EP2205723A1

EP2205723A1 - Method of initiating cardiomyocyte differentiation

Info

Publication number: EP2205723A1
Application number: EP08756942A
Authority: EP
Inventors: Mark Alexander Kirkland; Liem Thanh Vo
Original assignee: Cytomatrix Pty Ltd
Current assignee: Cytomatrix Pty Ltd
Priority date: 2007-06-14
Filing date: 2008-06-13
Publication date: 2010-07-14
Also published as: WO2008151387A1; AU2008261550A1

Abstract

In one embodiment the invention relates to 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. In another embodiment the invention relates to 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. In a further embodiment the invention relates to 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.

Description

METHOD OF INITIATING CARDIOMYOCYTE DIFFERENTIATION

FIELD OF THE INVENTION

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.

BACKGROUND OF THE INVENTION

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.

Restoration of blood flow to the heart muscle is the mainstay of treatment for ischaemic heart disease. Medical approaches include thrombolysis (dissolving the blood clot) and coronary artery stenting, while the most common surgical approach is coronary artery bypass surgery. However, no matter how effective these therapies are, heart muscle that has already been damaged will not be repaired. The heart is one of the few organs in the body that has limited or no ability to repair itself. Although a rare "cardiac stem cell" has been identified by some workers¹, this population of cells seems to have a limited capacity to generate new cardiomyocytes in response to ischaemic injury. As a result, the hearts of patients who have suffered a myocardial infarction are permanently impaired, and in many cases this leads to a loss of function sufficient to cause heart failure.

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.

Cellular therapies for heart failure are under active investigation around the world. 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 myotherapy² utilises skeletal muscle stem cells (known as satellite cells). These cells are isolated from a skeletal muscle biopsy and grown in the laboratory before being injected into the damaged areas of the heart. Phase II clinical studies have suggested an improvement in heart function, but complications have included an increased incidence of abnormal heart rhythms, necessitating the insertion of a cardiac pacemaker as part of the treatment. In addition, the skeletal muscle cells do not appear to integrate into the heart muscle, and remain as skeletal muscle cells.

Yamada et al³ have demonstrated that over expression of the homeoprotein CSX/Nkx2.5 in mesenchymal stem cells can induce them to differentiate into heart 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 fate⁴. 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 pancreas⁵'⁶'⁷, particularly when fused with the herpes simplex-derived transcriptional activator, VP 16⁸. Similarly, the homeoprotein

CSX/Nkx 2.5 (951 bp.) is an essential transcription factor in cardiac cell formation⁹. 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 manner³.

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 molecules¹⁰. Other transduction domains, such as the HIV TAT sequence, have been used experimentally to aid transmembrane delivery of homeoproteins¹ ' .

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 development¹² (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 synchrony.

It is with the above background in mind that the present invention has been conceived.

SUMMARY OF THE INVENTION According to one embodiment of the present invention there is provided 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.

According to another embodiment of the present invention there is provided 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.

According to a still further embodiment of the present invention there is provided 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. Alternatively the method is conducted in vivo within a mammalian organism.

According to a preferred embodiment of the invention the methods conducted are for treatment and/or prevention of heart failure or ischaemic heart disease.

In another embodiment of the invention 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".

In another preferred embodiment of the invention the responsive mammalian cells are mammalian cells other than cardiac cells. Preferably 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 stem cells, cord blood stem cells, bone marrow stem cells and peripheral blood stem cells.

In another preferred embodiment of the invention 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 & Russell¹³, the disclosure of which is included herein in its entirety by way of reference. For example, bacterial toxin permeabilisation can utilise streptolysin O and cell-permeable peptide vectors can include antennapedia/penetratin TAT and signal-peptide based sequences.

The 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.

According to another embodiment of the present invention there is provided 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.

DESCRIPTION OF THE FIGURES

The present invention will be further described, by way of example only, with reference to the figures wherein:

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). d) Spontaneously beating target cells after exposing to CSX-VP 16 containing media, stained with f-actin (green), dapi nuclear stain (blue) and MLC (red).

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))

• Beating cells - aggregates of cells picked from CSX-treated cultures

• Non-beating cells - Cells from CSX-treated cultures that were not beating.

• Primary SKM - Skeletal muscle cells, untreated • Differentiated SkM - Skeletal muscle cells differentiated in vitro to form myotubes.

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 ρGL4.2(ANP) is the pGL4.2 vector with ANP promoter

• CHO ρGL4.2(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

CSX-VP 16

• 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

DESCRIPTION OF SEQUENCE LISTINGS The invention will be further described with reference to the sequence listings, where:

SEQ ID NO. 1 - amino acid sequence of human CSX/Nkx2.5. (UniProt accession number P52952)

SEQ ID NO. 2 shows the amino acid sequence of CSX/Nkx2.5 from mus musculus.

(GenBank accession NM_008700)

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.

DETAILED DESCRIPTION OF THE INVENTION

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that the prior art forms part of the common general knowledge in Australia.

In a broad aspect of the invention, and as mentioned above, there is provided 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. For example, 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 stem cells, liver stem cells, breast stem cells, lung stem cells, muscle stem cells, heart stem cells, eye stem cells, bone marrow stem cells, mesenchymal stem cells, spleen stem cells, immune system stem cells, cord blood stem cells, bone marrow stem cells and peripheral blood stem cells. Of these cell types, there may be some that due to their cellular machinery and mechanisms may be preferred over others. For example, cell types adapted to produce other protein products or hormones may demonstrate particular suitability for cardiomyocyte differentiation, when treated according to the invention. In particularly preferred embodiments of the invention 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. In the treatment methods of the invention it is preferable, however, for cells used in treating a particular patient to be derived from an individual of the same species. In one embodiment, and to minimise problems associated with immune rejection, cells used to treat a particular patient are derived from the same patient.

By 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. Preferably, although not essentially, 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 al¹⁴. Regulation of CSX/Nkx2.5 gene, expression is further described by Nemur and Nemur¹⁵. 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. However, it is preferred that 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.

Throughout this specification the terms "isolated" and "purified" are intended to define that an agent is at least 50% by weight free from proteins, antibodies and naturally-occurring organic molecules with which it is endogenously associated. Preferably 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. In the case where the CSX/Nkx2.5 protein is produced by recombinant technology, 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. Such 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 ^π, Yamada et al³ Akazawa et al¹⁸ and Shirai et al¹⁹.

As indicated above it is included within the invention to introduce not only CSX/Nkx2.5 or other transcription factors into the cells being treated to initiate cardiomyocyte differentiation, but also to introduce, either in addition or in their place, functionally equivalent analogues, variants or fragments. By the phrase "functionally equivalent" it is intended to convey that the variant, analogue or fragment is also effective in initiating cardiomyocyte differentiation in the cells treated according to the invention and preferably a given quantity of the analogue, variant or fragment is at least 10%, preferably at least

30%, more preferably at least 50, 60, 80, 90, 95 or 99% as effective as an equivalent amount of CSX/Nkx2.5 or the transcription factor from which the analogue, variant or fragment is derived. Determination of the relative efficacy of the analogue, variant or fragment can readily be carried out by utilising a prescribed amount of the analogue, variant or fragment in the methods of the invention and then comparing cardiomyocyte differentiation achieved against the same amount of CSX/Nkx2.5 protein or transcription factor from which the analogue, fragment or variant is derived. Quantification of cardiomyocyte differentiation by cells treated in this regard can readily be determined by routine methods, as discussed above.

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. In particular, 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.

By reference to "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. Throughout this specification the terms "polypeptide", "peptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues. The terms apply equally to 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.

The term "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. For example, 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). Another agent that may be utilised in assisting introduction of proteins or other agents into the cells is the 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, β-galactasidase, caspase 3, caspase 8, grandzime B and fluorescent antibody complexes.

Examples of cell-permeant peptide vectors that can be utilised to introduce agents into cells include antennapedia/penetratin, TAT and signal-peptide based sequences as further discussed in Dunican & Doherty²⁰, the disclosure of which is included herein in its entirety by way of reference. In this regard it is noted that 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.

A further specific technique that can be utilised in introducing agents into the cells to be treated is Pro-Ject™ transfection using Pro-Ject™ reagent (Pierce, Rockford IL, USA). Pro-Ject™ is a cationic lipid-based carrier system that can be used to deliver biologically active proteins, peptides or antibodies into cells. Pro-Ject™ 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. The amount of 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. However, as a general guide 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 μg/ml per 10⁵ 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. For example, 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. As mentioned above, patients may for example be treated in an in vivo or indeed an in vitro fashion. By 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. In relation to in vitro applications of the treatment methods it is intended to convey that 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.

In in vivo embodiments of the invention the treatment agent may similarly be exposed to the cells into which it is intended to be introduced by a variety of conventional means. For example, 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. Indeed, specific endogenous cells within the patient can be subjected to electroporation permeabilisation to assist in cellular uptake of the treatment agent. For example, techniques and agents previously mentioned in the context of introducing the treatment agent into the cells to be treated can similarly be utilised for in vivo treatments, where these methods or agents are physiologically compatible and do not present an undue risk to general patient health. Naturally, the general state of health, sex, weight, age and pregnancy status of the patient would be considered by the skilled medical practitioner administering the treatment when optimising the particular treatment to meet individual patient needs.

In conjunction with in vivo aspects of the invention it is possible to conduct surgical or other intervention before or after exposure of cells to the treatment agent. For example, it is possible to relocate myoblast cells to a damaged region of the heart either before or after exposure to the treatment agent. Further details on the formulation of 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 Sciences²¹, the disclosure of which is included herein in its entirety by way of reference. As will be understood, 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. In the case of in vivo administration of cells together with the treatment agent, 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). Such 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, such as, for example, by intravenous, intramuscular, intradernal, intraperitoneal, and subcutaneous routes, 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. In the practice of this invention, 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.

In determining the effective amount of the cells or treatment agent to be administered in the treatment or prophylaxis of conditions owing to diminished or aberrant cardiomyocyte differentiation, the physician evaluates toxicities, transplantation reactions, progression of the disease, and the like. For administration, 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.

This invention relies upon routine techniques in the field of cell culture, and suitable methods can be determined by those of skill in the art using known methodology (see, e.g., Freshney et al ²²). In general, 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.

In a preferred embodiment, 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.

For the cells of the invention that are cultured under adherent conditions, 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 CO₂ 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). It is also to be understood that 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 that can be adopted are well recognised in the art, as for example outlined in Ausubel et al¹⁶ and Sambruck & Russell¹³.

It is to be recognised that the present invention has been described by way of example only and that modifications and/or alterations thereto which would be apparent to persons skilled in the art, based upon the disclosure herein, are also considered to fall within the spirit and scope of the invention.

The invention will now be further described with reference to the following non-limiting examples.

EXAMPLES

EXAMPLE l : INITIATING CARDIOMYOCYTE DIFFERENTIATION IN MOUSE

SKELETAL MUSCLE SATELLITE CELLS USING RECOMBINANT CSX-VP 16

Materials and Methods

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 37⁰C in a 5% CO₂ incubator in low (5%) oxygen.

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 Igκ 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.

Assay methods: Real time PCR: Total RNA was isolated with Trizol reagent (Invitrogen) from manually isolated beating cells (skeletal muscles cells exposed to CSX protein), non- beating cells (skeletal muscle cells treated with CHO conditioned media), primary skeletal muscle tissue, primary heart tissue and differentiated myotubes. RNA was extracted using illustraRNAspin minicolumns (GE Healthcare) which contains a Dnase I treatment to remove genomic DNA contamination. One microgram of total RNA was used for reverse transcription reaction with AMV reverse transcriptase (Promega) and random primers (Promega) according to manufacturers instructions. 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.

Visual assessment: Transdifferentiated cells showing spontaneous beating were photographed, recorded on film and counted.

Results

Beating cells: Small aggregates of cells spontaneously beating in synchrony were observed in treated but not control cultures (Figure 1 , n=6 experiments).

Immunohistochemistry: Treated cells expressed connexin-43, myosin light chain (MLC) and f-actin as assessed by immunostaining and confocal microscopy (Figures 2a, 2b and

2c). Control satellite cells did not express connexin-43 but express low level of MLC and f-actin (Figure 2d). Real time PCR: Beating aggregates from CSX-treated cultures, but not non-beating cells from the same culture or untreated control cells, expressed the cardiac-specific RNA for

MLC-2v, ANF, CSX and Troponin I (Fig 3).

EXAMPLE 2: PROTOCOLS FOR CSX/NKX2.5 ISOLATION, AMPLIFICATION. RECOMBINANT PRODUCTION AND USE

Materials and Methods

Examples of Primer sequences for CSX: 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 CSX₅ 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 ρSecTag/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.

Recombinant 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.

To test the ability of the recombinant proteins to induce differentiation of skeletal muscle stem cells into cardiac cells, 5% conditioned media (i.e. media taken from cultures of stably producing CHO cell lines) or purified recombinant protein (500 ng/ ml) was added to cultures of skeletal muscle stem cells from four different mice, using the same culture conditions described in Example 1, except that cells were cultured in 5% CO₂ and 5% O₂ throughout. Conditioned media or recombinant protein was added every 2 days, together with fresh culture medium.

Results The functionality of the constructs was confirmed using a luciferase reporter assay (Figure 4). 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. Minimal luciferase was detected in the negative control constructs (empty pGL4.2 construct, or ANP-pGL4.2 construct transfected into CHO cells that had not been stably transfected with CSX constructs), whereas a significant increase in signal was seen when this construct was transfected into the CSX producing lines. Unmanipulated CSX gave the strongest signal, followed by CSX-VP 16 the TAT-CSX.

To test the functionality of the purified recombinant protein, primary skeletal muscle cells were cultured in the presence of both conditioned media (as per example 1 above) or purified protein. After 2 days, cells were observed beating in all treated cultures, more so in those treated with purified recombinant protein. Negative controls were cells treated with CHO conditioned media (same cell line but without stably transfected CSX constructs), and CHO-HIS - CHO conditioned medium that had been passed through the Nickel Column purification process described above. After 8 days, cells treated with recombinant protein were observed to be rhythmically beating, with less seen in cultures treated with CSX-CM or CSX-VP 16 CM and no beating cells in the control cultures (Figure 5).

EXAMPLE 3: RESPONSE OF BEATING CELLS TO PHARMACOLOGICAL

AGENTS

Materials and Methods

Cultures containing aggregates of beating cells were examined for response to the beta agonist isoprenaline (from Abbott Australia Pty Ltd) and the beta antagonist Betaloc IV (Metoprolol tartrate from AstraZeneca). The tissue under investigation was focused under a light microscope, kept under constant temperature of 37⁰C and filmed at speed of 25 frames per second. Cumulative dose-responses to isoprenaline were assessed at doses of

• lxlO^"9M • 3xlO^"9M

• IxIO-⁸M

• 3xlO^"8M

• lxlO^"7M • 3xlO-⁷M

• IxIO-⁶M

The tissue was then washed twice with PBS₅ 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^"6M.

Results

Aggregates (n=10) showed increased contraction rate and force with the addition of isoprenaline above a dose of between 3 x lO^'8 and 1 x 10^"7 M. Addition of metoprolol tartrate caused the beating to slow down or cease. These results are consistent with the activity of cardiomyocyte cells.

ABBREVIATIONS

ATP adenosine triphosphate

CaI CSX-associated LIM protein

CSX/Nkx2.5 Cardiac-specific homeoprotein

DNA deoxyribose nucleic acid

DME Dulbecco's modified Eagles' medium

DTT dithiothreitol

EGF epidermal growth factor

FGF Fibroblast growth factor

FCS fetal calf serum

FRT flipase recognition target

GATA4 GATA-binding factor 4

GTP guanosine triphosphate

His Histidine

IGF-I Insulin-like growth factor 1

IgG immunoglobulin G

IgK Immunoglobulin kappa light chain moAb monoclonal antibody

NTP nucleotide triphosphate

PBS phosphate buffered saline

PEF polyethylene glycol

PMSF phenyl methyl sulfonyl fluoride

RAM-HRP rabbit anti-mouse horseradish peroxidase

RFLP restriction fragment length polymorphism

RIA radio-immuno assay

RPMI Roswell Park Memorial Institute

RT-PCR real time polymerase chain reaction

TAT Transactivator

TOPO Topoisomerase

TRITC tetramethyl rhodamine isothiocyanate

VP16 Viral; protein 16 REFERENCES

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² Smits PC. Myocardial repair with autologous skeletal myoblasts: a review of the clinical studies and problems. Minerva Carioangiol 52:525-35 (2004)

³ Yamada Y, Sakurada K, Takeda Y, Gojo S, Umezawa A. Single-cell-derived mesenchymal stem cells overexpressing Csx/Nkx2.5 and GATA4 undergo the stochastic cardiomyo genie fate and behave like transient amplifying cells. Exp. Cell Res. 313(4):698- 706.(2007)

⁴ Gehring WJ., Affolter M, Burglin T. Homeodomain proteins. Anna. Rev. Biochem. 1994;63:487-526.

⁵ Yamada S, Yamamoto Y, Nagasawa M, Hara A, Kodera T, Kojima I. In vitro transdifferentiation of mature hepatocytes into insulin-producing cells. Endocr. J. 2006 Dec;53(6):789-95. Epub 2006

⁶ Lu S, Wang WP, Wang XF et al. Heterogeneity in predisposition of hepatic cells to be induced into pancreatic endocrine cells by PDX-I. World J Gastroenterol. 2005 Apr 21;l l(15):2277-82

⁷ Noguchi H, Kaneto H, Weir GC. PDX-I protein containing its own antennapedia-like protein transduction domain can transduce pancreatic duct and islet cells. Diabetes 2003 Jul;52(7): 1732-7

⁸ Hideaki K, Yoshihisa N, Takeshi M, Taka-aki M, Munehide M, Masatsugu H, and Yoshimitsu Y. PDX-1/VP16 fusion protein, together with NeuroD or Ngn3, markedly induces insulin gene transcription and ameliorates glucose tolerance. Diabetes April 2005;54:1009-1021

⁹ Hideko K, Bora L, Schott JJ et al. Loss of function and inhibitory effects of human CSX/NKX2.5 homeoprotein mutations associated with congenital heart disease. The J of Clin. Invest. 2000; vol 106(2):299-307.

¹⁰ Amsellem S, Pflumio F, Bardinet D, et al. Ex vivo expansion of human hematopoitic stem cells by direct delivery of the HOXB4 homeoprotein-Technical reports. Nature Medicine 2003;Vol 9(11):1423-1427

¹¹ Kwon YD, Oh SK, Kim HS et al. Cellular manipulation of human embryonic stem cells by TAT-PDXl protein transduction. MoL Ther. 2005 Jul;12(l):28-32 ¹² Totko H et al. Csx/Nkx2-5 Is Required for Homeostasis and Survival of Cardiac Myocytes in the Adult Heart. J Biol Chem 24735-24743, 2002

¹³ Sambruck & Russell, Molecular Cloning: A laboratory manual, 3rd Edition, 2001, Cold Spring Harbour Laboratory Press, New York.

¹⁴ Turbay,D., Wechsler,S.B., Blanchard,K.M. and Izumo,S. Molecular cloning, chromosomal mapping, and characterization of the human cardiac- specific homeobox gene hCsx MoI Med. 2 (1), 86-96 (1996)

¹⁵ Nemer G, Nemer M. Regulation of heart development and function through combinatorial interactions of transcription factors. Ann Med. 2001 Dec;33(9):604-10

¹⁶ Ausubel et al, Current Protocols in Molecular Biology, John Wiley & Sons, New York, 2000.

¹⁷ Nemer G, Nemer M. Regulation of heart development and function through combinatorial interactions of transcription factors. Ann Med. 2001 Dec;33(9):604-10

¹⁸ Akazawa H et al. A novel LIM protein CaI promotes cardiac differentiation by association with CSX/NKX2-5. JCeIl Biol 164(3): 395-40, 2004

¹⁹ Shirai M et al. The Polycomb-group gene Rae28 sustains Nkx2.5/Csx expression and is essential for cardiac morphogenesis. JCl in Invest 110(2): 177- 184, 2002

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²¹ Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pennsylvania, USA

²² Freshney et al., Culture of Animal Cells (e. sup. rd. ed. 1994).

Claims

1. 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.

2. The method of claim 1 which is conducted in vivo within a mammalian organism.

3. The method of claim 1 which is conducted in vitro,

4. 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.

5. 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.

6. The method of claim 5 wherein introduction of CSX/Nkx2.5 protein and optionally other transcription factors or their functionally equivalent analogues, variants or fragments is conducted in vitro, with the cells subsequently being returned to the patient.

7. The method of claim 5 wherein introduction of CSX/Nkx2.5 protein and optionally other transcription factors or their functionally equivalent analogues, variants or fragments is conducted in vivo within a mammalian organism.

8. The method of any one of claims 1 to 7 wherein 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.

9. The method of any one of claims 1 to 8 wherein the responsive mammalian cells are mammalian cells other than cardiac cells.

10. The method of any one of claims 1 to 8 wherein 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 stem cells, cord blood stem cells, bone marrow stem cells and peripheral blood stem cells.

11. The method of any one of claims 1 to 10 wherein the CSX/Nkx2.5 protein or a functionally equivalent analogue, variant or fragment thereof and optional one or more other transcription factors is introduced utilising detergent, bacterial toxin or electroporation permeabilisation, lisosomal delivery or with the use of cell- permeant peptide vectors or polyethylene glycol (PEG). - ZI ¬

YI. The method of claim 11 wherein the bacterial toxin permeabilisation utilises streptolysin O.

13. The method of claim 11 wherein the cell-permeant peptide vectors include antennapedia/penetratin TAT and signal-peptide based sequences.

14. 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.

15. The agent of claim 15 further comprising one or more permeabilisation agents.

16. Use of CSX/Nkx2.5 protein or a functionally equivalent analogue, variant or fragment thereof in preparation of an agent for introduction into a responsive mammalian cell to initiate cardiomyocyte differentiation in the cell.

17. Use of CSX/Nkx2.5 protein or a functionally equivalent analogue, variant or fragment thereof in preparation of an agent for introduction into one or more responsive cells that have been removed from a patient suffering from or prone to suffer from heart failure or ischaemic heart disease and are being cultured in a suitable medium, wherein the introduction into the responsive cell/s initiates cardiomyocyte differentiation of the cells, which cells or cells derived from them are subsequently returned the to the patient.

18. Use of CSX/Nkx2.5 protein or a functionally equivalent analogue, variant or fragment thereof in preparation of an agent for introduction into one or more responsive cells of a patient suffering from or prone to suffer from heart failure or ischaemic heart disease, wherein the introduction into the responsive cell/s initiates cardiomyocyte differentiation of the cells.