subcutaneous, or other routes. It may also be administered by intranasal sprays or drops, rectal suppository and orally. It may also be administered into the epidermis or a mucosal surface using a gene-gun. The plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate and microencapsulation.

In another aspect, an inhibitor of ΔΝρ63 activity is used.

According to this aspect, said inhibitor of ΔΝρ63 activity is selected from the group consisting of small organic molecules, partial or complete ΔΝρ63 neutralizing antibodies or antibody fragments, and aptamers.

In one embodiment, the inhibitor of ΔΝρ63 activity may be a low molecular weight antagonist, e. g. a small organic molecule.

In a preferred embodiment, the inhibitor of ΔΝρ63 activity is the Janus kinase 2 (JAK2)/STAT3 inhibitor known as Tyrphostin AG490 or (E)-2-Cyano-3-(3,4- dihydrophenyl)-N-(phenylmethyl)-2-propenamide. Said Tyrphostin AG490 may be made by methods known in the art, for example, as described in the international patent application WO 98/06391. Briefly, Tyrphostin AG490 may be synthesized by Knoevenagel condensation of the appropriate benzaldehyde with malononitrile, the appropriate substituted amide, or other appropriate Knoevenagel condensation partner. In another embodiment, the inhibitor of ΔΝρ63 activity consists in an antibody (the term including antibody fragment) able to block ΔΝρ63 activity.

Antibodies directed against ΔΝρ63 may be raised according to known methods by administering the appropriate antigen or epitope to a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others. Various adjuvants known in the art can be used to enhance antibody production. Although antibodies useful in practicing the invention can be polyclonal, monoclonal antibodies are preferred. Monoclonal antibodies against ΔΝρ63 can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique originally described by Ko filer and Milstein (1975); the human B-cell hybridoma technique (Cote et al., 1983); and the EBV-hybridoma technique (Cole et al. 1985). Alternatively, techniques described for the production of single chain antibodies (see, e.g., U.S. Pat. No. 4,946,778) may be adapted to produce ΔΝρ63 single chain antibodies. Inhibitors of ΔΝρ63 activity useful in practicing the invention also include anti-ANp63 antibody fragments including but not limited to F(ab')₂ fragments, which may be generated by pepsin digestion of an intact antibody molecule, and Fab fragments, which may be generated by reducing the disulfide bridges of the F(ab')₂ fragments. Alternatively, Fab and/or scFv expression libraries may be constructed to allow rapid identification of fragments having the desired specificity to ΔΝρ63.

In another embodiment, the inhibitor of ΔΝρ63 activity is an aptamer directed against

ΔΝρ63. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena S.D., 1999. Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as Thioredoxin A of E. coli that are selected from combinatorial libraries by two hybrid methods (Colas et al., 1996). After raising aptamers directed against the ΔΝρ63 as above described, the skilled man in the art can easily select those blocking ΔΝρ63 activation. In another aspect, the invention relates to the use of an inhibitor of ΔΝρ63 expression and/or activity for increasing the cardiomyogenic differentiation of pluripotent cells.

Populations of cardiomyocyte-linease cells obtained according to a method of the invention and pharmaceuticals compositions thereof: In another aspect, the invention also relates to a population of cardiomyocyte-lineage cells obtainable by a method as defined above.

Advantageously, said population of cardiomyocyte-lineage cells is homogenous, i.e. it is not necessary to perform any sorting or selection to isolate the cardiomyocyte-lineage cells from other contaminating cells.

Typically, the population of cardiomyocyte-lineage cells according to the invention has a purity of at least 15%, preferably 20%, even more preferably 30%>.

The population of cardiomyocyte-lineage cells according to the invention may have a purity of at least 95%, preferably 99%, even more preferably 100%.

In one embodiment, the cardiomyocyte-lineage cells are cardiomyocytes.

The invention also provides a pharmaceutical composition comprising the population of cardiomyocyte-lineage cells according to the invention. The pharmaceutical composition may generally include one or more pharmaceutically acceptable and/or approved carriers, additives, antibiotics, preservatives, adjuvants, diluents and/or stabilizers. Such auxiliary substances can be water, saline, glycerol, ethanol, wetting or emulsifying agents, pH buffering substances, or the like. Suitable carriers are typically large, slowly metabolized molecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates, or the like. This pharmaceutical composition can contain additional additives such as mannitol, dextran, sugar, glycine, lactose or polyvinylpyrrolidone or other additives such as antioxidants or inert gas, stabilizers or recombinant proteins (e. g. human serum albumin) suitable for in vivo administration. Preferably, the pharmaceutical composition is stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi through the use of, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. Pharmaceutical compositions of the invention can be prepared by incorporating cardiomyocytes as described herein in a carrier or diluent and, as required, other ingredients enumerated above, followed by filtered sterilization.

As used herein, the term "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Screening methods:

Another aspect of the invention relates to a method for screening compounds having a cardioprotective or cardiotoxic effect wherein said method comprises the steps of:

a) culturing a population of cardiomyocyte- lineage cells of the invention in the presence of a test compound;

b) comparing the survival of the cells of step a) to that of a population of cardiomyocyte-lineage cells as defined above cultured in the absence of said test compound, wherein a survival of the cells cultured in the presence of said test compound is higher to the survival of the cells cultured in the absence of said test compound is indicative of a cardioprotective effect of said test compound.

Therapeutic methods and uses:

Yet another aspect of the invention relates to a population of cardiomyocyte-lineage cells of the invention for use in the treatment of a cardiac pathology and/or cardiac regeneration.

The invention also relates to a method for treating a cardiac pathology and/or cardiac regeneration comprising the step of administering a pharmaceutically effective amount of a population of cardiomyocyte-lineage cells of the invention to a patient in need thereof.

As used herein, the term "pharmaceutically effective amount" refers to any amount of cardiomyocytes according to the invention (or a population thereof or a pharmaceutical composition thereof) that is sufficient to achieve the intended purpose.

Effective dosages and administration regimens can be readily determined by good medical practice based on the nature of the pathology of the subject, and will depend on a number of factors including, but not limited to, the extent of the symptoms of the pathology and extent of damage or degeneration of the tissue or organ of interest, and characteristics of the subject (e.g., age, body weight, gender, general health, and the like).

For therapy, cardiomyocytes and pharmaceutical compositions according to the invention may be administered through intracardiac route (e.g., epicardial or intramyocardial). The dose and the number of administrations can be optimized by those skilled in the art in a known manner. Methods of administering the cardiomyocytes and pharmaceutical compositions of the invention to subjects, particularly human subjects include injection or implantation of the cells into target sites in the subjects, the cells of the invention can be inserted into a delivery device which facilitates introduction by, injection or implantation, of the cells into the subjects. Such delivery devices include tubes, e.g., catheters, for injecting cells and fluids into the body of a recipient subject. In a preferred embodiment, the tubes additionally have a needle, e.g., a syringe, through which the cells of the invention can be introduced into the subject at a desired location. The cardiomyocytes of the invention can be inserted into such a delivery device, e.g., a syringe, in different forms. For example, the cardiomyocytes can be suspended in a solution or embedded in a support matrix when contained in such a delivery device. As used herein, the term "solution" includes a carrier or diluent in which the cardiomyocytes of the invention remain viable. Carriers and diluents which can be used with this aspect of the invention include saline, aqueous buffer solutions, solvents and/or dispersion media. The use of such carriers and diluents is well known in the art. The solution is preferably sterile and fluid to the extent that easy syringability exists. For transplanting, cardiomyocytes is drawn up into a syringe and administrated to anesthetized transplantation recipients. Multiple injections may be made using this procedure.

As used herein, the terms "cardiac pathology" or "cardiac dysfunction" are used interchangeably and refer to any impairment in the heart's pumping function. This includes, for example, impairments in contractility, impairments in ability to relax (sometimes referred to as diastolic dysfunction), abnormal or improper functioning of the heart's valves, diseases of the heart muscle (sometimes referred to as cardiomyopathies), diseases such as angina pectoris and myocardial ischemia and infarction characterized by inadequate blood supply to the heart muscle, infiltrative diseases such as amyloidosis and hemochromatosis, global or regional hypertrophy (such as may occur in some kinds of cardiomyopathy or systemic hypertension), and abnormal communications between chambers of the heart.

As used herein, the term "cardiomyopathy" refers to any disease or dysfunction of the myocardium (heart muscle) in which the heart is abnormally enlarged, thickened and/or stiffened. As a result, the heart muscle's ability to pump blood is usually weakened. The etiology of the disease or disorder may be, for example, inflammatory, metabolic, toxic, infiltrative, fibroplastic, hematological, genetic, or unknown in origin. There are two general types of cardiomyopathies: ischemic (resulting from a lack of oxygen) and non- ischemic.

Ischemic cardiomyopathy is a chronic disorder caused by coronary artery disease (a disease in which there is atherosclerotic narrowing or occlusion of the coronary arteries on the surface of the heart). Coronary artery disease often leads to episodes of cardiac ischemia, in which the heart muscle is not supplied with enough oxygen-rich blood.

Non-ischemic cardiomyopathy is generally classified into three groups based primarily on clinical and pathological characteristics: dilated cardiomyopathy, hypertrophic cardiomyopathy and restrictive and infiltrative cardiomyopathy. In one embodiment, the cardiac pathology is selected from the group consisting of congestive heart failure, myocardial infarction, cardiac ischemia, myocarditis and arythmia.

In another embodiment, the cardiac pathology is a genetic disease such as Duchenne muscular dystrophy and Emery Dreiffuss dilated cardiomyopathy.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention. FIGURES:

Figure 1: ES cells were trans fected, during the first 2 days of differentiation into embryonic bodies, with control siR A or siR A specific for the TA- (Tf si-TAp63) or the ΔΝ- (Tf si-ANp63) p63 isoforms. At day 9 of differentiation, cells were collected, counted, specifically stained for expression of the Troponin T sarcomeric protein and analyzed by FACS. The graph represents the number of Troponin T-positive cells recovered from the different indicated culture conditions.

Figure 2: Human ES cells (line H9) have been stably infected with a lentivirus expressing a shRNA raised against the ΔΝρ63 transcript. The resulting cell lines are called H9shDN and the control cell lines (in which an empty lentivirus has been inserted) are called H9shCtl. (A) To confirm the stable expression of the shRNAs, the cell lines have been transiently trans fected with a DNp63 -expressing contruct. Protein extracts were collected after 4 days and analysed by Western blot using the p63-specific polyclonal antibody (4A4, santa cruz) at a dilution of 1 :200. Lane 1 : DNp63 transfection on H9 cells; lane 2: DNp63 transfection for 1 day on H9shDN; lane 3: DNp63 transfection for 1 day on H9shCtl; lane 4: DNp63 transfection for 2 days on H9shCtl; lane 5: DNp63 transfection for 2 days on H9shDN ane 6:_DNp63 transfection for 3 days on H9shDN. (B) H9shDN and H9shCtl cell lines were induced to differentiate into the cardiac fate as described (Leschik et al. 2008). At day 8, the resulting embryoid bodies (EB) were visualized under light microscope to monitor the number of beating area. The graph illustrates the results of three independent experiments.

EXAMPLES:

Material & Methods

EXEMPLE 1: Cardiomyogenic differentiation of mouse pluripotent cells: Derivation of mouse ES cells in cardiomyocytes: The mouse CGR8 ES cell line used in this study and the culture condition used have been described previously (Medawar et al. 2008). Cardiac differentiation of ES cells was induced as previously reported (Mery et al. 2005). Briefly, hanging drops containing 500 mouse ES cells in 20 μΐ of medium (+BMP2) are generated at day 0. At day 2, embryonic bodies (EBs) formed from aggregation of cells in hanging drops are put in suspension for 3 additional days. EBs are then put to adhere until analysis which is performed between day 8 and day 12.

siRNA transfection: ES cells were transfected into hanging drops, at day 0 of differentiation, with 20nM of siRNA in RNAi Max lipofectamine (Invitrogen) transfectant reagent.

Number of beating area: The number of beating area is counted between day 10 and day 12 (expressed as number of beating area/EB).

FACS analysis: The ES cells were stained (intracellular staining after dissociation and permeabilization) with mouse primary anti-troponinT (clone Ab-1, MS-295-PO Labvision), mouse anti-ptubulin (clone Tub 2-1, T4026, Sigma Aldrich). Mouse isotype control mAb (PharMingen) was used to set the background level of fluorescence. FITC-coupled Rabbit anti-mouse (Dakko A/S, country) or Tri-color-coupled goat anti-mouse (M35006, Caltag Laboratories) secondary antibodies were added. Samples were analyzed with CellQuest or DIVA BD's softwares on FACScan or FACScanto cytometers (Becton Dickinson).

EXEMPLE 2: Cardiomyogenic differentiation of human pluripotent cells:

Derivation of ItES cells in cardiomyocytes: hES cells stably expressing a sh-ANp63 construct were produced through lentiviral infection. For the hES cells, cardiomyocyte differentiation was performed according to a published protocol (Leschik et al. 2008).

Results

On murine ES cells: Specific inactivation of ΔΝρ63 enhanced slightly the number of beating area but significantly the size of the beating area (not shown) as illustrated by the increased percentage (Table 1) and number of Troponin-T-positive cells (Figure 1). Table 1: Increased differentiation of mouse ES cells in cardiomyocytes upon ΔΝρ63- specific siR A transfection.

Mouse ES cells transfected

with specific siRNA si-ctrl si-TAp63 si-ANp63

Number beating area/ EB

1.5 ± 0.1 0.3 ± 0.3 2.0 ± 0.6

(mean ± sd)

% Troponin-T-positive cells 14.7 ± 1.2 5.1 ± 0.5 19.5 ± 1.2 On human ES cells: Human ES cells (line H9) have been stably infected with a lentivirus expressing a shRNA raised against the ΔΝρ63 transcript. The resulting cell lines are called H9shDN and the control cell lines (in which an empty lentivirus has been inserted) are called H9shCtl. The efficiency of the shDNp63 lentivirus has been confirmed by Western blot analysis (Fig. 2a). Then, the H9shDN and H9shCtl were induced to differentiate into the cardiac fate through the formation of embryoid bodies (EB) (Leschik et al. 2008). At day 8, the number of beating area was carefully monitored for each EB as illustrated in Figure 2b.

REFERENCES:

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Claims

CLAIMS:

I . A method for obtaining a population of cardiomyocyte-lineage cells wherein said method comprises a step of culturing pluripotent cells in the presence of an inhibitor of ΔΝρ63 expression and/or activity. 2. The method according to claim 1, wherein an inhibitor of ΔΝρ63 expression is used.

3. The method according to claim 2, wherein said inhibitor of ΔΝρ63 expression is selected from the group consisting of antisense R A or DNA molecules, small inhibitory RNAs (siRNAs) and ribozymes.

4. The method according to claim 1, wherein an inhibitor of ΔΝρ63 activity is used. 5. The method according to claim 4, wherein said inhibitor of ΔΝρ63 activity is selected from the group consisting of small organic molecules, partial or complete ΔΝρ63 neutralizing antibodies or antibody fragments, and aptamers.

6. The method according to any of claims 1 to 5 wherein said pluripotent cells are human pluripotent cells. 7. The method according to claim 6, wherein said pluripotent cells are embryonic stem cells.

8. The method according to claim 6, wherein said pluripotent cells are induced pluripotent cells (iPS).

9. The method according to any of claims 1 to 8, wherein said cardiomyocyte-lineage cells are cardiomyocytes. 10. A population of cardiomyocyte-lineage cells obtainable by a method as defined in any one of claims 1 to 9.

I I . A pharmaceutical composition comprising a population of cardiomyocyte-lineage cells according to claim 10 and a pharmaceutically acceptable carrier or excipient.

12. The population of cardiomyocytes according to claim 10 or the pharmaceutical composition according to claim 11 for use in the treatment of a cardiac pathology and/or cardiac regeneration.

13. Use of an inhibitor of ΔΝρ63 expression and/or activity for increasing the cardiomyogenic differentiation of pluripotent cells.