WO2008151388A1

WO2008151388A1 - Methods of initiating insulin production

Info

Publication number: WO2008151388A1
Application number: PCT/AU2008/000863
Authority: WO
Inventors: Mark Kirkland; Nadine Blyth
Original assignee: Cytomatrix Pty Ltd
Priority date: 2007-06-15
Filing date: 2008-06-13
Publication date: 2008-12-18

Abstract

The present invention relates to methods of initiating insulin production in mammalian cells, which involve introducing into the cells pdx-1 protein or a functionally equivalent analogue, variant or fragment thereof which is in functional conjunction with both the VP16 transcriptional activator sequence and the TAT transduction domain. The invention also relates to methods of treatment or prevention of diabetes mellitus, and in particular of type 1 diabetes. The methods of treatment or prevention involve the initiation of insulin production in cells that would otherwise not produce insulin, or of increased insulin production in cells that normal would produce insulin. The methods of the invention can be conducted using the patient's own cells either in vivo, or in vitro, with the insulin producing cells then being returned to the patient.

Description

METHODS OF INITIATING INSULIN PRODUCTION

FIELD OF THE INVENTION

The present invention relates to methods of initiating insulin production in mammalian cells, which involve introducing into the cells pdx-1 protein or a functionally equivalent analogue, variant or fragment thereof which is in functional conjunction with both the VP 16 transcriptional activator sequence and the TAT transduction domain. The invention also relates to methods of treatment or prevention of diabetes mellitus, and in particular of type 1 diabetes. The methods of treatment or prevention involve the initiation of insulin production in cells that would otherwise not produce insulin, or of increased insulin production in cells that normal would produce insulin. The methods of the invention can be conducted using the patient's own cells either in vivo, or in vitro, with the insulin producing cells then being returned to the patient.

BACKGROUND OF THE INVENTION

Diabetes mellitus, which is a chronic disease characterised by abnormally high blood glucose levels, is a major healthcare issue, with some 12 million people (or roughly 6% of the population) in the United States diagnosed with the disease. It is also estimated that a further 10 million people in the United States may be undiagnosed diabetes sufferers. Not only does diabetes mellitus lead to blindness, kidney failure and nerve damage but it is also a key contributing factor to atherosclerosis, which may lead to stroke, coronary heart disease and other blood vessel diseases. Diabetes mellitus is a metabolic disorder that adversely affects the ability to produce and use insulin, which is a hormone necessary for regulation of blood glucose levels. Under normal circumstances an increase in blood glucose triggers insulin production, which results in glucose removal from the bloodstream and storage in the form of glucagon.

The two forms of diabetes mellitus are type 1 and type 2 diabetes. Type 1 diabetes is also referred to as insulin dependent diabetes mellitus or juvenile onset diabetes mellitus. This form of the disease is characterised by autoimmune attack of the pancreatic β-cells located within the islets of Langerhans, which are responsible for insulin production, thereby rendering the patient incapable of producing insulin. Type 2 diabetes, which is also referred to as non-insulin dependent diabetes mellitus or adult onset diabetes mellitus, is characterised by inappropriate levels of insulin production. In many cases, the cause of type 2 diabetes is sensitivity of cells (particularly fat and muscle cells) to insulin, with the result that unusually high levels of insulin are produced in an effort to register the signal to lower blood glucose levels.

Type 2 diabetes is treated at least initially by exercise and weight reduction (as it is linked to obesity) as well as with medications that may increase insulin production, decrease glucose removed from storage by the liver, increase cellular insulin sensitivity or decrease absorption of carbohydrates from the intestines, depending upon a patient's particular circumstances. As with type 2 diabetes sufferers, patients affected with diabetes type 1 will need to have diet and exercise closely monitored. However, type 1 diabetes mellitus patients rely on insulin medication for survival. While there have been a number of important developments in terms of insulin administration over recent years, including development of intranasal, transdermal and oral forms of insulin administration, these have to date proved largely ineffective or require further development before regulatory approval is likely to be secured. Therefore, most type 1 diabetes sufferers rely upon regular monitoring of glucose levels and administration of insulin by injection, or in some cases via the use of an insulin delivery pump. As can be imagined, these means of insulin delivery are time consuming and can be traumatic. More importantly, forgetting to take the medication or taking an incorrect dose can lead to serious and potentially life threatening consequences.

More recently, there has been considerable work devoted to transplantation of either whole pancreas or pancreatic islets of Langerhans as a means of returning to patients the ability to endogenously produce insulin. While these approaches offer some promise, transplant recipients must then be treated with immunosuppressing drugs that are known to have serious side effects. There is also a shortage of tissue available for transplantation. More recently, Taniguchi et al (1) have experimented with the controlled differentiation of adult pancreatic stem cells into insulin-producing cells, and in doing so have demonstrated that adenovirus-mediated expression of pdx-1 can activate the endogenous pdx-1 gene, leading to β-cell neogenesis and ductal proliferation. While offering some potential to allow endogenous production of insulin in type 1 diabetes patients, it is likely this production would be transient as the β-cells produced by this neogenesis approach would likely be subject to similar autoimmune attack to the patient's original β-cells. Furthermore, this approach requires adenoviral transfection of the pdx- 1 gene, and is therefore subject to the spectre of community safety and ethical concerns that relate to gene therapies. Other workers have similarly demonstrated that transfection of liver cells with pdx- 1 (2), or pdx- 1 modified by the addition of a transcriptional activation sequence, VP 16 (3), can induce transdifferentiation of liver cells into insulin producing cells. These results confirm the critical role of pdx-1 in controlling pancreatic endocrine differentiation. Further support has been obtained from two groups who used recombinant pdx-1 protein to induce pancreatic differentiation. Noguchi et (4) al exposed pancreatic ductal cells to recombinant pdx-1 protein produced in bacteria, and demonstrated that the cells expressed a range of beta-cell genes, including insulin. These cells were able to moderate streptozitocin-induced hyperglycaemia in mice. Kwon et al (5) fused pdx-1 to the TAT transduction domain and showed that this protein could up-regulate beta cell genes in embryonic stem cells, although they could not demonstrate efficacy of unmodified pdx-1 protein.

The present inventors have now determined that it is possible to initiate insulin production in cells that would usually not produce insulin and to increase insulin production in cells that usually do produce it, by introducing into such cells an effective amount of pdx-1 protein, or a functionally equivalent analogue, variant or fragment thereof in functional conjunction with (preferably, but not necessarily, fused with the pdx-1 protein, or a functionally equivalent analogue, variant or fragment thereof) both the VP 16 transcriptional activator sequence and the TAT transduction domain (hereinafter referred to as "TAT / pdx-1 / VP16"). While the role of pancreatic duodenal homeobox 1 (pdx-1) in pancreatic development (5) and β-cell neogenesis (1) is known, as well as the fact that pdx-1 transcription is regulated by a complex of transcription factors including pdx-1 itself and HNF- lα, HNF-3β, SPl, SP3, USFl and USF2 it has not previously been suggested that introduction of TAT / pdx-1 / VP 16 protein (as opposed to transfection of the pdx-1 gene) into cells other than pancreatic β-cells, optionally in conjunction with other relevant transcription factors, could be effective in initiating insulin production. It has also not previously been suggested that this insulin production could be sustained within the cells treated after the exposure to TAT / pdx-1 / VP 16, and nor that the insulin production could be glucose concentration dependent.

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 insulin production in a responsive but otherwise non-insulin producing mammalian cell, which comprises introducing into the cell an effective amount for initiating insulin production within the cell of pdx-1 protein or a functionally equivalent analogue, variant or fragment thereof which is in functional conjunction with both the VP 16 transcriptional activator sequence and the TAT transduction domain. 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 increasing insulin production in an insulin producing mammalian cell, which comprises introducing into the cell an effective amount for increasing insulin production within the cell of pdx-1 protein or a functionally equivalent analogue, variant or fragment thereof which is in functional conjunction with both the VP 16 transcriptional activator sequence and the TAT transduction domain. 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 diabetes mellitus, which comprises removing from the patient one or more responsive but otherwise non-insulin producing cells and culturing the cells in a suitable medium, introducing into the cells an effective amount for initiating insulin production within the cells of pdx-1 protein or a functionally equivalent analogue, variant or fragment thereof which is in functional conj unction with both the VP 16 transcriptional activator sequence and the TAT transduction domain, 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 diabetes mellitus, which comprises introducing into responsive but otherwise non-insulin producing cells of the patient an effective amount for initiating insulin production within the cells of pdx-1 protein or a functionally equivalent analogue, variant or fragment thereof which is in functional conjunction with both the VP 16 transcriptional activator sequence and the TAT transduction domain. Introduction of pdx-1 protein or a functionally equivalent analogue, variant or fragment thereof which is in functional conjunction with both the VP 16 transcriptional activator sequence and the TAT transduction domain, and optionally other transcription factors or their functionally equivalent analogues, variants or fragments may 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 another embodiment of the invention there is provided a method of insulin production which comprises introducing into cells of a cultured mammalian cell line an effective amount for initiating insulin production within the cells of pdx-1 protein or a functionally equivalent analogue, variant or fragment thereof which is in functional conjunction with both the VP 16 transcriptional activator sequence and the TAT transduction domain, and subsequently isolating insulin produced.

According to a preferred embodiment of the invention the methods conducted are for treatment and/or prevention of type 1 diabetes mellitus.

In another embodiment of the invention the pdx-1 protein or a functionally equivalent analogue, variant or fragment thereof which is in functional conjunction with both the VP 16 transcriptional activator sequence and the TAT transduction domain is introduced into the cells in conjunction with one or more other transcription factors selected from HNF- lα, HNF-3β, HNF- lβ, SPl, SP3, USFl and USF2, or their functionally equivalent analogues, variants or fragments. The combination of pdx-1 protein or a functionally equivalent analogue, variant or fragment thereof which is in functional conjunction with both the VP 16 transcriptional activator sequence and the TAT transduction domain (referred to generally as TAT / pdx-1 / VP 16) and optional other transcription factors or their functionally equivalent analogues, variants or fragments along with other optional components will for convenience by referred to herein as the "treatment agent".

In embodiments of the invention relating to increased production of insulin in cells that normally produce insulin the cells involved are preferably pancreatic β-cells. In another preferred embodiment of the invention the responsive but otherwise non-insulin producing mammalian cells are mammalian cells other than pancreatic β-cells. Preferably the responsive but otherwise non-insulin producing 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, 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 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 permeabilisation agents such as detergent, bacterial toxin, cell-permeant peptide vectors or polyethylene glycol (PEG) or permeabilising treatments such as electroporation permeabilisation or lisosomal delivery, each of which are techniques well known in the art as described in Sambruck & Russell (7), the disclosure of which is included herein in its entirety by way of reference. For example, bacterial toxin permeabilisation may utilise streptolysin O and cell-permeable peptide vectors may include antennapedia/penetratin

TAT and signal-peptide based sequences. In a preferred embodiment of the invention the pdx-1 protein or a functionally equivalent analogue, variant or fragment thereof is fused with both the VP 16 transcriptional activator sequence and the TAT transduction domain, or their functionally equivalent variants or fragments.

TAT / pdx-1 / VP 16 protein or optional other transcription factors or their functionally equivalent analogues or variants may be produced synthetically, recombinantly or may be isolated from mammalian cells.

According to another embodiment of the present invention there is provided an agent for increasing insulin production in an insulin producing mammalian cell or for initiating insulin production in a responsive but otherwise non-insulin producing mammalian cell which comprises pdx-1 protein or a functionally equivalent analogue, variant or fragment thereof which is in functional conjunction with both the VP 16 transcriptional activator sequence and the TAT transduction domain. Preferably the agent comprises pdx-1 protein or a functionally equivalent analogue, variant or fragment thereof fused with both the VP 16 transcriptional activator sequence and the TAT transduction domain. The agent may further comprise one or more of other transcription factors or their functionally equivalent analogues or variants, permeabilisation agents and physiologically acceptable carriers and/or diluents.

DESCRIPTION OF THE FIGURES

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

Figure 1 shows CHO cells stained for pdx-1 protein using anti-V5 antibody.

Figure 2 shows CHO cells stained for TAT-pdx-1 protein using anti-V5 antibody.

Figure 3 shows CHO cells stained for pdx-1 -VP 16 protein using anti-V5 antibody. Figure 4 shows CHO cells stained for TAT-pdx-l-VP16 protein using anti-V5 antibody.

Figure 5 shows control CHO cells stained using anti-V5 antibody.

Figure 6 shows a Western Blot of purified recombinant pdx-1 proteins stained using V5 antibody, wherein

Lane 1. Trichrom ranger Marker

Lane 2. PDX-I ~ 38kDa

Lane 3. PDX- 1 VP 16 ~ 47kDa

Lane 4. TAT PDX-I - 4OkDa

Lane 5. TAT PDX-I VP16 ~ 49kDa

Lane 6. Vector alone

Lane 7. Trichrom ranger Marker

Figure 7 shows Luminescence measurements of CHO cells transfected with the pGL reporter constructs and co-cultured with CHO cells stably transfected with PDX-I constructs, as below:

pGL construct PDXl construct

Lane 1 pGL4.20 containing PPI promoter PDXl

Lane 2 pGL4.20 containing PPI promoter PDX1-VP16

Lane 3 pGL4.20 containing PPI promoter TAT-PDX

Lane 4 pGL4.20 containing PPI promoter PAT-PDX 1 -VP 16

Lane 5 Vector only

Lane 6 pGL4.20, no promoter Vector only

Lane 7 pGL4.20 containing PPI promoter Vector only

Lane 8 pGL4.13 (Positive control) Vector only

Figure 8 shows Luminescence measurements of CHO cells stably transfected with vector only, PDX-I construct or PDXl -VP 16 construct, then transfected with the pGL reporter constructs, as below: pGL construct PDXl construct

Lane 1 pGL4.20, no promoter None

Lane 2 pGL4.20 containing PPI promoter None

Lane 3 pGL4.20 containing PPI promoter Vector only

Lane 4 pGL4.20 containing PPI promoter PDXl

Lane 5 pGL4.20 containing PPI promoter PDX 1 -VP 16

Lane 6 pGL4.20 containing PPI promoter TAT-PDX

Lane 7 pGL4.20 containing PPI promoter TAT-PDX 1 -VP 16

Lane 8 pGL4.13 (Positive control) Vector only

Figure 9 shows. RTPCR measurement of insulin mRNA expression by hepatocytes cultured in the presence of CHO conditioned medium containing PDX-I, PDXl- VP 16, TAT-PDX or TAT-PDXl -VP 16 proteins, or no recombinant protein ("Vector"). Figure 10 shows RTPCR measurement of GLUT2 mRNA expression by hepatocytes cultured in the presence of CHO conditioned medium containing PDX-I, PDXl- VP 16, TAT-PDX or TAT-PDXl -VP 16 proteins, or no recombinant protein ("Vector").

DESCRIPTION OF SEQUENCE LISTINGS

SEQ ID NO 1 shows the amino acid sequence of human pdx-1.

SEQ ID NO 2 shows the amino acid sequence oϊmus muscularis pdx-1.

SEQ ID NO 3 shows the amino acid sequence of mus muscularis TAT-pdx-1.

SEQ ID NO 4 shows the amino acid sequence of mus muscularis pdx-l-VP16.

SEQ ID NO 5 shows the amino acid sequence of mus muscularis TAT-pdx-1 -VP 16.

SEQ ID NO 6 shows the sequence of herpesvirus VP 16.

SEQ ID NO 7 shows the sequence of HIV TAT.

SEQ ID NO 8 shows the amino acid sequence of human TAT-pdx-1 -VP 16.

SEQ ID NO 9 shows the amino acid sequence of human HNF lα.

SEQ ID NO 10 shows the amino acid sequence of human HNF3α.

SEQ ID NO 11 shows the amino acid sequence of human SP 1.

SEQ ID NO 12 shows the amino acid sequence of human SP3.

SEQ ID NO 13 shows the amino acid sequence of human USFl. SEQ ID NO 14 shows the amino acid sequence of human USF3.

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 one broad aspect of the invention, and as mentioned above, there is provided a method of initiating insulin production in a responsive but otherwise non-insulin producing mammalian cell. By the phrase "responsive but otherwise non-insulin producing mammalian cell", it is intended to encompass mammalian cells other than pancreatic β- cells, which in unmodified mammalian systems are the only insulin producing cells. For example, types of mammalian cells that may be treated according to the invention to initiate insulin production 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 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 insulin production, when treated according to the invention. In particularly preferred embodiments of the invention the cells utilised are stem cells, as referred to above, hepatocytes or fibroblasts. The cells utilised according to the invention may be derived from any of a variety of mammalian organisms, including, but not limited to humans, primates such as chimpanzees, gorillas, baboons, orang-utans, 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. Most preferably, and to minimise problems associated with immune rejection, cells used to treat a particular patient will be derived from the same patient.

By the phrase "initiating insulin production" 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 insulin production (or increase insulin production) as a result of the treatment according to the invention. Preferably, although not essentially, the insulin production of the cells concerned will be glucose dependent such that following the treatment according to the invention the treated cells, or cells derived from them, will produce insulin or increase insulin production, following exposure to glucose. Preferably the level of insulin production will increase if the treated cells are exposed to increasing glucose concentrations. Cellular insulin production may for example be detected by immunohistochemistry, by the use of insulin specific stains or other insulin binding and readily detectable compounds, by radioimmunoassay or real time PCR which more particularly monitors insulin gene expression. At least in the case of radioimmunoassay and real time PCR it is possible to quantify the levels of insulin production in a particular population of cells. In another broad aspect of the invention, and as also mentioned above, there is provided a method of increasing insulin production in cells, such as pancreatic β-cells. By "increasing production" it is intended to mean that the level of production of insulin in a population of cells in increased over the levels produced prior to the exposure to the treatment agent. For example the increase in production may be by at least 5%, preferably at least 10%, 20%, 30% 50%, 75% or at least 100%, 200% or 500%. In preferred embodiments of the invention the increase in insulin production is at least about a 10- or 20-fold increase.

A key aspect of the present invention is the introduction into the cell or cells in which insulin production is to be initiated of pdx-1 protein or a functionally equivalent analogue, variant or fragment thereof in functional conjunction with both the VP 16 transcriptional activator sequence and the TAT transduction domain ("TAT / pdx-1 / VP 16"). By "functional conjunction" it is intended to convey that the elements are provided in a form that permits them to interact in a functional manner; namely to be taken up by the cells and to initiate or increase insulin production within those cells. Two or three of the elements may be fused to form a single protein molecule. They can equally be provided separately in the same formulation or may be fused or bound to other elements such as other active agents, delayed release substrates or matrices or other proteins or peptides.

Pancreatic duodenal homeobox 1 (pdx-1) is an orphan homeo domain protein known to be important in pancreas development, and which has also been referred to in some reports as insulin promoter factor 1 (IPF 1). The pdx-1 gene is localised on human chromosome 13 and the nucleotide sequence of the gene has been reported by Ohlsson et al (7). Regulation of pdx-1 gene expression is further described by Melloul et al (6). The disclosures of these papers are included herein in their entirety, by way of reference.

TAT / pdx-1 / VP 16 may 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, nutrients or active compounds. However, it is preferred that the elements of TAT / pdx-1 / VP 16 are at least to some extent isolated or purified from other components of a cytoplasmic extract from which they 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 (8), 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 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 may include one or more of hepatocyte nuclear factor (HNF) 3-β, HNF-I α, SPl, SP3, USF 1 and USF 2, as for example referred to in Melloul et al (6).

As indicated above it is included within the invention to introduce not only pdx-1 or other transcription factors into the cells being treated, 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 or increasing insulin production 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 pdx-1 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 insulin production achieved against the same amount of pdx-1 protein or transcription factor from which the analogue, fragment or variant is derived. Quantification of insulin production 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 or regulatory sequences and to encompass the deletion of sequences within the normal protein sequence 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 treatment agent may 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 may be introduced by using permeabilisation agents such as utilising detergent, bacterial toxin or by using 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 may 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 (9). 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 may be utilised to introduce agents into cells include antennapedia/penetratin, TAT and signal-peptide based sequences as further discussed in Dunican & Doherty (10), the disclosure of which is included herein in its entirety by way of reference. Pdx-1 itself contains an intrinsic cell-permeant sequence, homologous to the anennapedia/penetratin domain. In a preferred embodiment, therefore, the recombinant pdx-1 protein or analogue or variant thereof will enter the cell without the use of additional cell permeabilisation methods as described above. The HIV-derived TAT sequence is both a cell permeant peptide and a transcriptional activator. Therefore, the addition of a TAT sequence to the pdx-1 protein enhances both its cell entry and its activity. The VP 16 sequence, derived from Herpes simplex virus, is an additional transcriptional activator which enhances activity of the recombinant protein. Therefore, when pdx-1 protein is in functional conjunction with both the TAT and VP 16 sequences there is shown to be both enhanced cell entry and transcriptional activity, which could not have been predicted before the work by the present inventors. The amount of TAT / pdx-1 / VP 16 or of the treatment agent introduced into the cells in which insulin production is intended to be initiated (or increased) and which is effective for insulin production, 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 insulin production within the cell of TAT / pdx-1 / VP 16 may fall within the range of 1 ng/ml to 1 microgram per ml.

The initiation or increase of insulin production in mammalian cells that may be achieved through the present invention may be utilised in both therapeutic and prophylactic contexts. For example, mammalian and particularly human patients identified as possessing risk factors for development of diabetes mellitus, and particularly type 1 diabetes, may 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 diabetes mellitus, particularly type 1 diabetes, may 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 insulin production 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 insulin production 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 insulin production 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 endoscopic retrograde cholangiopancreatography (ERCP) type techniques can readily be adapted for implantation of cells treated according to the invention. This technique involves introduction of a duodenoscope via the mouth to the duodenum and subsequently into the papilla of Vater and then into the bile ducts to access the liver or the pancreatic duct to access the pancreas.

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, may be injected into the appropriate tissue or organ, may be applied to the skin 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 may 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 may 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.

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 Sciences (11), the disclosure of which is included herein in its entirety by way of reference. As will be understood, physiologically or 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.

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 conventional physiologically acceptable carriers and diluents such as antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient. Aqueous and non-aqueous sterile suspensions that can include carriers and diluents such as 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. In particular the invention included controlled or delayed release formulations of the treatment agent that allow for sustained release of the treatment agent in the vicinity of the cells to be treated. Such formulations are well known in the art and are described in detail in Remington's Pharmaceutical Sciences (1 1).

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 insulin production, 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- insulin production 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 (12)). 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 10³ to 1 x 10⁷ 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 are used for cell cultures. 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%. The oxygen concentration may be the same as that in the atmosphere (approximately 20%), or may, through the use of special incubators, be at a lower concentration that more closely matches the physiological oxygen in tissues. The preferred oxygen concentration will depend on the cells being cultured, but may typically be one of 1 %, 2%, 5%, 10%, 15% or 20%.

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 is used in the methods of the invention. The culture medium is usually buffered to maintain the cells at a pH preferably from 1.2-1 A. Other supplements to the media include, e.g., antibiotics, amino acids, sugars, and growth factors such as hepatocyte growth factor/scatter factor, epidermal growth factor, insulin-like growth factor or fibroblast growth factor or a variant thereof.

In another aspect of the invention it is possible to produce insulin in mammalian cells, and preferably in a stable cell line by exposing cells from the cell line to the treatment agent and then by isolating insulin so produced. In this aspect of the invention cells may be drawn from the variety of mammalian cells mentioned above and may be cultured in accordance with the cell culture conditions also explained above. A "stable" cell line or culture is one that can grow in vitro for an extended period of time, such as for at least about 50 cell divisions, or for about six months, more preferably for at least about 150 cell divisions, or at least about 10 months, and more preferably for at least about a year.

It is also to be understood that the pdx-1 or other transcription factors or their functionally equivalent analogues or variants, TAT and VP 16 that are included within the treatment agent may be chemically synthesised, recombinantly produced or isolated from mammalian cells. Chemical synthesis, recombinant production and isolation techniques that may be adopted are well recognised in the art, as for example outlined in Ausubel et al (8) and Sambruck & Russell (9).

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 1: PREPARATION OF MAMMALIAN CELLS SECRETING

RECOMBINANT PDXl AND VARIANT PDXl PROTEINS Materials and Methods Pdx-1 was cloned from a mouse cDNA library by standard methods. Three variants were also cloned - TAT-pdx-1, pdx-1 -VP 16, and TAT-pdx-l-VP16 (where the notation "TAT- pdx-l-VP16" indicates fusion of the three elements). All these sequences were then cloned into a Gateway Entry Vector (Invitrogen) which allows the sequences to be inserted into multiple destination vectors. The pdx-1 sequences were also cloned into the pSecTag/FRT/V5-His-TOPO vector using the TOPO cloning system (Invitrogen). This vector adds an Ig kappa secretory signal at the 5' end, and adds a V5 tag and a poly His tail at the 3' ane of the sequence. The vector also includes sequences that enable the clone to be inserted into a known site in an FRT cell line to maximise protein production (Invitrogen).

Results

AU sequences were cloned into FRT-CHO cells to create stable cell lines. Production of protein by these cells was confirmed by immunohistochemistry using anti-V5 antibodies (Figure 1-5). Production of recombinant protein was confirmed by western blot (Figure 6).

EXAMPLE 2: BINDING OF RECOMBINANT PDXl PROTEINS TO THE

PREPROΓNSULΓN i GENE PROMOTER

Materials and Methods

The preproinsulin I gene promoter sequence was amplified from genomic mouse DNA using PCR. This sequence was then inserted into a cloning site of the pGL4.20 luciferase reporter gene vector (Invitrogen) according to the manufacturer's instructions. In brief, this vector drives expression of the luciferase gene upon binding of a transcription factor to the inserted promoter sequence. This vector was then transfected using lipofectamine into

CHO cells, which were then co-cultured with stable cell lines transfected with pdx-1 constructs. Controls included - CHO cells transfected with empty pSecTag/FRT/V5-His-

TOPO vector, pGL4.20 vector alone (no preproinsulin I promoter sequence inserted), pGL4.20 vector containing promoter co-cultured with CHO cells not expressing protein, and a positive control consisting of vector pGL4.13, which has constitutive expression of the luciferase gene.

Alternatively, the reporter gene constructs were transfected directly into CHO cells stably transfected with pdx-1 constructs or control cells.

Culture conditions: All cells were cultured in RPMI medium with 10% Fetal Calf serum (FCS) at 37°C in a 5% CO₂ incubator.

Assay methods: Luciferase activity was measured using a luciferase detection kit (Invitrogen) according to the manufacturer's instructions, and quantitated using a luminometer.

Results and Discussion Figure 7 shows the luminescence readings for co-culture of CHO cells secreting the various pdx-1 proteins, compared to controls. These data demonstrate that the CHO cells are secreting recombinant protein, which is then being taken up by co-cultured target cells and is initiating expression of the reporter gene. The most effective protein was TAT-pdx- 1-VP16.

Figure 8 shows luminescence results for direct transfection of stable cell lines producing pdx-1 proteins. Since these results are dependent on the transcriptional activity of the proteins irrespective of their ability to enter target cells, this experiment demonstrates that constructs containing the VP 16 domain are more transcriptionally active than those without.

Taken together, these two experiments indicate that VP 16 enhances transcriptional activity, while TAT enhances cell entry, and that the combination of the two domains gives maximal activity in a co-culture setting. EXAMPLE 3: INITIATING INSULIN PRODUCTION IN MOUSE LIVER CELLS

USING CHO-CONDITIONED MEDIA CONTAINING TAT-PDXl -VP 16 Materials and Methods Freshly isolated primary hepatocytes were co-cultured with CHO cell lines stably transfected with pdx-1 constructs, or with control CHO cells. After 72 hours culture, the cells were harvested and placed in RNAsol. RNA was isolated and cDNA was synthesised using standard methods. Results and Discussion Gene expression was measured by real time PCR using Sybr Green and primers specific for the mouse Insulin 1 gene (Figure 9) and the mouse Glut2 gene (Figure 10).

These results indicate that hepatocytes co-cultured with CHO cells secreting the TAT-pdx- 1-VP16 construct showed up-regulation of both the Insulin 1 and the GLUT2 genes, which are both prerequisites for conversion of hepatocytes into insulin-producing cells.

ABBREVIATIONS

ATP adenosine triphosphate

CHO Chinese hamster ovary cells

DNA deoxyribose nucleic acid

DME Dulbecco's modified Eagles' medium

DTT dithiothreitol

ERCP endoscopic retrograde cholangio-pancreatography

FCS fetal calf serum

FITC Fluorescene isothiocyante

GTP guanosine triphosphate

HNF- lα hepatocyte nuclear factor- lα

HNF-3β hepatocyte nuclear factor-3β

IgG immunoglobulin G

IPF l insulin promoter factor 1 moAb monoclonal antibody

NTP nucleotide triphosphate

PBS phosphate buffered saline

PCR Polymerase chain reaction

PEF polyethylene glycol

PMSF Phenoxymethylsulfonylfluoride

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

TRITC tetramethyl rhodamine isothiocyanate

USFl Upstream transcription factor 1

USF2 Upstream transcription factor 2 REFERENCES

1. H. Ttaniguchi et al, Gene Therapy 2002 (10), 15-23: beta-cell neogenesis induced by adenovirus-mediated gene delivery of transcription factor pdx-1 into mouse pancreas - Adenoviral delivery of pdx-1 into pancreas via common bile duct led to islet neogenesis (abs only seen)

2. Zalzman M, Gupta S, Giri RK, Berkovich I, Sappal BS, Karnieli O, Zern MA, Fleischer N, Efrat S: Reversal of hyperglycemia in mice by using human expandable insulin-producing cells differentiated from fetal liver progenitor cells. Proc Natl Acad Sci USA 100:7253-7258, 2003

3. Horb HE, Shen C-N, Tosh D, Slack JMW. Experimental conversion of liver to pancreas. Curr Biol 13:105-115, 2003

4. Noguchi, H., Kaneto, H., Weir, G. C, and Bonner-Weir, S. (2003). PDX-1 protein containing its own antennapedia-like protein transduction domain can transducer pancreatic duct and islet cells. Diabetes 52: 1732 - 1737.

5 Kwon YD, Oh SK, Kim HS, Ku S-Y, Kim SH,Young Min Choi YM, Moon SY.

(2005) Cellular Manipulation of Human Embryonic Stem Cells by TAT-PDXl Protein Transduction. MoI Ther 12:28-32

6 Melloul et al, Diabetes 51 (suppl 3) s320, 2002: Regulation of pώc-l gene expression - 3 critical upstream promoter elements PHl, 2 and 3. Bound by HNF- 3β, HNF-I α and PDX-1 cooperatively (i.e. PDX-1 seems to act as an autoregulator). Other TF's of relevance include SPl, SP3, Beta2 and USF.

7. Ohlsson H, Karlsson K, Edlund T, IPFl, A homeo domain-containing transactivator of the insulin gene, EMBO J 1993, Vol. 12, pp. 4251-4259. 8. Ausubel et al, Current Protocols in Molecular Biology, John Wiley & Sons, New York, 2000.

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

10. Dunican, D. J., Doherty P., "Designing cell-permeant phospho peptides to modulate intracellular signalling pathways", Biopolymers (Peptide Science), Vol. 60, pp. 45- 60 (2001).

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

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

SEQUENCE LISTING

SEQ ID NO. 1 shows the amino acid sequence of Human PDX-I (Genbank code P52945).

MNGEEQYYAA TQLYKDPCAF QRGPAPEFSA SPPACLYMGR QPPPPPPHPF PGALGALEQG

SPPDISPYEV PPLADDPAVA HLHHHLPAQL ALPHPPAGPF PEGAEPGVLE EPNRVQLPFP WMKSTKAHAW KGQWAGGAYA AEPEENKRTR TAYTRAQLLE LEKEFLFNKY ISRPRRVELA

VMLNLTERHI KIWFQNRRMK WKKEEDKKRG GGTAVGGGGV AEPEQDCAVT SGEELLALPP PPPPGGAVPP AAPVAAREGR LPPGLSASPQ PSSVAPRRPQ EPR

SEQ ID NO. 2 shows the amino acid sequence of mus muscularis PDX-I (Genbank code NM_008814). +_{+ + + + +}

MNSEEQYYAA TQLYKDPCAF QRGPVPEFSA NPPACLYMGR QPPPPPPPQF TSSLGSLEQG SPPDISPYEV PPLASDDPAG AHLHHHLPAQ LGLAHPPPGP FPNGTEPGGL EEPNRVQLPF PWMKSTKAHA WKGQWAGGAY TAEPEENKRT RTAYTRAQLL ELEKEFLFNK YISRPRRVEL AVMLNLTERH IKIWFQNRRM KWKKEEDKKR SSGTPSGGGG GEEPEQDCAV TSGEELLAVP PLPPPGGAVP PGVPAAVREG LLPSGLSVSP QPSSIAPLRP QEPR

SEQ ID NO. 3 shows the amino acid sequence of mus muscularis TAT-PDX-I with leading Ig-kappa secretory signal and trailing V5 tag and poly-His sequence to enable purification . _{+ + + + + +}

GDPSWLATME TDTLLLWVLL LWVPGSTGDA AQPARRARRT KLALYGRKKR RQRRRNSEEQ YYAATQLYKD PCAFQRGPVP EFSANPPACL YMGRQPPPPP PPQFTSSLGS LEQGSPPDIS PYEVPPLASD DPAGAHLHHH LPAQLGLAHP PPGPFPNGTE PGGLEEPNRV QLPFPWMKST KAHAWKGQWA GGAYTAEPEE NKRTRTAYTR AQLLELEKEF LFNKYISRPR RVELAVMLNL TERHIKIWFQ NRRMKWKKEE DKKRSSGTPS GGGGGEEPEQ DCAVTSGEEL LAVPPLPPPG GAVPPGVPAA VREGLLPSGL SVSPQPSSID MTGSLSTKGE LGTELGSEGK PIPNPLLGLD STRTGHHHHH H

SEQ ID NO. 4 shows the amino acid sequence of mus muscularis PDX-I -VP 16 with leading Ig-kappa secretory signal and trailing V5 tag and poly-His sequence to enable purification + _{+ + + + +}

GDPSWLATME TDTLLLWVLL LWVPGSTGDA AQPARRARRT KLALNSEEQY YAATQLYKDP

CAFQRGPVPE FSANPPACLY MGRQPPPPPP PQFTSSLGSL EQGSPPDISP YEVPPLASDD PAGAHLHHHL PAQLGLAHPP PGPFPNGTEP GGLEEPNRVQ LPFPWMKSTK AHAWKGQWAG

GAYTAEPEEN KRTRTAYTRA QLLELEKEFL FNKYISRPRR VELAVMLNLT ERHIKIWFQN RRMKWKKEED KKRSSGTPSG GGGGEEPEQD CAVTSGEELL AVPPLPPPGG AVPPGVPAAV

REGLLPSGLS VSPQPSSIDM TGSLSTAPPT DVSLGDELHL DGEDVAMAHA DALDDFDLDM

LGDGDSPGPG FTPHDSAPYG ALDTADFEFE QMFTDALGID EYGGLEPLEL KGELGTELGS

EGKPIPNPLL GLDSTRTGHH HHHH

SEQ ID NO. 5 shows the amino acid sequence of mus muscularis TAT-PDX-I- VP 16 with leading Ig-kappa secretory signal and trailing V5 tag and poly-His sequence to enable purification

+ + + + + +

GDPSWLATME TDTLLLWVLL LWVPGSTGDA AQPARRARRT KLALYGRKKR RQRRRNSEEQ YYAATQLYKD PCAFQRGPVP EFSANPPACL YMGRQPPPPP PPQFTSSLGS LEQGSPPDIS PYEVPPLASD DPAGAHLHHH LPAQLGLAHP PPGPFPNGTE PGGLEEPNRV QLPFPWMKST KAHAWKGQWA GGAYTAEPEE NKRTRTAYTR AQLLELEKEF LFNKYISRPR RVELAVMLNL TERHIKIWFQ NRRMKWKKEE DKKRSSGTPS GGGGGEEPEQ DCAVTSGEEL LAVPPLPPPG GAVPPGVPAA VREGLLPSGL SVSPQPSSID MTGSLSTAPP TDVSLGDELH LDGEDVAMAH ADALDDFDLD MLGDGDSPGP GFTPHDSAPY GALDTADFEF EQMFTDALGI DEYGGLEPLE LKGELGTELG SEGKPIPNPL LGLDSTRTGH HHHHH

SEQ ID NO. 6 shows the amino acid sequence of VP 16.

APPTDVSLGD ELHLDGEDVA MAHADALDDF DLDMLGDGDS PGPGFTPHDS APYGALDTAD FEFEQMFTDA LGIDEYGGLE PLEL

SEQ ID NO. 7 shows the amino acid sequence of TAT. +_{+ + + + +} D PYGRKKRRQ RRR

SEQ ID NO. 8 shows the amino acid sequence of human TAT-PDX-I -VP 16

+ + + + + +

GDPSWLATME TDTLLLWVLL LWVPGSTGDA AQPARRARRT KLALYGRKKR RQRRRNGEEQ

YYAATQLYKD PCAFQRGPAP EFSASPPACL YMGRQPPPPP PHPFPGALGA LEQGSPPDIS

PYEVPPLADD PAVAHLHHHL PAQLALPHPP AGPFPEGAEP GVLEEPNRVQ LPFPWMKSTK

AHAWKGQWAG GAYAAEPEEN KRTRTAYTRA QLLELEKEFL FNKYISRPRR VELAVMLNLT ERHIKIWFQN RRMKWKKEED KKRGGGTAVG GGGVAEPEQD CAVTSGEELL ALPPPPPPGG

AVPPAAPVAA REGRLPPGLS ASPQPSSMTG SLSTAPPTDV SLGDELHLDG EDVAMAHADA

LDDFDLDMLG DGDSPGPGFT PHDSAPYGAL DTADFEFEQM FTDALGIDEY GGLEPLELKG

ELGTELGSEG KPIPNPLLGL DSTRTGHHHH HH SEQ ID NO. 9 shows the amino acid sequence of Human HNF lα (Genbank code NP_000536).

MVSKLSQLQT ELLAALLESG LSKEALIQAL GEPGPYLLAG EGPLDKGESC GGGRGELAEL PNGLGETRGS EDETDDDGED FTPPILKELE NLSPEEAAHQ KAWETLLQE DPWRVAKMVK SYLQQHNIPQ REWDTTGLN QSHLSQHLNK GTPMKTQKRA ALYTWYVRKQ REVAQQFTHA GQGGLIEEPT GDELPTKKGR RNRFKWGPAS QQILFQAYER QKNPSKEERE TLVEECNRAE CIQRGVSPSQ AQGLGSNLVT EVRVYNWFAN RRKEEAFRHK LAMDTYSGPP PGPGPGPALP AHSSPGLPPP ALSPSKVHGV RXGQPATSET AEVPSSSGGP LVTVSTPLHQ VSPTGLEPSH SLLSTEAKLV SAAGGPLPPV STLTALHSLE QTSPGLNQQP QNLIMASLPG VMTIGPGEPA SLGPTFTNTG ASTLVIGLAS TQAQSVPVIN SMGSSLTTLQ PVQFSQPLHP SYQQPLMPPV

QSHVTQSPFM ATMAQLQSPH ALYSHKPEVA QYTHTGLLPQ TMLITDTTNL SALASLTPTK QVFTSDTEAS SESGLHTPAS QATTLHVPSQ DPAGIQHLQP AHRLSASPTV SSSSLVLYQS SDSSNGQSHL LPSNHSVIET FISTQMASSS Q

SEQ ID NO. 10 shows the amino acid sequence of Human HNF3α (Genbank code

P55317). + + + + + +

MLGTVKMEGH ETSDWNSYYA DTQEAYSSVP VSNMNSGLGS MNSMNTYMTM NTMTTSGNMT PASFNMSYAN PALGAGLSPG AVAGMPGGSA GAMNSMTAAG VTAMGTALSP SGMGAMGAQQ AASMMNGLGP YAAAMNPCMS PMAYAPSNLG RSRAGGGGDA KTFKRSYPHA KPPYSYISLI TMAIQRAPSK MLTLSEIYQW IMDLFPYYRQ NQQRWQNSIR HSLSFNDCFV KVARSPDKPG KGSYWTLHPD SGNMFENGCY LRRQKRFKCE KQPGAGGGGG SGSGGSGAKG GPESRKDPSG ASNPSADSPL HRGVHGKTGQ LEGAPAPGPA ASPQTLDHSG ATATGGASEL KTPASSTAPP ISSGPGALAS VPASHPAHGL APHESQLHLK GDPHYSFNHP FSINNLMSSS EQQHKLDFKA YEQALQYSPY GSTLPASLPL GSASVTTRSP IEPSALEPAY YQGVYSRPVL NTS

SEQ ID NO. 11 shows the amino acid sequence of Human SPl (Genbank code

XP_028606). +_{+ + + + +}

MSDQDHSMDE MTAWKIEKG VGGNNGGNGN GGGAFSQARS SSTGSSSSTG GGGQESQPSP LALLAATCSR IESPNENSNN SQGPSQSGGT GELDLTATQL SQGANGWQII SSSSGATPTS KEQSGSSTNG SNGSESSKNR TVSGGQYWA AAPNLQNQQV LTGLPGVMPN IQYQVIPQFQ TVDGQQLQFA ATGAQVQQDG SGQIQIIPGA NQQIITNRGS GGNIIAAMPN LLQQAVPLQG LANNVLSGQT QYVTNVPVAL NGNITLLPVN SVSAATLTPS SQAVTISSSG SQESGSQPVT SGTTISSASL VSSQASSSSF FTNANSYSTT TTTSNMGIMN FTTSGSSGTN SQGQTPQRVS

GLQGSDALNI QQNQTSGGSL QAGQQKEGEQ NQQTQQQQIL IQPQLVQGGQ ALQALQAAPL SGQTFTTQAI SQETLQNLQL QAVPNSGPII IRTPTVGPNG QVSWQTLQLQ NLQVQNPQAQ

TITLAPMQGV SLGQTSSSNT TLTPIASAAS IPAGTVTVNA AQLSSMPGLQ TINLSALGTS GIQVHPIQGL PLAIANAPGD HGAQLGLHGA GGDGIHDDTA GGEEGENSPD AQPQAGRRTR REACTCPYCK DSEGRGSGDP GKKKQHICHI QGCGKVYGKT SHLRAHLRWH TGERPFMCTW SYCGKRFTRS DELQRHKRTH TGEKKFACPE CPKRFMRSDH LSKHIKTHQN KKGGPGVALS VGTLPLDSGA GSEGSGTATP SALITTNMVA MEAICPEGIA RLANSGINVM QVADLQSINI SGNGF SEQ ID NO. 12 shows the amino acid sequence of Human SP3 (Genbank code Q02447).

MTAPEKPVKQ EEMAALDVDS GGGGGGGGGH GEYLQQQQQH GNGAVAAAAA AQDTQPSPLA

LLAATCSKIG PPSPGDDEEE AAAAAGAPAA AGATGDLASA QLGGAPNRWE VLSATPTTIK

DEAGNLVQIP SAATSSGQYV LPLQNLQNQQ IFSVAPGSDS SNGTVSSVQY QVIPQIQSAD GQQVQIGFTG SSDNGGINQE SSQIQIIPGS NQTLLASGTP SANIQNLIPQ TGQVQVQGVA

IGGSSFPGQT QWANVPLGL PGNITFVPIN SVDLDSLGLS GSSQTMTAGI NADGHLINTG

QAMDSSDNSE RTGERVSPDI NETNTDTDLF VPTSSSSQLP VTIDSTGILQ QNTNSLTTSS

GQVHSSDLQG NYIQSPVSEE TQAQNIQVST AQPWQHLQL QESQQPTSQA QIVQGITPQT IHGVQASGQN ISQQALQNLQ LQLNPGTFLI QAQTVTPSGQ VTWQTFQVQG VQNLQNLQIQ NTAAQQITLT PVQTLTLGQV AAGGAFTSTP VSLSTGQLPN LQTVTVNSID SAGIQLHPGE NADSPADIRI KEEEPDPEEW QLSGDSTLNT NDLTHLRVQV VDEEGDQQHQ EGKRLRRVAC TCPNCKEGGG RGTNLGKKKQ HICHIPGCGK VYGKTSHLRA HLRWHSGERP FVCNWMYCGK RFTRSDELQR HRRTHTGEKK FVCPECSKRF MRSDHLAKHI KTHQNKKGIH SSSTVLASVE AARDDTLITA GGTTLILANI QQGSVSGIGT VNTSATSNQD ILTNTEIPLQ LVTVSGNETM E

SEQ ID NO. 13 shows the amino acid sequence of Human USFl (Genbank code NP_009053). + + + + + +

MKGQQKTAET EEGTVQIQEG AVATGEDPTS VAIASIQSAA TFPDPNVKYV FRTENGGQVM YRVIQVSEGQ LDGQTEGTGA ISGYPATQSM TQAVIQGAFT SDDAVDTEGT AAETHYTYFP STAVGDGAGG TTSGSTAAW TTQGSEALLG QATPPGTGQF FVMMSPQEVL QGGSQRSIAP RTHPYSPKSE APRTTRDEKR RAQHNEVERR RRDKINNWIV QLSKIIPDCS MESTKSGQSK GGILSKACDY IQELRQSNHR LSEELQGLDQ LQLDNDVLRQ QVEDLKNKNL LLRAQLRHHG LEWIKNDSN

SEQ ID NO. 14 shows the amino acid sequence of Human USF3 (Genbank code AAB51179).

MDMLDPGLDP AASATAAAAA SHDKGPEAEE GVELQEGGDG PGAEEQTAVA ITSVQQAAFG DHNIQYQFRT ETNGGQVTYR WQVTDGQLD GQGDTAGAVS WSTAAFAGG QQAVTQVGVD

GAAQRPGPAA ASVPPGPAAP FPLAVIQNPF SNGGSPAAEA VSGEARFAYF PASSVGDTTA

VSVQTTDQSL QAGGQFYVMM TPQDVLQTGT QRTIAPRTHP YSPKIDGTRT PRDERRRAQH

NEVERRRRDK INNWIVQLSK IIPDCNADNS KTGASKGGIL SKACDYIREL RQTNQRMQET

FKEAERLQMD NELLRQQIEE LKNENALLRA QLQQHNLEMV GEGTRQ

Claims

1. A method of initiating insulin production in a responsive but otherwise non-insulin producing mammalian cell, which comprises introducing into the cell an effective amount for initiating insulin production within the cell of pdx-1 protein or a functionally equivalent analogue, variant or fragment thereof which is in functional conjunction with both the VP 16 transcriptional activator sequence and the TAT transduction domain, or functionally equivalent variants or fragments thereof.

2. A method of increasing insulin production in an insulin producing mammalian cell, which comprises introducing into the cell an effective amount for increasing insulin production within the cell of pdx-1 protein or a functionally equivalent analogue, variant or fragment thereof which is in functional conjunction with both the VP 16 transcriptional activator sequence and the TAT transduction domain, or functionally equivalent variants or fragments thereof.

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

4. The method of either claim 1 or claim 2 which is conducted in vitro within a mammalian organism.

5. A method of treatment of a patient suffering from or prone to suffer from diabetes mellitus, which comprises removing from the patient one or more responsive but otherwise non-insulin producing cells and culturing the cells in a suitable medium, introducing into the cells an effective amount for initiating insulin production within the cells of pdx-1 protein or a functionally equivalent analogue, variant or fragment thereof which is in functional conjunction with both the VP 16 transcriptional activator sequence and the TAT transduction domain or functionally equivalent variants or fragments thereof, and subsequently returning the cells or cells derived from them to the patient.

6. A method of treatment of a patient suffering from or prone to suffer from diabetes mellitus, which comprises introducing into responsive but otherwise non-insulin producing cells of the patient an effective amount for initiating insulin production within the cells of pdx-1 protein or a functionally equivalent analogue, variant or fragment thereof which is in functional conjunction with both the VP 16 transcriptional activator sequence and the TAT transduction domain or functionally equivalent variants or fragments thereof.

7. The method of claim 6 wherein introduction of pdx-1 protein or a functionally equivalent analogue, variant or fragment thereof which is in functional conjunction with both the VP 16 transcriptional activator sequence and the TAT transduction domain, 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.

8. The method of claim 6 wherein introduction of pdx-1 protein or a functionally equivalent analogue, variant or fragment thereof which is in functional conjunction with both the VP 16 transcriptional activator sequence and the TAT transduction domain, and optionally other transcription factors or their functionally equivalent analogues, variants or fragments is conducted in vivo within a mammalian organism.

9. A method of insulin production which comprises introducing into cells of a cultured mammalian cell line an effective amount for initiating insulin production within the cells of pdx-1 protein or a functionally equivalent analogue, variant or fragment thereof which is in functional conjunction with both the VP 16 transcriptional activator sequence and the TAT transduction domain or functionally equivalent variants or fragments thereof, and subsequently isolating insulin produced.

10. The method of any one of claims 1 to 9 wherein the pdx-1 protein or a functionally equivalent analogue, variant or fragment thereof which is in functional conjunction with both the VP 16 transcriptional activator sequence and the TAT transduction domain or functionally equivalent variants or fragments thereof is introduced into the cells in conjunction with one or more other transcription factors.

11. The method of claim 10 wherein the one or more other transcription factors is selected from HNF-lα, HNF-3β, HNF-lβ, SPl, SP3, USFl and USF2, or their functionally equivalent analogues, variants or fragments.

12. The method of claim 2 wherein the cells are pancreatic β-cells.

13. The method of any one of claims 1 and 5 to 9 wherein the responsive but otherwise non-insulin producing 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, 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 stem cells, spleen stem cells, immune system stem cells, cord blood stem cells, bone marrow stem cells and peripheral blood stem cells.

14. The method of any one of claims 1 to 13 wherein the pdx-1 protein or a functionally equivalent analogue, variant or fragment thereof which is in functional conjunction with both the VP 16 transcriptional activator sequence and the TAT transduction domain or functionally equivalent variants or fragments thereof is introduced utilising permeabilisation agents or permeabilising treatments.

15. The method of claim 14 wherein the permeabilisation agents or permeabilising treatments are selected from one or more of detergent, bacterial toxin, cell-permeant peptide vectors, polyethylene glycol (PEG), electroporation permeabilisation and lisosomal delivery.

16. The method of any one of claims 1 to 15 wherein the pdx-1 protein or a functionally equivalent analogue, variant or fragment thereof is fused with both the VP 16 transcriptional activator sequence and the TAT transduction domain.

17. An agent for increasing insulin production in an insulin producing mammalian cell or for initiating insulin production in a responsive but otherwise non-insulin producing mammalian cell which comprises pdx-1 protein or a functionally equivalent analogue, variant or fragment thereof which is in functional conjunction with both the VP 16 transcriptional activator sequence and the TAT transduction domain or functionally equivalent variants or fragments thereof.

18. The agent of claim 17 which comprises pdx-1 protein or a functionally equivalent analogue, variant or fragment thereof fused with both the VP 16 transcriptional activator sequence and the TAT transduction domain or functionally equivalent variants or fragments thereof.

19. The agent of either claim 17 or claim 18 further comprising one or more of other transcription factors or their functionally equivalent analogues or variants, permeabilisation agents and physiologically acceptable carriers and/or diluents.

20. Use of pdx-1 protein or a functionally equivalent analogue, variant or fragment thereof which is in functional conjunction with both the VP 16 transcriptional activator sequence and the TAT transduction domain, or functionally equivalent variants or fragments thereof in preparation of an agent for introduction into a responsive but otherwise non-insulin producing mammalian cell to initiate insulin production in the cell.

21. Use of pdx-1 protein or a functionally equivalent analogue, variant or fragment thereof which is in functional conjunction with both the VP 16 transcriptional activator sequence and the TAT transduction domain, or functionally equivalent variants or fragments thereof in preparation of an agent for introduction into an insulin producing mammalian cell to increase insulin production in the cell.

22. Use of pdx-1 protein or a functionally equivalent analogue, variant or fragment thereof which is in functional conjunction with both the VP 16 transcriptional activator sequence and the TAT transduction domain, or functionally equivalent variants or fragments thereof in preparation of an agent for introduction into one or more a responsive but otherwise non-insulin producing mammalian cells that have been removed from a patient suffering from or prone to suffer from insufficient insulin production and are being cultured in a suitable medium, wherein the introduction into the responsive cell/s initiates insulin production of the cells, which cells or cells derived from them are subsequently returned the to the patient.

23. Use of pdx-1 protein or a functionally equivalent analogue, variant or fragment thereof which is in functional conjunction with both the VP 16 transcriptional activator sequence and the TAT transduction domain, or functionally equivalent variants or fragments thereof in preparation of an agent for introduction into one or more insulin producing mammalian cells that have been removed from a patient suffering from or prone to suffer from insufficient insulin production and are being cultured in a suitable medium, wherein the introduction into the responsive cell/s increases insulin production of the cells, which cells or cells derived from them are subsequently returned the to the patient.

24. Use of pdx-1 protein or a functionally equivalent analogue, variant or fragment thereof which is in functional conjunction with both the VP 16 transcriptional activator sequence and the TAT transduction domain, or functionally equivalent variants or fragments thereof in preparation of an agent for introduction into cells of a cultured mammalian cell line in order to produce insulin in the cells.

25. The use of claim 24 wherein the insulin produced in the cells is subsequently isolated.