WO2008151387A1 - Procédé d'amorçage de la différenciation des cardiomyocytes - Google Patents

Procédé d'amorçage de la différenciation des cardiomyocytes Download PDF

Info

Publication number
WO2008151387A1
WO2008151387A1 PCT/AU2008/000862 AU2008000862W WO2008151387A1 WO 2008151387 A1 WO2008151387 A1 WO 2008151387A1 AU 2008000862 W AU2008000862 W AU 2008000862W WO 2008151387 A1 WO2008151387 A1 WO 2008151387A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
csx
protein
stem cells
cell
Prior art date
Application number
PCT/AU2008/000862
Other languages
English (en)
Inventor
Mark Alexander Kirkland
Liem Thanh Vo
Original Assignee
Cytomatrix Pty Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from AU2007903204A external-priority patent/AU2007903204A0/en
Application filed by Cytomatrix Pty Ltd filed Critical Cytomatrix Pty Ltd
Priority to EP08756942A priority Critical patent/EP2205723A1/fr
Priority to AU2008261550A priority patent/AU2008261550A1/en
Publication of WO2008151387A1 publication Critical patent/WO2008151387A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/34—Muscles; Smooth muscle cells; Heart; Cardiac stem cells; Myoblasts; Myocytes; Cardiomyocytes
    • 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
    • C12N2501/60—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
    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention relates to methods of initiating cardiomyocyte differentiation in mammalian cells which involve introducing into the cells CSX/Nkx2.5 protein or a functionally equivalent analogue, variant or fragment thereof.
  • the invention also relates to methods of treatment or prevention of heart disease, and in particular of heart failure.
  • the methods of treatment or prevention involve the initiation of cardiomyocyte differentiation in cells that would otherwise differentiate along other lineages.
  • the patient's own cells can be used either in vivo, or in vitro with differentiated cardiomyocyte cells then being returned to the patient.
  • Congestive heart failure as a result of ischaemic heart disease or other causes, is a major healthcare issue, with some 20 million people (or roughly 10.9% of the population) in the United States being affected with heart disease. It remains the most common cause of mortality in western countries. Heart failure and malignancy together account for more than half of all deaths over the age of 45. Heart disease alone accounted for approximately 700,000 deaths in the US in 2001. On average, 246 deaths per 100,000 people were heart- related.
  • Ischaemic heart disease caused by atherosclerosis and/or thrombosis in the coronary arteries, results in heart attack (myocardial infarction) in over 900,000 patients per year in the US, of whom over 300,000 will die before reaching hospital.
  • Ischaemic heart disease is the most common cause of congestive heart failure.
  • Other causes include cardiomyopathy, which can be a primary disease or can be secondary to other systemic illnesses.
  • Heart failure is defined as a failure of the heart to effectively pump blood at a rate sufficient to meet the metabolic needs of tissues.
  • Loss of effective contractile heart muscle is a common feature of many forms of heart failure. Loss of blood supply to the heart muscle causes ischaemic damage and death of heart muscle cells (cardiomyocytes), leading ultimately to scarring and loss of contractility.
  • Established heart failure can be treated medically (for example, diuretics to decrease blood volume and lower blood pressure), but frequently is progressive.
  • Various approaches have been proposed to improve heart muscle function, including mechanical external cardiac assist devices, various stem cell therapies and even heart transplant.
  • Coronary artery bypass surgery can improve the symptoms of heart failure by improving the blood flow to, and function of, ischaemic heart tissue.
  • Embryonic stem cells can differentiate into functional heart cells, though there are a number of technical as well as ethical barriers to their use in the clinic.
  • Four major studies have examined the ability of bone marrow stem cells to improve outcomes in patients with ischaemic heart disease. Although these studies were initiated because of animal experiments that suggested that bone marrow stem cells could differentiate into heart cells, and so contribute to heart muscle repair, these clinical studies in humans have shown mixed results. While there appears to be some global improvement in heart function, this has not necessarily translated into symptomatic improvement. Importantly, the benefits seen appear to be due to the bone marrow cells contributing to new blood vessel growth rather than to new heart muscle.
  • Skeletal myotherapy 2 utilises skeletal muscle stem cells (known as satellite cells).
  • CSX/Nkx2.5 is a member of a class of transcription factor proteins called homeobox proteins.
  • Homeobox proteins comprise a large family of transcription factors that regulate embryogenesis and determine tissue fate 4 . This family of proteins includes both the archetypal HOX cluster genes that are differentially regulated during segmentation of the embryo, and a large family of over 200 structurally related homeobox proteins, all of which share a highly conserved 60-amino acid DNA binding sequence called the Homeodomain,
  • the non-HOX homeobox genes are key regulators of tissue identity. Adenovirus-mediated transfection with these homeobox genes can induce transdifferentiation - for example, transfection of hepatocytes with the pancreatic homeobox Pdx-1 can cause them to transdifferentiate into exocrine and endocrine pancreas 5 ' 6 ' 7 , particularly when fused with the herpes simplex-derived transcriptional activator, VP 16 8 . Similarly, the homeoprotein
  • CSX/Nkx 2.5 (951 bp.) is an essential transcription factor in cardiac cell formation 9 .
  • a recent study has shown that adenoviral transfection of mesenchymal stem cells with the
  • CSX gene can induce transdifferentiation of mesenchymal stem cells into cardiac cells in a stochastic manner 3 .
  • the consensus homeodomain sequence also includes a transduction domain, Penetratin, allowing the protein to cross the cell membrane, and it has been suggested that homeoproteins may act as cell-cell signalling molecules 10 .
  • Other transduction domains such as the HIV TAT sequence, have been used experimentally to aid transmembrane delivery of homeoproteins 1 ' .
  • the present inventors have now determined that it is possible to initiate differentiation of skeletal myoblasts into cardiomyocyte-like cells, by introducing into such cells an effective amount of CSX/Nkx2.5 protein or a functionally equivalent analogue, variant or fragment thereof. While the role of cardiac homeobox 1 (CSX/Nkx2.5) in cardiac development 12 (4) is known, as well as the fact that CSX/Nkx2.5 transcription is regulated by a complex of transcription factors including CSX/Nkx2.5 itself and Rae28, Gata4, CaI and Nkx2.6/Tix, it has not previously been suggested that introduction of the CSX/Nkx2.5 protein (as opposed to transfection of the CSX/Nkx2.5 gene) into cells, optionally in conjunction with other relevant transcription factors, could be effective in initiating cardiomyocyte differentiation. It has also not previously been suggested that this cardiomyocyte differentiation could be sustained by the cells treated after the exposure to CSX/Nkx2.5, and nor that the cardiomyocytes produced could spontaneously beat in
  • a method of initiating cardiomyocyte differentiation in a responsive mammalian cell which comprises introducing into the cell an effective amount for initiating cardiomyocyte differentiation within the cell of CSX/Nkx2.5 protein or a functionally equivalent analogue, variant or fragment thereof.
  • the method may be conducted in vivo within a mammalian organism or may be conducted in vitro.
  • a method of treatment of a patient suffering from or prone to suffer from heart failure or ischaemic heart disease which comprises removing from the patient one or more responsive cells and culturing the cells in a suitable medium, introducing into the cells an effective amount for initiating cardiomyocyte differentiation of CSX/Nkx2.5 protein or a functionally equivalent analogue, variant or fragment thereof and subsequently returning the cells or cells derived from them to the patient.
  • a method of treatment of a patient suffering from or prone to suffer from heart failure or ischaemic heart disease which comprises introducing into responsive cells of the patient an effective amount of CSX/Nkx2.5 protein or a functionally equivalent analogue, variant or fragment thereof.
  • Introduction of CSX/Nkx2.5 protein and optionally other transcription factors or their functionally equivalent analogues, variants or fragments can be conducted in vitro with the cells subsequently being returned to the patient.
  • the method is conducted in vivo within a mammalian organism.
  • the methods conducted are for treatment and/or prevention of heart failure or ischaemic heart disease.
  • the CSX/Nkx2.5 protein or a functionally equivalent analogue, variant or fragment thereof is introduced into the cells in conjunction with one or more other transcription factors selected from Rae28, Gata4, CaI, KLF13, Hex and Nkx2.6/Tix, or their functionally equivalent analogues, variants or fragments.
  • the combination of CSX/Nkx2.5 and optional other transcription factors or their functionally equivalent analogues, variants or fragments along with other optional components will for convenience be referred to herein as the "treatment agent".
  • the responsive mammalian cells are mammalian cells other than cardiac cells.
  • the responsive mammalian cells are selected from one or more of hepatocytes, fibroblasts, endothelial cells, B cells, T cells, dendritic cells, keratinocytes, adipose cells, epithelial cells, epidermal cells, chondrocytes, cumulus cells, neural cells, glial cells, astrocytes, cardiac cells, oesophageal cells, skeletal muscle cells, skeletal muscle satellite melanocytes, hematopoietic cells, osteocytes, macrophages, monocytes, mononuclear cells or stem cells including embryonic stem cells, embryonic germ cells, adult brain stem cells, epidermal stem cells, skin stem cells, pancreatic stem cells, kidney stem cells, liver stem cells, breast stem cells, lung stem cells, muscle stem cells, heart stem cells, eye stem cells, bone stem cells, spleen stem cells, immune system
  • the treatment agent is introduced utilising detergent, bacterial toxin or electroporation permeabilisation, lisosomal delivery or with the use of cell-permeant peptide vectors or polyethylene glycol (PEG), each of which are techniques well known in the art as described in Sambruck & Russell 13 , the disclosure of which is included herein in its entirety by way of reference.
  • bacterial toxin permeabilisation can utilise streptolysin O
  • cell-permeable peptide vectors can include antennapedia/penetratin TAT and signal-peptide based sequences.
  • CSX/Nkx2.5 or optional other transcription factors or their functionally equivalent analogues or variants can be produced recombinantly or can be isolated from mammalian cells.
  • an agent for initiating cardiomyocyte differentiation in a responsive mammalian cell which comprises CSX/Nkx2.5 protein or a functionally equivalent analogue, variant or fragment thereof, one or more other transcription factors and one or more physiologically acceptable carriers and/or diluents.
  • Such an agent may further comprise one or more permeabilisation agents.
  • Fig. 1 shows a bar graph of the frequency of beating aggregates seen in control cultures compared to cultures containing CSX protein (mean of 6 experiments).
  • Fig. 2 shows confocal microscope images of treated and untreated cells, as follows: a) Negative control (dapi staining of nuclei) b) Negative control - untreated cells showing low level staining with f- actin (green) and MLC (red). c) Aggregate of spontaneously beating target cells after exposing to CSX-
  • VP 16 containing media stained with f-actin (red), dapi nuclear stain (blue) and cpnnexin 43 (green).
  • Fig 3 shows a bar graph demonstrating the expression of cardiac-specific RNA (CSX, Mlc-v2 and Troponin I) by beating cells, but not by non-beating cells from the same cultures or untreated control cells (P ⁇ 0.05 for beating cells vs other groups for all genes (ANOVA))
  • Fig. 4 shows a bar graph of luminescence generated in the CSX reporter assay by the transfection of CHO cell lines using the pGL4.2-ANP construct, where: • CHO pGL4.2(neg) is the pGL4.2 vector without a promoter insert
  • CHO ⁇ GL4.2(ANP+Vector) is the pGL4.2 vector with ANP promoter transfected into CHO cells
  • CHO pGL4.13 (pos) is a positive control vector
  • CSX ANP is the stable cell line expressing CSX transfected with the pGL4.2(ANP) vector
  • CSX-VP 16 ANP is the stable cell line expressing CSX-VP 16 transfected with the pGL4.2(ANP) vector
  • CSX-TAT ANP is the stable cell line expressing CSX-TAT transfected with the pGL4.2(ANP) vector.
  • Fig 5 shows a bar graph of the number of beating cells observed in each high power field (HPF), where
  • CHO CM is media conditioned by CHO cells (not transfected)
  • CHO-CSX is media conditioned by CHO cells transfected with CSX
  • CHO-CSX-VP 16 is media conditioned by CHO cells transfected with
  • CHO-HIS is CHO CM passed down a nickel column to purify poly-HIS proteins (negative control)
  • CHO-CSX is CHO-CSX passed down a nickel column to purify CSX protein
  • CHO-CSX-VP 16 is CHO-CSX-VP 16 passed down a nickel column to purify CSX-VP 16 protein
  • SEQ ID NO. 2 shows the amino acid sequence of CSX/Nkx2.5 from mus musculus.
  • SEQ ID NO. 3 shows the amino acid sequence of human Rae28,.(also known as Polyhomeotic-like protein 1). (UniProt accession number P78364)
  • SEQ ID NO. 4 shows the amino acid sequence of human Gata4. (UniProt accession number P43694)
  • SEQ ID NO. 5 shows the amino acid sequence of human Nkx2.6/Tix. (GenBank accession number XM_939389)
  • SEQ ID NO. 6 shows the amino acid sequence of human CaI (also known as Filamin-binding LIM protein 1). (UniProt accession number Q8WUP2)
  • SEQ ID NO. 7 shows the amino acid sequence of KLF13.
  • SEQ ID NO. 8 shows the amino acid sequence of Hex.
  • SEQ ID NO. 9 shows the amino acid sequence of VP 16.
  • SEQ ID NO . 10 shows the amino acid sequence of TAT.
  • SEQ ID NO. 11 shows the nucleic acid sequence of the mouse ANP gene promoter region product used in reporter studies.
  • a method of initiating cardiomyocyte differentiation in a responsive mammalian cell By the phrase "responsive mammalian cell", it is intended to encompass mammalian cells other than cardiac cells.
  • types of mammalian cells that can be treated according to the invention to initiate cardiomyocyte differentiation include hepatocytes, fibroblasts, endothelial cells, B cells, T cells, dendritic cells, keratinocytes, adipose cells, epithelial cells, epidermal cells, chondrocytes, cumulus cells, neural cells, glial cells, astrocytes, cardiac cells, oesophageal cells, muscle cells, melanocytes, hematopoietic cells, osteocytes, macrophages, monocytes, mononuclear cells or stem cells including embryonic stem cells, embryonic germ cells, adult brain stem cells, epidermal stem cells, skin stem cells, pancreatic stem cells, kidney
  • the cells utilised are stem cells, as referred to above, hepatocytes, skeletal muscle cells, mesenchymal stem cells, bone marrow stem cells or fibroblasts.
  • the cells utilised according to the invention can be derived from any of a variety of mammalian organisms, including, but not limited to humans, primates such as chimpanzees, gorillas, baboons, orangutans, laboratory animals such as mice, rats, guinea pigs, rabbits, domestic animals such as cats and dogs, farm animals such as horses, cattle, sheep, goats or pigs or captive wild animals such as lions, tigers, elephants, buffalo, deer or the like.
  • cells used in treating a particular patient it is preferable, however, for cells used in treating a particular patient to be derived from an individual of the same species.
  • cells used to treat a particular patient are derived from the same patient.
  • the phrase "initiating cardiomyocyte differentiation” it is intended to convey that as a result of the treatment conducted at least some, preferably at least 0.1 %, more preferably at least 1%, still more preferably at least 5%, particularly preferably at least 10% and more preferably at least 20, 30, 40, 60, 80 or 90% of the mammalian cells treated according to the invention will commence cardiomyocyte differentiation as a result of the treatment according to the invention.
  • the cardiomyocyte differentiation of the cells concerned is associated with spontaneous rhythmic contraction of the cells.
  • Cellular cardiomyocyte differentiation can, for example, be detected by immunohistochemistry, by the use of specific stains for cardiac proteins or other detectable compounds, by radioimmunoassay or real time PCR, which more particularly monitors cardiac gene expression. At least in the case of radio-immunoassay and real time PCR it is possible to quantify the levels of cardiac gene expression in a particular population of cells.
  • a key aspect of the present invention is the introduction into the cell or cells in which cardiomyocyte differentiation is to be initiated of CSX/Nkx2.5 protein or a functionally equivalent analogue, variant or fragment thereof.
  • Cardiac homeobox (CSX/Nkx2.5) is an orphan homeo domain protein known to be important in cardiac development.
  • the CSX/Nkx2.5 gene is localised on human chromosome 5 and the nucleotide sequence of the gene has been reported by Turbay et al 14 . Regulation of CSX/Nkx2.5 gene, expression is further described by Nemur and Nemur 15 . The disclosures of these papers are included herein in their entirety, by way of reference.
  • the CSX/Nkx2.5 protein can be introduced into the cells being treated in combination with one or more other components of what is referred to herein as the "treatment agent", including for example nucleic acids or proteins such as DNA methyl transferases, histone deacetylases, histones, nuclear laminins, transcription factors, activators, repressors, growth factors, hormones or cytokines as well as other agents such as detergents, salt solutions, compatible solvents, buffers, demethylating agents, histone deacetylation agents, nutrients or active compounds.
  • the CSX/Nkx2.5 protein or analogue or variant thereof is at least to some extent isolated or purified from other components of a cytoplasmic extract from which it may be obtained.
  • an agent is at least 50% by weight free from proteins, antibodies and naturally-occurring organic molecules with which it is endogenously associated.
  • the agent is at least 75% and more preferably at least 90%, 95% or 99% by weight pure.
  • a substantially pure agent may be obtained by chemical synthesis, separation of the agent from natural sources or production of the agent in a recombinant host cell that does not naturally produce the agent.
  • Agents may be purified using standard techniques such as for example those described by Ausubel et al 16 , the disclosure of which is incorporated herein in its entirety by way of reference.
  • the agent is preferably at least 2, 5 or 10 times as pure as the starting material from which it is derived, as measured using polyacrylamide gel electrophoresis, column chromatography, optical density, HPLC analysis or western analysis.
  • Preferred methods of purification include immuno precipitation, column chromatography such as immuno affinity chromatography, magnetic bead immuno affinity chromatography and panning with a plate-bound antibody.
  • the protein can be purified by virtue of specific sequences incorporated into the protein, for example, through Nickel column affinity in the case where the protein has 6 or more histidine amino acids incorporated into the sequence.
  • the treatment agent introduced into the cells to be treated may also include one or more other transcription agents or their functionally equivalent analogues, variants or fragments.
  • transcription agents can include one or more of Rae28, Gata4, CaI, Hex, KLF 13 and Nkx2.6/Tix, as for example referred to Nemur and Nemur ⁇ , Yamada et al 3 Akazawa et al 18 and Shirai et al 19 .
  • Variants are intended to encompass proteins having amino acid sequence differing from the protein from which they are derived by virtue of the addition, deletion or substitution of one or more amino acids to result in an amino acid sequence that is preferably at least 60%, more preferably at least 80%, particularly preferably at least 85, 90, 95, 98, 99 or 99.9% identical to the amino acid sequence of the original protein.
  • the variants specifically include polymorphic variants and interspecies homologues.
  • the term "variants” is intended to encompass the inclusion in the protein of additional functional sequences, such as the transcriptional activator sequence VP 16 derived from the herpes simplex virus or the TAT sequence derived from the Human Immunodeficiency virus. It is also intended to encompass the deletion of sequences within the normal CSX/Nkx2.5 sequences so as to alter the distribution and metabolism of the protein, such as, for example, PEST sequences associated with protein metabolism and destruction.
  • Analogues are compounds that may or may not be proteins or peptides (and can for example be small organic compounds) that are functionally equivalent to the protein of which they are a fragment.
  • fragments it is intended to encompass fragments of a protein that are of at least 10, preferably at least 20, more preferably at least 30, 40 or 50 amino acids in length and which are of course functionally equivalent to the protein of which they are a fragment.
  • polypeptide peptide
  • protein protein
  • amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to both naturally and non-naturally occurring amino acid polymers.
  • amino acid refers to naturally occurring and synthetic amino acids as well as amino acid analogues and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproaline, gamma-carboxyglutamate, and O-phosphoserene.
  • amino acid analogues refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, that is a carbon that is bound to a hydrogen, a carboxyl group, an amino group and an R group, e.g., homoserene, norlusene, methianene sulfoxide and methanene methyl sulphonian. Such analogues have modified R groups (e.g. norlusene) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but retain a function similar to that of a naturally occurring amino acid.
  • the CSX/Nkx2.5 protein or functionally equivalent analogue, variant or fragment thereof, optionally in conjunction with other components such as transcription factors (ie. the treatment agent) can be introduced into the cells to be treated according to the invention by a variety of different means.
  • the treatment agent to be introduced into the cells can be introduced by utilising detergent, bacterial toxin or electroporation techniques for increasing permeabilisation of the cell. To some extent these methods introduce repairable pores, voids or weaknesses into the cellular membrane which allow the agents to pass across the membrane.
  • An example of a detergent that can be utilised to achieve permeabilisation is digitonin, and streptolysin O is a bacterial toxin commonly used in this manner.
  • Electroporation of a plasma membrane is a technique commonly used for introduction of foreign DNA during cell transfections, but can also be used for introduction of proteins. This method introduces large size and temporary openings in the plasma membrane which allows free diffusion of extra-cellular components into the cells, without the requirement for active uptake. Electroporation parameters may be tested and optimised for the specific type of cell being treated and the particular protein or proteins being introduced. Electroporation techniques are well known in the art and are further described in detail in Sambruck & Russell (7).
  • BioPorter® protein delivery reagent (Gene Therapy Systems, Inc.) which is a unique lipid based formulation that allows the delivery of proteins, peptides or other bioactive molecules into a broad range of cell types. It interacts non-covalently with the protein creating a protective vehicle for immediate delivery into cells.
  • the reagent fuses directly with the plasma membrane of the target cell.
  • the extent of introduction can be monitored by TRITC-conjugated antibody uptake during the treatment. This is easily detected using low light fluorescence on living cells.
  • Molecules that have been successfully introduced in this manner into various cell types include high and low molecular weight dextran sulphate, ⁇ -galactasidase, caspase 3, caspase 8, grandzime B and fluorescent antibody complexes.
  • CSX/Nkx 2.5 includes within the homeobox domain a sequence homologous to antennapedia/penetratin, which enables entry of the protein into the cell in the absence of additional cell-permeant peptide vectors.
  • Pro-JectTM transfection using Pro-JectTM reagent (Pierce, Rockford IL, USA).
  • Pro-JectTM is a cationic lipid-based carrier system that can be used to deliver biologically active proteins, peptides or antibodies into cells.
  • Pro-JectTM Reagent/protein complexes attach to negatively charged cell surfaces and enter the cell either by directly fusing with the plasma membrane or by endocytosis and subsequent fusion with the endosome.
  • CSX/Nkx2.5 protein or its analogues, variants or fragments introduced into the cells in which cardiomyocyte differentiation is intended to be initiated and which is effective for cardiomyocyte differentiation can readily be optimised by persons skilled in the art.
  • the effective amount will, however, vary depending upon the technique adopted for introducing the agent into the cells and may also depend upon the types and species of cell utilised, cell culture conditions, use of other transcription factors and indeed whether the method is conducted in vivo or in vitro.
  • effective amounts for initiating cardiomyocyte differentiation within the cell of CSX/Nkx2.5 protein or functionally equivalent analogue, variant or fragment thereof will in one embodiment fall within the range of 0.1 - 10 ⁇ g/ml per 10 5 target cells.
  • the initiation of cardiomyocyte differentiation in mammalian cells that can be achieved through the present invention can be utilised in both therapeutic and prophylactic contexts.
  • mammalian and particularly human patients identified as possessing risk factors for development of heart failure or myocardial ischaemia can be treated according to methods of the invention in a prophylactic fashion to prevent or slow onset of the disease or minimise its severity.
  • Patients diagnosed as suffering from heart failure or myocardial ischaemia can of course also be treated utilising methods of the invention.
  • patients may for example be treated in an in vivo or indeed an in vitro fashion.
  • in vivo treatment it is intended to mean that methods of initiating cardiomyocyte differentiation in mammalian cells are conducted upon these cells while they are located within the organism concerned.
  • mammalian cells preferably those derived from an organism of the same species, and particularly preferably derived from the particular patient concerned, are exposed to the treatments according to the invention in an in vitro or cell culture setting. After exposure of the cells to the treatment agent to induce cardiomyocyte differentiation the cells so treated, or progeny cells ultimately derived from them, are returned to the patient.
  • Cells can readily be removed from patients for conducting in vitro aspects of the invention by routine techniques such as by biopsy of the appropriate tissue or organ or extraction of cell containing fluid from the patient. The cells obtained can then be cultured under appropriate cell culture conditions, as will be further explained. Similarly, cells in which cardiomyocyte differentiation has been initiated can be introduced to the patient by a variety of conventional means, such as for example by intravenous, intra-arterial, intramuscular, transdermal, intraperitoneal or direct injection into an organ using a physiologically compatible suspension of the treated cells. It is also possible to surgically implant the cells into a desired location within the organism, possibly by utilising endoscopic techniques to minimise patient trauma. For example, cells can be introduced into and around an area of myocardial ischemia or scarring by transthoracic injection under radionuclide or other imaging guidance.
  • the treatment agent may similarly be exposed to the cells into which it is intended to be introduced by a variety of conventional means.
  • the treatment agent possibly in conjunction with one or more physiologically compatible permeabilisation agents, can be injected into the appropriate tissue or organ, may be applied to the heart or another tissue or organ using a patch or matrix or may be applied or injected to a suitable tissue or organ in conjunction with a liposomal delivery system.
  • specific endogenous cells within the patient can be subjected to electroporation permeabilisation to assist in cellular uptake of the treatment agent.
  • injectable formulations which can be utilised for preparation of injectable cell suspensions and treatment agents, as well as preparation of other pharmaceutical forms for delivery of treatment agents according to the invention are explained in detail in Remington's Pharmaceutical Sciences 21 , the disclosure of which is included herein in its entirety by way of reference.
  • pharmaceutically acceptable carriers and formulations are determined in part by the particular agent, compound or composition being administered (e.g., the cell or treatment agent), as well as by the particular method used to administer the formulation.
  • the carriers can include slow release agents that deliver a dose of the treatment agent to the cells in a controlled fashion over time (hours, days or weeks as necessary).
  • controlled release carriers include polymers, lipid formulations, and other biodegradable or non-biodegradable materials as are well know in the pharmaceutical field.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain physiologically acceptable (especially pharmaceutically acceptable) carriers and diluents such as antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilisers, thickening agents, stabilisers, and preservatives.
  • compositions can be administered, for example, by direct surgical transplantation, intraportal administration, intravenous infusion, or intraperitoneal infusion.
  • Injection solutions and suspensions can be prepared from sterile powders, granules, and tablets.
  • the dose of cells or treatment agent administered to a patient, in the context of the present invention should be sufficient to effect a beneficial therapeutic response in the patient over time.
  • the dose will be determined by the efficacy of the particular cells or treatment agent employed and the condition of the patient, as well as the body weight or surface area of the patient to be treated.
  • the size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects in a particular patient.
  • cells of the present invention can be administered in amount effective to provide improved and preferably normalised glucose responsive-cardiomyocyte differentiation and normalised glucose levels to the subject. Administrations according to the invention can be accomplished via single or divided doses.
  • the cell culture environment includes consideration of such factors as the substrate for cell growth, cell density and cell contact, the gas phase, the medium and temperature.
  • the cells are grown in suspension as three dimensional aggregates. Suspension cultures can be achieved by using, e.g., a flask with a magnetic stirrer or a large surface area paddle, or on a plate that has been coated to prevent the cells from adhering to the bottom of the dish. In a preferred embodiment, the cells are grown in Costar dishes that have been coated with a hydrogel to prevent them from adhering to the bottom of the dish.
  • plastic dishes, flasks, roller bottles, or microcarriers in suspension are used.
  • Other artificial substrates can be used such as glass and metals.
  • the substrate is often treated by etching, or by coating with substances such as collagen, chondronectin, fibronectin, and laminin.
  • the type of culture vessel depends on the culture conditions, e.g., multi-well plates, petri dishes, tissue culture tubes, flasks, roller bottles, and the like.
  • Cells are grown at optimal densities that are determined empirically based on the cell type. For example, a typical cell density for .beta.lox5 cultures varies from 1 x 10 to 1 x 10 cells per ml. Cells are passaged when the cell density is above optimal.
  • Cultured cells are normally grown in an incubator that provides a suitable temperature, e.g., the body temperature of the animal from which is the cells were obtained, accounting for regional variations in temperature. Generally, 37°C is the preferred temperature for cell culture. Most incubators are humidified to approximately atmospheric conditions.
  • Important constituents of the gas phase are oxygen and carbon dioxide. Typically, atmospheric oxygen tensions (20%) are used for cell cultures, though for some cell types lower oxygen concentrations of 10%, 5% or 2% are preferred. Culture vessels are usually vented into the incubator atmosphere to allow gas exchange by using gas permeable caps or by preventing sealing of the culture vessels. Carbon dioxide plays a role in pH stabilisation, along with buffer in the cell media and is typically present at a concentration of 1-10% in the incubator. The preferred CO 2 concentration typically is 5%.
  • Defined cell media are available as packaged, premixed powders or presterilised solutions. Examples of commonly used media include DME, RPMI 1640, Iscove's complete media, or McCoy's Medium (see, e.g., GibcoBRL/Life Technologies Catalogue and Reference Guide; Sigma Catalogue). Typically, low glucose DME or RPMI 1640 are used in the methods of the invention. Defined cell culture media are often supplemented with 5-20% serum, typically heat inactivated, e.g., human, horse, calf, and fetal bovine serum. Typically, 10% fetal calf serum or human serum is used in the methods of the invention.
  • the culture medium is usually buffered to maintain the cells at a pH preferably from 7.2- 7.4.
  • Other supplements to the media include, e.g., antibiotics, amino acids, sugars, and growth factors such as hepatocyte growth factor/scatter factor (HGF), Insulin-like growth factor- 1 (IGF-I), members of the fibroblast growth factor (FGF) family, members of the bone morphogenic protein (BMP) family, and epidermal growth factor (EGF).
  • HGF hepatocyte growth factor/scatter factor
  • IGF-I Insulin-like growth factor- 1
  • FGF fibroblast growth factor
  • BMP bone morphogenic protein
  • EGF epidermal growth factor
  • the CSX/Nkx2.5 or other transcription factors or their functionally equivalent analogues, variants or fragments that may comprise or be included within the treatment agent can be chemically synthesised, recombinantly produced or isolated from mammalian cells. Chemical synthesis, recombinant production and isolation techniques
  • Target cells Skeletal muscle satellite cells (stem cells) from adult mouse skeletal muscle were isolated from 3 -week old mouse thigh muscle by collagenase and dispase digestion and cultured in skeletal muscle growth media
  • Cardiac Cells Normal mouse cardiomyocytes were isolated from the hearts of adult mice by collagenase and dispase treatment. Culture conditions: All cells were cultured in DMEM medium with 2% Fetal Calf serum (FCS), 0.5mg/ml Fetuin, 0.5mg/ml bovine serum albumin (BSA), 0.39ug/ml dexamethasone, and 2 ng/ml EGF at 37 0 C in a 5% CO 2 incubator in low (5%) oxygen.
  • FCS Fetal Calf serum
  • BSA bovine serum albumin
  • EGF 2 ng/ml EGF
  • Recombinant protein production The sequence of mouse CSX/Nkx2.5 was cloned and sequenced from normal mouse heart tissue. A second variant was then made by fusing the HIV-TAT sequence to the 3' end of the CSX/Nkx2.5 sequence, and a third variant was generated by fusing the Herpesvirus VP 16 sequence to the 5' end of the CSX/Nkx2.5 sequence. These clones were then inserted into the pSecTag/FRT/V5-His-T0P0 vector using standard methods. This vector includes an Ig ⁇ secretory signal, allowing the protein to be secreted into the medium.
  • the recombinant proteins also have a V5 tag to allow identification and tracking of the protein, and a His sequence to enable purification on a nickel column.
  • the CSX/Nkx2.5 clone and its variants were then stably transfected into Chinese hamster ovary (CHO) cells.
  • Treating satellite cells using recombinant protein Supernatant from the CHO cell line stably transfected with the CSX-VP16 sequence ("CSX- VP16 conditioned medium") was added to cultures of mouse skeletal muscle satellite cells daily for three weeks. Treated and control satellite cells were examined by immunostaining and RT-PCR for expression of cardiac specific genes, and were examined for morphological changes.
  • Real-time PCR was performed with SYBR Green PCR Master Mix and TaqMan Universal PCR Master Mix (Applied Biosystems) and analyzed with the 7700 real-time PCR instrument (Applied Biosystems).
  • TaqMan Gene Expression Assays were purchased from Applied Biosystems. Real time PCR was used to detect the level of heart specific genes such as MLC 2v and ANF in samples. Results are expressed as arbitrary gene units.
  • Immunohistochemistry Treated target cells and untreated control cells were fixed in 4% paraformaldehyde and stained with fluorescently labelled anti-connexin 43 and anti-ANF antibodies as well as other markers indicating contractile properties such as f-actin.
  • EXAMPLE 2 PROTOCOLS FOR CSX/NKX2.5 ISOLATION, AMPLIFICATION. RECOMBINANT PRODUCTION AND USE
  • CSX forward primer 5' ttc ccc age cct gcg etc aca 3' CSX reverse primer: 5'cca gga teg gat gcc gtg cag c 3' Specific primers for CSX 5 CSX-tat, CSX-VP 16 and CSX-tat-VP16 were used to create cDNA from mouse genomic DNA. These PCR products were purified and cloned into ⁇ SecTag/FRT/V5-His-TC)PO vector (Invitrogen). The vectors were inserted into Top 10 E. CoIi cells by heat shock treatment.
  • Cells containing the correct inserts were selected by resistance to antibiotics. Correct sequence orientation was checked by restriction enzymes and sequenced to detect mutations. Clones containing the correct sequences were transfected into FIp-In transfectable CHO cells (Invitrogen). Transfected cells were selected on the basis of Hygromycin resistance and colonies were selected for expansion in vitro. Secreted proteins were collected in the supernatant and confirmed by western blotting by using anti v5 antibodies with standard molecular weight markers. Once the molecular weights were confirmed, the secreted proteins were purified using Nickel columns and quantitated by colorimeter reading against protein standards.
  • CSX and CSX-VP 16 proteins were tested for functionality using the pGL reporter system (Invitrogen).
  • the promoter region for the Atrial Naturetic Protein (ANP) gene (SEQ ID No. 9) was cloned by PCR and was inserted into the pGL4.2 vector. The vector was then amplified in bacteria, isolated and then transfected into CHO cell lines stably transfected with the CSX, CSX-VP16 and TAT-CSX constructs. Luminescence was measured 48 hours later using a Tecan luminometer.
  • 5% conditioned media i.e. media taken from cultures of stably producing CHO cell lines
  • purified recombinant protein 500 ng/ ml
  • CHO cells stably transfected with CSX, CSX-VPl 6 or TAT-CSX constructs were transfected with the ANP-pGL4.2 construct, or with control constructs.
  • Unmanipulated CSX gave the strongest signal, followed by CSX-VP 16 the TAT-CSX.
  • tissue was then washed twice with PBS 5 the location of tissue was marked with permanent marker and returned to the tissue culture incubator for 15 minutes recovery.
  • the tissue was then treated with the beta blocker, metoprolol tartrate, at a dose of 1x10 "6 M.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Cell Biology (AREA)
  • Veterinary Medicine (AREA)
  • Genetics & Genomics (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Medicinal Chemistry (AREA)
  • Cardiology (AREA)
  • Immunology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Virology (AREA)
  • Rheumatology (AREA)
  • Marine Sciences & Fisheries (AREA)
  • Microbiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Vascular Medicine (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

Dans un mode de réalisation, l'invention porte sur un procédé d'amorçage de la différenciation des cardiomyocytes dans une cellule sensible de mammifère. Ce procédé consiste à introduire dans la cellule une quantité efficace pour amorcer la différenciation des cardiomyocytes dans la cellule de la protéine CSX/Nkx2.5 ou d'un analogue, variant ou fragment fonctionnellement équivalent de celle-ci. Dans un autre mode de réalisation, l'invention porte sur un procédé de traitement d'un patient souffrant ou susceptible de souffrir d'une insuffisance cardiaque ou d'une cardiopathie ischémique. Ce procédé consiste à prélever une ou plusieurs cellules sensibles sur un patient; à cultiver les cellules dans un milieu approprié; à introduire dans les cellules une quantité efficace pour amorcer la différenciation des cardiomyocytes de la protéine CSX/Nkx2.5 ou d'un analogue, variant ou fragment fonctionnellement équivalent de celle-ci; et à réinsérer chez le patient, les cellules ou des cellules issues de celles-ci. Dans un autre mode de réalisation, l'invention porte sur un procédé de traitement d'un patient souffrant ou susceptible de souffrir d'une insuffisance cardiaque ou d'une cardiopathie ischémique qui consiste à introduire dans des cellules sensibles du patient une quantité efficace de la protéine CSX/Nkx2.5 ou d'un analogue, variant ou fragment fonctionnellement équivalent de celle-ci.
PCT/AU2008/000862 2007-06-14 2008-06-13 Procédé d'amorçage de la différenciation des cardiomyocytes WO2008151387A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP08756942A EP2205723A1 (fr) 2007-06-14 2008-06-13 Procédé d'amorçage de la différenciation des cardiomyocytes
AU2008261550A AU2008261550A1 (en) 2007-06-14 2008-06-13 Method of initiating cardiomyocyte differentiation

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AU2007903204 2007-06-14
AU2007903204A AU2007903204A0 (en) 2007-06-14 Methods

Publications (1)

Publication Number Publication Date
WO2008151387A1 true WO2008151387A1 (fr) 2008-12-18

Family

ID=40129149

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/AU2008/000862 WO2008151387A1 (fr) 2007-06-14 2008-06-13 Procédé d'amorçage de la différenciation des cardiomyocytes

Country Status (3)

Country Link
EP (1) EP2205723A1 (fr)
AU (1) AU2008261550A1 (fr)
WO (1) WO2008151387A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011139688A2 (fr) 2010-04-28 2011-11-10 The J. David Gladstone Institutes Procédés de génération de cardiomyocytes
US9828585B2 (en) 2011-08-30 2017-11-28 The J. David Gladstone Instututes Methods for generating cardiomyocytes

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20010016193A1 (en) * 1998-05-30 2001-08-23 Engler Robert L. Methods of altering cardiac cell phenotype
WO2004019767A2 (fr) * 2002-08-29 2004-03-11 Baylor College Of Medicine Cellules derivees du coeur pour une reparation cardiaque

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20010016193A1 (en) * 1998-05-30 2001-08-23 Engler Robert L. Methods of altering cardiac cell phenotype
WO2004019767A2 (fr) * 2002-08-29 2004-03-11 Baylor College Of Medicine Cellules derivees du coeur pour une reparation cardiaque

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
AKAZAWA H. ET AL.: "A novel LIM proteinCal promotes cardiac differentiation by association with CSX/NKx2-5", THE JOURNAL OF CELL BIOLOGY, vol. 164, no. 3, 2 February 2004 (2004-02-02), pages 395 - 405, XP008132803 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011139688A2 (fr) 2010-04-28 2011-11-10 The J. David Gladstone Institutes Procédés de génération de cardiomyocytes
EP2563907A2 (fr) * 2010-04-28 2013-03-06 The J. David Gladstone Institutes Procédés de génération de cardiomyocytes
JP2013524837A (ja) * 2010-04-28 2013-06-20 ザ ジェイ． デヴィッド グラッドストーン インスティテューツ 心筋細胞を発生させるための方法
EP2563907A4 (fr) * 2010-04-28 2014-03-05 David Gladstone Inst Procédés de génération de cardiomyocytes
US9517251B2 (en) 2010-04-28 2016-12-13 The J. David Gladstone Institutes Methods for generating cardiomyocytes
US9517250B2 (en) 2010-04-28 2016-12-13 The J. David Gladstone Institutes Methods for generating cardiomyocytes
US9828585B2 (en) 2011-08-30 2017-11-28 The J. David Gladstone Instututes Methods for generating cardiomyocytes

Also Published As

Publication number Publication date
EP2205723A1 (fr) 2010-07-14
AU2008261550A1 (en) 2008-12-18

Similar Documents

Publication Publication Date Title
US20110190730A1 (en) Methods of inducing pluripotency involving oct4 protein
US20110190729A1 (en) Methods of inducing pluripotency involving sox2 protein
Yoshida et al. Maturation of human induced pluripotent stem cell-derived cardiomyocytes by soluble factors from human mesenchymal stem cells
Song et al. Transfection of mesenchymal stem cells with the FGF-2 gene improves their survival under hypoxic conditions
JP5684963B2 (ja) 筋肉由来前駆細胞、その組成物、および治療法を使用した軟部組織および骨の増大および肥厚
US20210145893A1 (en) Compositions to amplify cardiac stem cells in vitro and in viv
SG177467A1 (en) Pre-natal mesenchymal stem cells
WO2009027563A1 (fr) Population de cellules souches adultes issues du tissu adipeux cardiaque et son utilisation dans la régénération cardiaque
US20100209404A1 (en) Enhanced method for producing stem-like cells from somatic cells
JP5395672B2 (ja) 心臓病理学を処置するための筋肉由来の細胞およびその作製法および使用法
US20200345851A1 (en) Epicardial-derived paracrine factors for repairing cardiac tissue
Hamdi et al. Efficacy of epicardially delivered adipose stroma cell sheets in dilated cardiomyopathy
US20180071341A1 (en) Bone augmentation utilizing muscle-derived progenitor compositions, and treatments thereof
JP7165979B2 (ja) 骨格筋細胞及びその誘導方法
EP2205723A1 (fr) Procédé d'amorçage de la différenciation des cardiomyocytes
WO2008151388A1 (fr) Procédés d'amorçage de la production d'insuline
JP5687059B2 (ja) 筋肉誘導前駆体組成物を利用した骨格筋の強化およびその処置
US20230212570A1 (en) Cell-penetrating peptide-microrna conjugates for intracellular cell delivery
Tanaka et al. The synthetic peptide SVVYGLR promotes cell motility of myogenic cells and facilitates differentiation in skeletal muscle regeneration
US20110223138A1 (en) Mesenchymal stem cells that express increased amounts of anti-apoptotic proteins
JP2022026709A (ja) ＴＧＦ－βを含む組織の線維化抑制剤

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 08756942

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 2008261550

Country of ref document: AU

ENP Entry into the national phase

Ref document number: 2008261550

Country of ref document: AU

Date of ref document: 20080613

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 2008756942

Country of ref document: EP