WO2011080261A1 - Procédé améliorant la différentiation cardiomyogène de cellules souches pluripotentes - Google Patents

Procédé améliorant la différentiation cardiomyogène de cellules souches pluripotentes Download PDF

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WO2011080261A1
WO2011080261A1 PCT/EP2010/070786 EP2010070786W WO2011080261A1 WO 2011080261 A1 WO2011080261 A1 WO 2011080261A1 EP 2010070786 W EP2010070786 W EP 2010070786W WO 2011080261 A1 WO2011080261 A1 WO 2011080261A1
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cells
δνρ63
inhibitor
cardiomyocyte
pluripotent cells
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PCT/EP2010/070786
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Daniel Aberdam
Matthieu Rouleau
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INSERM (Institut National de la Santé et de la Recherche Médicale)
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0657—Cardiomyocytes; Heart cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/155—Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • 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 ⁇ 63 expression and/or activity.
  • stem 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.
  • 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 ( ⁇ 63).
  • TAp63 amino -terminal trans-activation domain
  • ⁇ 63 amino -terminal trans-activation domain
  • Both the TAp63 and ⁇ 63 transcripts are also alternatively spliced at the 3' end producing proteins with unique C-termini that are designated as ⁇ , ⁇ and ⁇ isoforms.
  • 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 ⁇ 63 expression and/or activity.
  • the invention also relates to the use of an inhibitor of ⁇ 63 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.
  • step 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.
  • 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.
  • 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.
  • 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.
  • ⁇ 63 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.
  • the TP63 gene a member of the TP53 gene family, encodes several isoforms with (TAp63) or without ( ⁇ 63) a typical transactivation domain in N-terminal by using two different promoters.
  • alternative splicing within the 3' end results in several C-terminus isoforms called ⁇ , ⁇ , ⁇ for ⁇ 63.
  • ⁇ 63 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 ⁇ 63 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 ⁇ 63 isoform.
  • a compound must reduce the expression of the ⁇ 63 protein by at least 60%.
  • inhibitor of ⁇ 63 activity refers to any compound, natural or synthetic, which results in a decreased activation of the ⁇ 63 signaling pathway, which is the series of molecular signals generated as a consequence of ⁇ 63 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 ⁇ 63 -specific exons.
  • ES cells will be committed to epidermal differentiation and thus will become green. Therefore any compound that will inhibit ⁇ 63 expression will significantly reduce EGFP production as detected by UV microscopy and FACS analysis.
  • inhibitors of ⁇ 63 activity are selective for ⁇ 63 as compared with TAp63.
  • selective it is meant that the affinity of the inhibitor for ⁇ 63 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 ⁇ 63 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- ⁇ , 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 ⁇ 63 may be quantified by measuring the activity of ⁇ 63 (or TAp63) in the presence a range of concentrations of said inhibitor in order to establish a dose-response curve.
  • small organic molecule refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals.
  • 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.
  • treating refers 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.
  • a subject denotes a mammal, such as a rodent, a feline, a canine and a primate.
  • a subject according to the invention is a human.
  • 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 ⁇ 63 expression and/or activity.
  • the cardiomyocyte-lineage cells are cardiomyocytes.
  • pluripotent cells 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.
  • the pluripotent cells are human pluripotent cells.
  • the pluripotent cells are non-human pluripotent cells, such as mouse cells.
  • the pluripotent cells are stem cells.
  • said stem cells are embryonic stem cells.
  • the pluripotent cells are human embryonic stem cells (hES cells).
  • 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).
  • 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.
  • the pluripotent cells are non-human embryonic stem cells, such a mouse stem cells.
  • the pluripotent cells are induced pluripotent stem cells (iPS).
  • iPS induced pluripotent stem cells
  • 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.
  • 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).
  • 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.
  • adult somatic cells are transfected with viral vectors, such as retroviruses, which comprises Oct3/4, Sox2, Klf4, and c-Myc genes.
  • hES cells or human iPS cells may be selected from master cell banks that may be constituted in a therapeutic purpose.
  • hES cells or human iPS may be selected to avoid or limit immune rejection in a large segment of the human population.
  • 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 ⁇ 63 expression and/or activity in any culture medium capable of promoting the growth and the differentiation of pluripotent cells into cardiomyocyte-lineage cells.
  • pluripotent cells are cultured in a cardiomyogenic differentiation culture medium comprising factors inducing the differentiation of pluripotent cells into cardiomyocyte-lineage cells.
  • 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.
  • 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.
  • BMP2 cardiogenic morphogen bone morphogenetic protein 2
  • SU5402 a FGF receptor inhibitor
  • pluripotent cells are cultured with an inhibitor of ⁇ 63 expression and/or activity in a culture medium allowing spontaneous cardiomyogenic differentiation.
  • CDM chemically defined media
  • serum-free refers to a culture medium containing no added serum.
  • feeder-free refers to culture medium containing no added feeder cells.
  • 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.
  • 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.
  • FBS Foetal Bovine Serum
  • BSA Bovine Serum Albumin
  • 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).
  • PVA polyvinyl alcohol
  • BSA Bovine Serum Albumin
  • CDM-PVA refers to a chemically defined medium comprising polyvinyl alcohol (PVA) instead of bovine or human serum albumin.
  • an appropriate CDM according to the invention may consist of 50 % IMDM (Invitrogen, Cergy, France) and 50% F12 NUT MIX (Invitrogen), supplemented with 7 ⁇ g/ml of insulin (Roche, Sandhofer, Germany), 15 ⁇ g/ml of transferrin (Roche), 450 ⁇ 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 ⁇ 63 expression and/or activity shall be carried out for the necessary time required for the production of cardiomyocyte-lineage cells.
  • the culture of pluripotent cells with an inhibitor of ⁇ 63 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.
  • the culture medium may be renewed, partly or totally, at regular intervals.
  • the culture medium may be replaced with fresh culture medium of the invention every other day for 10 days.
  • an inhibitor of ⁇ 63 expression is used.
  • said inhibitor of ⁇ 63 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 ⁇ 63 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 ⁇ 63 mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of ⁇ 63, and hence ⁇ 63 activity, in a cell.
  • antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence encoding ⁇ 63 can be synthesized, e.g., by conventional phosphodiester techniques and administered e.g., by intravenous injection or infusion.
  • Small inhibitory RNAs can also function as inhibitors of ⁇ 63 gene expression for use in the invention.
  • ⁇ 63 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 ⁇ 63 gene expression is specifically inhibited (i.e. RNA interference or RNAi).
  • dsRNA small double stranded RNA
  • RNAi RNA interference
  • 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).
  • sequence of the mouse si-ANp63 RNA is represented by SEQ ID NO: 1.
  • sequence of the human sh-ANp63 RNA is represented by SEQ ID NO: 2.
  • Ribozymes can also function as inhibitors of ⁇ 63 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 ⁇ 63 mRNA sequences are thereby useful within the scope of the invention.
  • 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.
  • antisense oligonucleotides and ribozymes useful as inhibitors of ⁇ 63 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.
  • 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 ⁇ 63.
  • 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.
  • 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.
  • retrovirus such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus
  • retrovirus such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus
  • adenovirus adeno
  • 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.
  • adeno-viruses and adeno-associated viruses 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.
  • 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.
  • 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.
  • 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.
  • Plasmids may be delivered by a variety of parenteral, mucosal and topical routes.
  • 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.
  • 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.
  • an inhibitor of ⁇ 63 activity is used.
  • said inhibitor of ⁇ 63 activity is selected from the group consisting of small organic molecules, partial or complete ⁇ 63 neutralizing antibodies or antibody fragments, and aptamers.
  • the inhibitor of ⁇ 63 activity may be a low molecular weight antagonist, e. g. a small organic molecule.
  • the inhibitor of ⁇ 63 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.
  • the inhibitor of ⁇ 63 activity consists in an antibody (the term including antibody fragment) able to block ⁇ 63 activity.
  • Antibodies directed against ⁇ 63 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.
  • 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.
  • antibodies useful in practicing the invention can be polyclonal, monoclonal antibodies are preferred.
  • Monoclonal antibodies against ⁇ 63 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).
  • techniques described for the production of single chain antibodies may be adapted to produce ⁇ 63 single chain antibodies.
  • Inhibitors of ⁇ 63 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.
  • 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.
  • Fab and/or scFv expression libraries may be constructed to allow rapid identification of fragments having the desired specificity to ⁇ 63.
  • the inhibitor of ⁇ 63 activity is an aptamer directed against
  • 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 ⁇ 63 as above described, the skilled man in the art can easily select those blocking ⁇ 63 activation.
  • the invention relates to the use of an inhibitor of ⁇ 63 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.
  • 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.
  • 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%.
  • 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.
  • 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.
  • 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.
  • 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.
  • step 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.
  • 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.
  • 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).
  • 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.
  • delivery devices include tubes, e.g., catheters, for injecting cells and fluids into the body of a recipient subject.
  • 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.
  • a needle e.g., a syringe
  • the cardiomyocytes of the invention can be inserted into such a delivery device, e.g., a syringe, in different forms.
  • the cardiomyocytes can be suspended in a solution or embedded in a support matrix when contained in such a delivery device.
  • 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.
  • cardiomyocytes is drawn up into a syringe and administrated to anesthetized transplantation recipients. Multiple injections may be made using this procedure.
  • cardiomy 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.
  • 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.
  • cardiomyopathies 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.
  • the cardiac pathology is selected from the group consisting of congestive heart failure, myocardial infarction, cardiac ischemia, myocarditis and arythmia.
  • the cardiac pathology is a genetic disease such as Duchenne muscular dystrophy and Emery Dreiffuss dilated cardiomyopathy.
  • FIGURES
  • FIG. 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 ⁇ - (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.
  • FIG. 2 Human ES cells (line H9) have been stably infected with a lentivirus expressing a shRNA raised against the ⁇ 63 transcript. The resulting cell lines are called H9shDN and the control cell lines (in which an empty lentivirus has been inserted) are called H9shCtl.
  • H9shDN the control cell lines (in which an empty lentivirus has been inserted)
  • H9shCtl the control cell lines
  • 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.
  • 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 ⁇ 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.
  • EBs embryonic bodies
  • 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:
  • hES cells stably expressing a sh-ANp63 construct were produced through lentiviral infection.
  • cardiomyocyte differentiation was performed according to a published protocol (Leschik et al. 2008).
  • % Troponin-T-positive cells 14.7 ⁇ 1.2 5.1 ⁇ 0.5 19.5 ⁇ 1.2
  • Human ES cells (line H9) have been stably infected with a lentivirus expressing a shRNA raised against the ⁇ 63 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.

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Abstract

La présente invention concerne un procédé d'obtention d'une population de cellules de la lignée des cardiomyocytes. Ce procédé comporte une étape de mise en culture de cellules souches pluripotentes, en particulier des cellules souches pluripotentes humaines, en présence d'un inhibiteur de l'expression et/ou de l'activité de ΔNp63.
PCT/EP2010/070786 2009-12-28 2010-12-28 Procédé améliorant la différentiation cardiomyogène de cellules souches pluripotentes WO2011080261A1 (fr)

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US11421229B2 (en) 2015-02-20 2022-08-23 Baylor College Of Medicine p63 inactivation for the treatment of heart failure

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11421229B2 (en) 2015-02-20 2022-08-23 Baylor College Of Medicine p63 inactivation for the treatment of heart failure
US12031135B2 (en) 2015-02-20 2024-07-09 Baylor College Of Medicine p63 inactivation for the treatment of heart failure

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