AU783594B2 - Method of prophylaxis and treatment of diabetes - Google Patents

Description

WO 00/62862 PCT/AU00/00318

-I-

METHOD OF PROPHYLAXIS AND TREATMENT OF DIABETES FIELD OF THE INVENTION The present invention relates generally to a method of prophylaxis and treatment of diabetes and agents useful for same. More particularly, the present invention contemplates the transplantation of insulin producing cells to protect against or ameliorate the symptoms associated with diabetes. Even more particularly, the present invention provides a method of preventing diabetes or reducing or ameliorating the effects of diabetes by transplanting cells genetically altered to express insulin, and still more particularly, transplanting liver cells genetically altered to express insulin.

BACKGROUND OF THE INVENTION Bibliographic details numerically referred to in this specification are collected at the end of the description.

Insulin dependant diabetes mellitus (also referred to as type 1 diabetes or juvenile onset diabetes due to its appearance in childhood or early adolescence) is a debilitating autoimmune condition caused by the selective destruction of insulin producing 3 cells in the islets of the pancreas. The infiltration of the pancreatic islets of Langerhans with lymphocytes results in regions of inflammation often referred to as insulitis. Autoreactive T cells have been implicated in destruction of the 3 cells.

The absence of treatment for diabetes can be fatal while poorly controlled diabetes leads to the appearance of complications such as diabetic glomerulosclerosis, wherein the kidneys are irreversibly damaged leading to renal failure. Treatment of type 1 diabetic individuals, and also individuals exhibiting severe symptoms of type 2 diabetes, is by daily insulin injection to replace the insulin which the damaged 3 cells are no longer able to produce.

However. it is often not possible to achieve normalisation of blood glucose levels.

WO 00/62862 PCT/AU00/00318 -2- To obtain normal blood glucose levels, attempts have been made to replace the missing 13 cells by transplantation of a whole pancreas, the islets from an adult pancreas or insulin producing fetal pancreatic cells. While transplantation of whole pancreas is of benefit in reversing diabetes in humans, with 76% one year graft survival its success is limited mostly to those people who have renal failure. Allografted adult human islets are capable of reversing diabetes but the success rate is low Transplantation of fetal pancreatic tissue is an alternative in respect of which little progress has been made to date due, inter alia, to ethical considerations.

Accordingly, there is a need to develop alternative and more effective methods of regulating glucose levels in diabetic patients. Further, techniques such as transplantation of whole pancreas or fetal pancreatic cells are cost prohibitive and limited by the availability of donor tissue. Therefore, methods of treatment which also overcome these difficulties are needed.

In work leading up to the present invention, the inventors have successfully transplanted genetically altered liver cells which synthesise, store and secrete insulin in response to glucose stimulation, thereby normalising blood glucose levels of the diabetic recipients.

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

The subject specification contains nucleotide sequence information prepared using the programme PatentIn Version 2.0, presented herein after the bibliography. Each nucleotide sequence is identified in the sequence listing by the numeric indicator <201> followed by the sequence identifier (eg. <210>1, <2 10>2. etc). The length, type of sequence (DNA, etc) and source organism for each nucleotide sequence are indicated by information WO 00/62862 PCT/AU00/00318 provided in the numeric indicator fields <211>, <212> and <213>. respectively.

Nucleotide sequences referred to in the specification are defined by the information provided in numeric indicator field <400> followed by the sequence identifier (eg.

<400>1, <400>2, etc).

One aspect of the present invention is directed to a method of preventing, reducing or otherwise ameliorating diabetes in a subject, said method comprising introducing into said subject an effective number of cells wherein said cells produce insulin or a derivative, homologue or a mimetic thereof.

Another aspect of the present invention more particularly provides a method of preventing.

reducing or otherwise ameliorating diabetes in a subject, said method comprising introducing into said subject an effective number of cells, which cells have been transfected with a nucleic acid molecule encoding insulin or a derivative, homologue or mimetic thereof wherein said cells produce insulin or a derivative, homologue or mimetic thereof.

Yet another aspect of the present invention provides a method of preventing, reducing or otherwise ameliorating diabetes in a subject, said method comprising introducing into said subject an effective number of hepatocytes, which hepatocytes have been transfected with a nucleic acid molecule encoding insulin or a derivative, homologue or mimetic thereof.

Still yet another aspect of the present invention provides a method of preventing, reducing or otherwise ameliorating diabetes in a subje:ct, said method comprising introducing into said subject an effective number of Huh7ins cells wherein said cells produce insulin or a derivative, homologue or mimetic thereof.

Accordingly, another aspect of the present invention provides a method for the treatment and/or prophylaxis of a condition characterised by aberrant, unwanted or otherwise inappropriate functional activity or levels of a molecule in a subject, which molecule is modulatable by insulin or derivative, homologue or mimetic thereof, said method WO 00/62862 PCT/AU00/00318 -4comprising administering to said subject an effective number of cells wherein said cells product insulin or a derivative, homologue or a mimetic thereof.

Another aspect of the present invention provides a method for the treatment and/or prophylaxis of a condition characterised by aberrant, unwanted or otherwise inappropriate levels of glucose and/or insulin or derivative or equivalent thereof in a subject said method comprising administering to said subject an effective number of cells wherein said cells produce insulin or a derivative, homologue or a mimetic thereof.

Still another aspect of the present invention provides a method for the treatment and/or prophylaxis of a condition characterised by aberrant, unwanted or otherwise inappropriate levels of glucose and/or insulin or derivative or equivalent thereof in a subject said method comprising administering to said subject an effective number of cells, which cells have been transfected with a nucleic acid molecule encoding insulin or a derivative, homologue or mimetic thereof, wherein said cells produce insulin or a derivative, homologue or mimetic thereof.

Another aspect of the present invention contemplates a method of modulating insulin levels in a subject said method comprising introducing into said subject an effective number of cells wherein said cells produce insulin or a derivative, homologue or mimetic thereof.

Yet another aspect of the present invention contemplates a method of modulating glucose levels in a subject said method comprising introducing into said subject an effective number of cells wherein said cells produce insulin or a derivative, homologue or mimetic thereof.

Still another aspect of the present invention relates to the use of cells which produce insulin in the manufacture of a medicament for the treatment of a condition characterised by aberrant, unwanted or otherwise inappropriate functional activity or levels of a WO 00/62862 PCT/AU00/00318 molecule in a subject, which molecule is modulatable by insulin or derivative. homologue or mimetic thereof.

Still another aspect of the present invention relates to cells which produce insulin, as hereinbefore defined.

WO 00/62862 PCT/AU00/00318 -6- BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is an image of a Northern blot analysis of GLUT 2 in HEP G2 (lane Huh7 (lane Huh7ins/clone 2 (lane 3) and Huh7ins/clone 9. (Lane 4 run simultaneously on a separate gel).

Figure 2 is an image of Western blot analysis of GLUT 2 in HEP G2 (lane Huh7 control cells (lane Huh7ins cells, clone 2 (lane Huh7ins cells, clone 9 (lane 4).

(Lanes 1 and 2 run simultaneously on separate gel).

Figure 3 is an image of RT/PCR expression of human glucokinase (hGLK) 155 bp product in HEP G2 cells (lane Huh 7 cells (lane Huh7ins cells (clone 2, transfected with insulin cDNA, lane Huh7ins cells (clone 9, transfected with insulin cDNA, lane 4), human liver (lane 5) and human skin fibroblasts (lane 6).

Figure 4 is an image of light micrographs of Huh7 cells (negative controls) and (b) Huh7ins cells (clone 2) immunochemically stained for insulin and counterstained with horse radish peroxidase/Mayer's haemotoxylin. Granular red, positive staining in cytoplasm of Huh7ins cells. (Magnifications X 348).

Figure 5 (a and b) Transmission electron micrograph of the ultrastructure of Huh7ins cells. Demonstrates a cluster of Huh7ins cells expressing the characteristic P cell granule with an electron dense core and an electron lucent halo surrounded by an outer limiting memorane (Bar=2 gm). At higher magnification an organised secretory apparatus is seen within the cytoplasm: Golgi apparatus and rough endoplasmic reticulum. Bar=l im. Immunoelectronmicrograph with a 10 nm gold probe directed against an insulin primary antibody. Localisation of insulin is demonstrated within the characteristic insulin granules (arrow). The density of the granules is lower than seen in a b due to the removal of osmium during antigen retreival performed on routinely processed cells. Nucleus Bar=0.5 pm. Immunoelectronmicrograph of Huh7ins cells using a technique to maximise antigen preservation. A 10 nm gold probe was WO 00/62862 PCT/AU00/00318 -7directed against insulin primary antibody and demonstrates the expression of insulin within the cytoplasmic granules. Large vacuoles are evident in this section. Bar =1.2 glm, inset 40,000X, granule is 280 nm.

Figure 6 is a graphical representation of stimulation by 20 mM glucose, 10 mM theophylline, 10 mM calcium and 20 mM glucose 10 mM theophylline on the acute regulated release ofimmunoreactive (pro)insulin. Huh7ins cells were incubated in the basal medium for 2 consecutive 1 hr periods to stabilize the basal secretion of insulin.

Monolayers were exposed to the stimulus for 1 hr (solid bars); corresponding cells were treated throughout with basal medium (unfilled bars). n: number of experiments; B l: first basal period; B2: second basal period; B: basal period following stimulation; S: stimulus.

Values are expressed as means S.E.

Figure 7 is a graphical representation of (pro)insulin secretion of Huh7ins cells in response to varying concentrations of glucose (0-20 mM). Huh7ins cells (clone 2) were incubated in the basal medium for 2 consecutive 1 hr periods to stabilize the basal secretion of insulin. Monolayers were exposed to the 0-20 mM glucose for 1 hr, S.E. mean.

Figure 8 is a graphical representation of(pro)insulin synthesis of Huh7ins cells in response to varying concentrations of glucose (0-20 mM), n=4. S.E. mean.

Figure 9 is a graphical representation of the blood glucose levels of mice transplanted with HUH7-ins cells. Data are presented in all mice or in selected mice The cells were placed either beneath the renal capsule (n 8) or subcutaneously (n Grafts placed beneath the renal capsule functioned more efficiently than those implanted subcutaneously. In all cases the elevated blood glucose values first became normal and then subnormal. Removal of the graft resulted in an immediate rise in the blood glucose levels WO 00/62862 PCT/AU00/00318 -8- Figure 10 is a graphical representation of the oral glucose tolerance test in mice transplanted with HUH7-ins cells when random blood glucose levels were normal, 3.7 0.8 mM. The test was conducted in fasted mice with 16.7 pmol per g body weight of a 1.67 M glucose solution being injected into the stomach through polyethylene tubing swallowed by the mice.

Figure 11 is an image of the analysis of HUH7-ins cells implanted beneath the renal capsule.

A. Histology. (Pro)insulin is stained darkly in this picture. Stain is immunoperoxidase with an anti-insulin antibody. Bar is 30 pim. B-D. Ultrastructure. Bar is 1 Vpm.

Electron dense granules are present in the cytoplasm of the cells in 3 different arrangements.

B. in close association with a well developed Golgi apparatus and endothelium Nucleus C. within the rough endoplasmic reticulum (arrow). Golgi apparatus nucleus D. adjacent to vacuoles. This contrasts with the pattern in the neighbouring cell which contains granules but no vacuoles. Nucleus Figure 12 is a graphical representation of high performance liquid chromatographic analysis of acid ethanol extracts of transplanted HUH7-ins cells. Arrows designate the elution positions of: 1- intact human insulin; 2- carboxy extended diarginyl-insulin; 3split 31, 32 human proinsulin; 4- split 64, 65 human proinsulin; 5- intact human proinsulin. The profile is representative of 4 qualitatively similar experiments.

Figure 13 is a graphical representation of chronic (pro)insulin secretion and storage of cells isolated from grafts removed diabetic SCID mice, the blood glucose levels of which had been normalized by the grafts. SC1 and SC2 represent two different mice from which grafts were removed. Clone 2 is the HUH7-ins cells prior to transplantation.

WO 00/62862 PCT/AU00/00318 -9- Figure 14 is a graphical representation of acute (pro)insulin secretion in response to mM glucose from cells isolated from grafts removed diabetic SCID mice, the blood glucose levels of which had been normalized by the grafts. SC1 and SC2 represent two different mice from which grafts were removed. Cells were exposed to basal medium both before ("Basal 1" and "Basal and after ("Basal addition of 20 mM glucose Note that levels of the hormone rose when glucose was added and declined when glucose was removed.

WO 00/62862 PCT/AU00/00318 DETAILED DESCRIPTION OF THE INVENTION The present invention is predicated, in part, of the development of genetically altered cells which synthesise, store and secrete insulin in response to glucose stimulation. The inventors have further developed a method of reducing, ameliorating or preventing diabetes by transplanting, into diabetic recipients, cells which have been genetically altered to produce insulin.

Accordingly, one aspect of the present invention is directed to a method of preventing, reducing or otherwise ameliorating diabetes in a subject, said method comprising introducing into said subject an effective number of cells wherein said cells produce insulin or a derivative, homologue or a mimetic thereof.

Reference to "diabetes" should be understood as a reference to a condition in which insufficient levels of insulin are produced to maintain biologically normal glucose levels.

This may be due to congenital defects in the pancreatic islet cells, the onset of an autoimmune response directed to the pancreatic 13 cells (for example type 1 diabetes/IDDM, gestational diabetes or slowly progressive IDDM which is also referred to as latent autoimmune diabetes in adults), defects in the functioning of the pancreatic islet cells caused by environmental factors such as diet or stress (for example type 2 diabetes/adult onset diabetes), damage to the pancreatic islet cells such as, but not limited to, as caused by physical injury, the degeneration of pancreatic islet cells due to non autoimmune conditions or as a side effect due to the onset or treatment of an unrelated disease condition.

Reference to "preventing, reducing or otherwise ameliorating" diabetes in a subject should be understood as a reference to the prevention, reduction or amelioration of any one or more symptoms of diabetes via the production of insulin. Symptoms of diabetes include, but are not limited, to abnormal glucose levels or glucose level regulation, abnormal insulin levels, thirst, frequent urination, weight loss, blurred vision, headache and abdominal pain. It should be understood that the method of the present invention may WO 00/62862 PCT/AU00/00318 -11either reduce the severity of any one or more symptoms or eliminate the existence of any one or more symptoms. For example, the method of the present invention may either fully or partially normalise glucose levels in a diabetic individual. Although complete normalisation is most desirable, partial normalisation is nevertheless useful, for example, to reduce the risk of a type I diabetic individual succumbing to a diabetic coma. The method of the present invention extends to preventing the onset of any one or more symptoms of diabetes. For example, in individuals who are predisposed to the development of diabetes, whose pancreatic islet cells are gradually degenerating or who have suffered acute and irreparable injury to pancreatic islet cells, the method of the present invention may be employed to restore insulin production prior to the occurrence of any one or more symptoms of diabetes.

Reference to "insulin" should be understood as a reference to all forms of insulin including, but not limited to, precursor forms (for example, proinsulin), split products or partially cleaved proinsulin (for example des 32,33 insulin and des 64,65 insulin), mature insulin (for example, the product obtained following cleavage of proinsulin) the a or P chain of insulin in isolation or various isoforms of insulin due to the translation of mRNA splice variants. For example, the Huh7ins cell line produces proinsulin as the bioactive product since liver cells do not naturally express the enzymes PC2 or PC3 which cleave proinsulin to insulin. Also encompassed in the scope of the present invention are derivatives, homologues or mimetics of insulin which exhibit one or more of the functional activities of insulin.

The term "derivatives" includes fragments, parts, portions, mutants or mimetics of insulin.

Derivatives include one or more insertions, deletions or substitutions of amino acids.

Amino acid insertional derivatives include amino and/or carboxylic terminal fusions as well as intrasequence insertions of single or multiple amino acids. Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site. Random insertion is also possible with suitable screening of the resulting product. Deletional variants are characterised by the removal of one or more amino acids from the sequence. Substitutional amino acid variants are those in which at WO 00/62862 PCT/AU00/00318 -12least one residue in the sequence has been removed and a different residue inserted in its place. Additions to amino acid sequences include fusions with other peptides or polypeptides. This may be desirable, for example, to facilitate the co-expression of both proinsulin and a cleavage enzyme.

The derivatives of insulin also include fragments having particular biologically active regions of insulin. A "mimetic" should be understood as a molecule which exhibits at least some of the biological activity of insulin. It should be understood that the mimetic may be a non-insulin molecule identified, for example, via natural product screening.

Preferably the insulin is human insulin, however, the invention also extends to homologues of human insulin. A homologue of insulin as contemplated herein includes insulin from non human species. Accordingly, "homologues" of insulin include forms of insulin which are normally found in a species other than the species which is the subject of treatment.

Reference to a cell which produces insulin or a derivative, homologue or mimetic thereof should be understood either as a reference to a cell which has been engineered to produce insulin (wherein in its natural state that cell would not produce insulin) or to a cell which has been engineered to produce increased levels of insulin (wherein in its natural state that cell would produce low levels of insulin). The cell may be engineered to produce insulin or increased levels of insulin by any suitable method. Preferably, the cell is transfected with a nucleic acid molecule encoding insulin or a derivative, homologue or mimetic thereof. Even more preferably, the cell is permanently transfected with cDNA or genomic DNA encoding insulin or a derivative, homologue or mimetic thereof. However, transplantation of cells which transiently express a nucleic acid molecule encoding insulin may be useful in certain circumstances, for example, where the individual will only temporarily exhibit symptoms of diabetes due to the temporary down regulation of the activity of their cells. This may be of use, for example, in the treatment of transient conditions such as gestational diabetes. The present invention should also be understood to extend to the use of cells in which, rather than transfecting a nucleic acid molecule encoding insulin into the cell, an endogenous but unexpressed genomic insulin gene is WO 00/62862 PCT/AU00/00318 -13switched on, that is, expression of the gene is induced or even up-regulated where the gene is not expressed in sufficiently high levels.

Accordingly, the present invention more particularly provides a method of preventing, reducing or otherwise ameliorating diabetes in a subject, said method comprising introducing into said subject an effective number of cells, which cells have been transfected with a nucleic acid molecule encoding insulin or a derivative, homologue or mimetic thereof, wherein said cells produce insulin or a derivative, homologue or mimetic thereof.

The cells of the present invention are preferably eucaryotic, non-islet cells which produce insulin either constitutively and/or as a result of stimulation which leads to expression of the transfected nucleic acid molecule encoding insulin or a derivative, homologue or mimetic thereof. Accordingly, reference to "produce" is a reference to the expression (being the transcription and translation) of an insulin encoding nucleic acid molecule.

Without limiting the present invention to any one theory or mode of action, the insulin or derivative, homologue or mimetic thereof is released into the extracellular environment either via secretion of soluble insulin by the cell or via anchoring of the insulin molecules to cell-surface molecules. Preferably, the insulin is secreted. It should be understood that following expression of the nucleic acid molecule encoding insulin, the insulin which is produced may be stored intracellularly for a period of time prior to its release. For example, the cell may store insulin intracellularly where, upon glucose stimulation, the stored insulin is released and/or the expression of insulin is up regulated. This cell may also constitutively express and secrete insulin.

The nucleic acid molecule which is utilised to transfect the cells of the present invention is preferably from a human, however, it may also be derived from any other suitable source including, for example, primates, livestock animals, laboratory test animals or captive wild animals. The subject nucleic acid molecule may be any suitable form of nucleic acid molecule including, for example, a genomic, cDNA or ribonucleic acid molecule which encodes the subject insulin or derivative, homologue or mimetic thereof.

WO 00/62862 PCT/AU00/00318 -14- Preferably, the cell which is utilised in the method of the present invention is one which can produce insulin in response to glucose or some other stimulus. Accordingly, cells which either naturally or as a result of genetic modification express the high capacity glucose transporter, GLUT 2 and the high capacity glucose phosphorylation enzyme glucokinase are particularly preferred since the GLUT 2 transporter and glucokinase comprise the key elements of the "glucose sensing system" which regulates insulin release from pancreatic cells in response to small external nutrient changes. Further, cells which are naturally adapted to protein secretion due to the existence of secretory granules and regulatory pathways capable of processing prohormones are particularly desirable.

It should be understood that the "cells" which are introduced into an individual in accordance with the method of the present invention may be in any suitable form. For example, they may be a population of cells existing as a single cell suspension or as a tissue sample such as a tissue graft. It is within the scope of the present invention that only some of the cells which are introduced into an individual may actually produce insulin. In this regard, reference to an "effective number of cells" should be understood as a reference to the total number of cells required to be introduced such that the number of insulin producing cells is sufficient to produce levels of insulin which achieve the object of the invention, being the reduction, reversal or amelioration of diabetes.

The cells may be donated from any suitable source. For example, the cells may be isolated from an individual or from an existing cell line. The cells may be primary cells or secondary cells. A primary cell is one which has been isolated from an individual. A secondary cell is one which, following its isolation has undergone some form of in vitro manipulation such as immortalisation or the preparation of a cell line. In this regard, the process of the present invention may be "syngeneic" meaning that the donor cells are of the same MHC phenotype as the recipient (for example, cells may be removed from the individual to be treated whereby they are transfected and returned to that individual or the cells may be isolated from an identical twin). The process of the present invention may be "allogeneic" wherein the donor cells are of the same species as the recipient but are MHC incompatible (for example, where the cells are isolated from a human cell line of an WO 00/62862 PCT/AU00/00318 unrelated individual). Finally, the process of the present invention may be "xenogeneic" meaning that the donor cells were isolated from a different species to that of the recipient.

(For example, where pig cells are introduced into a human recipient).

The cells may be introduced into an individual by any suitable method. For example, cell suspensions may be introduced by direct injection or inside a blood clot whereby the cells are immobilised in the clot thereby facilitating transplantation. Routes of administration include but are not limited to intravenously, intraperitioneal, subcutaneously, intracranial, intradermal, intramuscular, intraocular, intrathecal, intracerebrally, intranasally, infusion, orally, rectally, via iv drip, patch and implant. Subcutaneous routes are particularly preferred. They may also be introduced by surgical implantation. This may be necessary, for example, where the cells exist in the form of a tissue graft or where the cells are derived from a tumour cell line and therefore must be encapsulated prior to transplanting.

The site of transplant may be any suitable site, for example, subcutaneously or, where the donor cells are liver cells, under the renal capsule. Without limiting the present invention to any one theory or mode of action, where cells are administered as an encapsulated cell suspension, the cells will coalesce into a mass. It should also be understood that the cells may continue to divide following transplantation.

In a preferred embodiment, the cells are liver cells (hepatocytes) since liver cells express GLUT 2 and glucokinase.

According to this preferred embodiment the present invention provides a method of preventir.g, reducing or otherwise ameliorating diabetes in a subject, said method comprising introducing into said subject an effective number of hepatocytes, which hepatocytes have been transfected with a nucleic acid molecule encoding insulin or a derivative, homologue or mimetic thereof, wherein said hepatocytes produce insulin or a derivative, homologue or mimetic thereof.

Even more preferably, said hepatocytes are from the glucose-responsive insulin secreting hepatoma cell line Huh7ins, which has been developed by the inventors. The cells of this WO 00/62862 PCT/AU00/00318 -16cell line have been transfected with insulin cDNA and are capable of storing insulin and expressing and secreting insulin in response to glucose stimulation. Although the Huh7ins cells exemplified herein rely on endogenous expression of GLUT 2, it is within the scope of the present invention to induce the subject Huh7ins cells to over express GLUT 2 for the purpose of increasing their glucose responsiveness. Without limiting the mode of action of the present invention in any way, the Huh7ins cells produce insulin in response to glucose stimulation in response to increased levels of glucose in the circulation of an individual) but once the stimulus has been removed, they return to basal levels of constitutive secretion of insulin. Huh7ins cells have the ability to package insulin into a form of limiting membrane a releasable compartment) until regulated release occurs.

In a most preferred embodiment, the present invention provides a method of preventing, reducing or otherwise ameliorating diabetes in a subject, said method comprising introducing into said subject an effective number of Huh7ins cells wherein said cells produce insulin or a derivative, homologue or mimetic thereof.

In accordance with the method of the present invention, other proteinaceous or nonproteinaceous molecules may be coadministered either with the introduction of the insulinproducing cells or during insulin production by the transplanted cells. By "coadministered" is meant simultaneous administration in the same formulation or in different formulations via the same or different routes or sequential administration via the same or different routes. By "sequential" administration is meant a time difference of from seconds, minutes, hours or days between the transplantation of these cells and the administration of the proteinaceous or non-proteinaceous molecules or the onset of insulin production and the administration of the proteinaceous or non-proteinaceous molecule.

For example, it may be necessary to co-administer the enzyme PC2 or PC3 to facilitate cleavage of proinsulin to insulin. Alternatively, where the transplanted cells constituitively release insulin, it may be necessary inhibit the proliferation of these cells once blood glucose levels have been normalised in recipients. This will avoid the occurrence of hypoglycaemia. This may be achieved, for example, utilising the tetracycline conditional gene expression system whereby insulin synthesis is inhibited when tetracycline is given WO 00/62862 PCT/AU00/00318 -17to the host when this gene is stimulated. Where the transplanted cells are either allogeneic or xenogeneic, relative to the recipient, it may be necessary to co-administer immunosuppressive drugs to minimise the risk of immunological rejection. It should be understood that co-administration is in no way limited to these examples.

In a related aspect of the present invention, the subject undergoing treatment or prophylaxis may be any human or animal in need of therapeutic or prophylactic treatment.

In this regard, reference herein to "treatment" and "prophylaxis" is to be considered in its broadest context. The term "treatment" does not necessarily imply that a mammal is treated until total recovery. Similarly, "prophylaxis" does not necessarily mean that the subject will not eventually contract a disease condition. Accordingly, treatment and prophylaxis include amelioration of the symptoms of a particular condition or preventing or otherwise reducing the risk of developing a particular condition. The term "prophylaxis" may be considered as reducing the severity of onset of a particular condition.

"Treatment" may also reduce the severity of an existing condition or the frequency of acute attacks.

In a preferred embodiment the subject of the treatment is a mammal and still more preferably a human. Although the present invention is exemplified utilising a murine model, this is not intended as a limitation on the application of the method of the present invention to other species, in particular, humans.

Although the method of the present invention is particularly suited to the treatment or prophylaxis of diabetes, it is not to be understood as being limited to the t:eatment of this condition. Rather, the method of the present invention can be utilised to treat any condition characterised by aberrant, unwanted or otherwise inappropriate functional activity or levels of a molecule which is directly or indirectly modulatable by insulin, such as but not limited to, the levels of glucose and/or insulin or derivative or equivalent thereof. Reference to "aberrant, unwanted or otherwise inappropriate" functional activity or levels of such a molecule (for example, glucose and/or insulin) should be understood as a reference to either permanently or transiently abnormal levels or activities of these WO 00/62862 PCT/AU00/00318 -18molecules or to physiologically normal levels or activities of one or both of these molecules, which levels or activities are nevertheless unwanted or otherwise inappropriate.

It should be understood that a molecule which is "directly" modultable by insulin is one which the subject insulin associates or otherwise interacts with to up-regulate, downregulate or otherwise modulate its functional activity or levels or to in any way alter its structural or other phenotypic, molecular or other physical features. Increasing insulin levels, per se, should be understood to fall within the context of this definition. A molecule which is "indirectly" modulatable by insulin is one which is modulated (in the context described above) by a proteinaceous or non-proteinaceous molecule other than insulin, which other proteinaceous or non-proteinaceous molecule is directly or indirectly modulated by said insulin. Accordingly, the present invention extends to the modulation of the functional activity or levels of a given molecule via an insulin induced cascade of regulatory steps.

Accordingly, another aspect of the present invention provides a method for the treatment and/or prophylaxis of a condition characterised by aberrant, unwanted or otherwise inappropriate functional activity or levels of a molecule in a subject, which molecule is modultable by insulin or derivative, homologue or mimetic thereof, said method comprising administering to said subject an effective number of cells wherein said cells produce insulin or a derivative, homologue or mimetic thereof.

Preferably, said molecule is glucose or insulin or derivative or equivalent thereof.

Accordingly, in a preferred embodiment of the present invention provides a method for the treatment and/or prophylaxis of a condition characterised by aberrant, unwanted or otherwise inappropriate levels of glucose and/or insulin or derivative or equivalent thereof in a subject said method comprising administering to said subject an effective number of cells wherein said cells produce insulin or a derivative, homologue or a mimetic thereof WO 00/62862 PCT/AU00/00318 -19- More particularly, the present invention provides a method for the treatment and/or prophylaxis of a condition characterised by aberrant, unwanted or otherwise inappropriate levels of glucose and/or insulin or derivative or equivalent thereof in a subject said method comprising administering to said subject an effective number of cells, which cells have been transfected with a nucleic acid molecule encoding insulin or a derivative, homologue or mimetic thereof, wherein said cells produce insulin or a derivative, homologue or mimetic thereof.

Preferably, said cells are hepatocytes and still more preferably Huh7ins cells.

Another aspect of the present invention contemplates a method of modulating insulin levels in a subject said method comprising introducing into said subject an effective number of cells wherein said cells produce insulin or a derivative, homologue or mimetic thereof.

Preferably, said modulation is up-regulation.

More preferably, said cells have been transfected with a nucleic acid molecule encoding insulin or a derivative, homologue or mimetic thereof. Even more preferably, said cells are hepatocytes and still more preferably Huh7ins cells.

Yet another aspect of the present invention contemplates a method of modulating glucose levels in a subject said method comprising introducing into said subject an effective number of cells wherein said cells produce insulin or a derivative, homologue or mimetic thereof.

Preferably said modulation is down-regulation.

More preferably, said cells have been transfected with a nucleic acid molecule encoding insulin or a derivative, homologue or mimetic thereof. Even more preferably, said cells are hepatocytes and still more preferably Huh7ins cells.

WO 00/62862 PCT/AU00/00318 Still another aspect of the present invention relates to the use of cells which produce insulin in the manufacture of a medicament for the treatment of a condition characterised by aberrant, unwanted or otherwise inappropriate functional activity or levels of a molecule in a subject, which molecule is modulatable by insulin or derivative, homologue or mimetic thereof.

Preferably said molecule is insulin and/or glucose or derivative or equivalent thereof.

Preferably, said condition is diabetes.

More preferably, said cells have been transfected with a nucleic acid molecule encoding insulin or a derivative, homologue or mimetic thereof. Even more preferably, said cells are hepatocytes and still more preferably Huh7ins cells.

Still another aspect of the present invention relates to cells which produce insulin or a derivative, homologue or mimetic thereof, as hereinbefore defined.

Preferably said cells are utilised in the methods hereinbefore defined.

More preferably, said cells are transfected with a nucleic acid molecule encoding insulin or a derivative, homologue or mimetic thereof. Even more preferably said cells are hepatocytes. Still more preferably said transfected hepatocytes are Huh7ins.

Further features of the present invention are more fully described in the following non limiting Examples: WO 00/62862 PCT/AU00/00318 -21 EXAMPLE 1 GENERATION OF A HUMAN HEPATOMA CELL LINE HUH7INS Materials and Methods Plasmid construction The full-length 0.6-kb human insulin cDNA was cloned into the multi-cloning site of pRcCMV (cytomegalovirus promoter) which expresses resistance to the eukaryocidal antibiotic neomycin/ G418. Huh7 cells were transfected with 20 ig of the recombinant plasmid and vector. Transfection was accomplished by electroporation at 400V and 500 pF in a Biorad gene pulser at a cell concentration of 5 X 10 6 cells per cuvette, in Dulbecco's Modification of Eagles's medium (DMEM) (Trade Biosciences, Melbourne, Australia) containing no fetal calf serum (FCS). To obtain stable transfectants of Huh7 cells containing insulin cDNA, 48 hr later 0.55 mg/ml of the G418 antibiotic (Gibco Laboratories, Grand Island, NY, USA) was added to the culture medium. Medium plus drug was changed every 2-3 days. After 3-4 weeks of selection, colonies were picked and screened for production of insulin by radioimmunoassay (RIA), Selected clones were expanded into mass cultures and maintained in G418-containing media throughout the course of all experiments.

Cell culture Huh7 cells were grown as monolayers in DMEM supplemented with 1U% FCS (Trace Biosciences, Melbourne, Australia) and Huh7ins cells were grown in the presence of 0.55 mg/ml G418 in 5% CO 2 in air at 37°C.

Cell extraction and radioimmunoassays (RIA) Samples of culture medium were collected for estimation of human (pro)insulin by radioimmunoassay (RIA) as described previously The RIA was carried out using WO 00/62862 PCT/AU00/00318 -22guinea pig insulin antibody (Lilly Research Laboratories, Indianapolis, IN, USA) and 125Ilabelled insulin prepared by the chloramine-T method. Insulin content of the cells was determined after overnight extraction in acid-ethanol.

RNA preparation and Northern (RNA) blot Total cellular RNA was isolated by the guanine thiocyanate method Northern blot analysis was carried out as previously described Blots were hybridized with 32

P-

labelled human GLUT 2.

Immunohistochemical analysis Immunohistochemical analysis was performed to confirm the storage or insulin in Huh7ins cells. Huh7 and Huh7ins cells were collected after typsinization and fixed in 10% formalin at 4°C for 2 hours. They were then washed in phosphate buffered saline (PBS) and loaded onto slides coated with poly-L-lysine (Sigma, St. Louis, MI, USA). The endogenous peroxidase was inactivated by treatment of cells with 3% hydrogen peroxide. Prior to antibody labeling, 10% goat serum was applied to the samples to prevent non-specific antibody labeling. The cells were incubated with the primary antibody [guinea pig, antirabbit immunoglobulin (1:500)] for 30 min at room temperature. This was followed by sequential labeling with biotinylated anti-rabbit immunoglobulin and steptavidinperoxidase conjugate. Coloration was produced with substrate solution containing hydrogen peroxidase and -amino-9-ethylcarbazole (AEC). For the negative control, primary antibody was emitted by replacing the antibody with PBS. Pig pancreas was immunohistochemically stained to serve as the positive control. After immunostaining the nuclei were counterstained with Mayer's haemotoxylin (Sigma). All immunoglobulins and AEC substrate were purchased from Dako (Santa Barbara, CA, USA).

WO 00/62862 PCT/AU00/00318 -23- Membrane preparation, gel electrophoresis and Western blotting Cell membranes were prepared from HEP G2, Huh7 and Huh7ins cells by suspending cells in ice-cold buffer (20 mM K 2

HPO

4 1 mM EDTA and 110 mM KCL) and sonicating cells on ice. Supernatants were prepared by centrifugation at 16,000 g in a refrigerated microfuge. Protein concentration in the supernatant was determined by the method of Bradford and 5 lg of protein was suspended in an equal volume of 2X sample buffer (100 mM Tris, 4%SDS, 0.2% bromophenol blue, 20% glycerol, 10% p-mercaptoethanol, pH 6.80, heated at 95°C for 5 min, and electrophoresed using 16% Tris-glycine gels (Novex, San Diego, CA). Proteins were transferred to PVDF membranes (Bio Rad, Hercules, CA, USA) and blocked with 4% dry milk in Tris-buffered saline with Tween (TBST: 10 mM Tris, 150 mM NAC1, 0.05% Tween 20, pH The blot was incubated overnight with primary antibody to human GLUT 2 (1:500) (gift from Prof. M. Permutt, Washington University, St. Louis, MI, USA) in TBST plus 1% bovine serum albumin (BSA) and bands visualised using the ECL chemiluminescent Western blotting kit (Amersham International, Amersham, Bucks, UK).

Analysis of gene expression by RT/PCR.

Total RNA was isolated by the guanidinium thiocyanate method For RT/PCR analysis RNA (I g) was reverse transcribed using Moloney murine leukemia virus reverse transcriptase and random hexaneucleotide primers (15) (Perkin/Elmer, Norwalk, IN, USA).

The PCR reaction was performed using a cDNA amount representing 100ng total RNA and 1 unit of Amplitaq polymerase (Perkin/Elmer, Norwalk, IN, USA) in a reaction mix containing 10 mM Tris-HCI (pH 50 mM KCI, 1.5 mM MgCl 2 0.01% gelatin, 250 uM dNTP's and 20 pmol of each primer. The PCR was performed in a Corbett Thermal Sequencer: following an initial denaturation step of 2 minutes at 94C, a 35 cycle program consisting of 94 0 C for 45 sec. 61 C for 1 min and 72 0 C for 45 sec was performed.

This was followed by a final cycle at 72 0 C for 5 min, prior to cooling to 4°C. Following PCR, a 10 pl aliquot was subjected to 12% polyacrylamide gel electrophoresis, prior to ethidium bromide staining and photography. The gene specific human glucokinase (gift WO 00/62862 PCT/AU00/00318 -24from Prof. Alan Permutt, Washington University, St. Louis, MI, USA) forward PCR primer was 5' CTACTTGAAACAAATCGCCC and the reverse PCR primer was 3'CCTCCTCCTGGACTTCTTCC (<400>2) (primers synthesized by Gibco Laboratories, Grand Island, NY, USA). This primer pair generated a 155 bp PCR product from bp 244-379 of the published human glucokinase sequence Several negative control reactions were included in each experiment. Some of the negative controls contained water instead of cDNA, and other control reaction mixtures were prepared without the addition of RNA. All necessary precautions against contamination of PCRs were rigorously observed.

Electron microscopy After trypsinization, a single cell suspension of control Huh7 cells and Huh7ins cells were fixed for 1 hour at 4 0 C in 0.5% glutaraldehyde in 0.1M Cacodylate buffered at pH 7.4, followed by 1% osmium tetroxide at room temperature for 20 minutes. A block stain was carried out in aqueous 2% uranyl acetate at room temperature for 1 hour. Tissue was dehydrated in solutions of graded alcohol and embedded in Durcopan (Fluka, Switzerland).

Ultra-thin sections (80 nm) were cut on a Reichert Ultracut-S and counter-stained with Reynold's lead citrate and examined at 80 kV on a JEOL 100C electron microscope.

Images were generated using Adobe Photoshop 4.01. Granule size was measured directly from the image.

For immunoelectronmicroscopy (IEM), two different post-embedding immunogold procedures were used to confirm the localization of intracellular insulin. Single cell suspensions of control Huh 7, Huh7ins and NIT-I cells (mouse insulinoma cell line, used as a positive control) were analyzed. Firstly, an antigen unmasking technique previously described (17) was modified and performed on routinely processed material as stated above. Briefly, the sections were incubated in 50% sodium metaperiodate at 50 0 C for minutes followed by 0.01M sodium citrate buffer pH 6.0 at 95°C for 10 minutes and a minute cooling period. Non specific binding was blocked using 1% goat serum for minutes at room temperature. A goal anti guinea pig 10nm (1:50) primary antibody WO 00/62862 PCT/AU00/00318 (Zymed, San Francisco, USA) incubated overnight at 4 0 C. Primary antibody was replaced by PBS to assess the level of non specific binding of the gold probe. Sections were counter-stained with Reynold's lead citrate prior to examination. Secondly, a technique devised for antigen preservation and antibody penetration was used. Pelleted cells were fixed in a microwave (80 watts for 5 min) in 10% buffered formalin. The cells were partially dehydrated in 70% ethanol and embedded in LR-White (Probing and Structure, Brisbane, Australia). Ultrathin (100-120nm) sections were mounted on nickel grids and incubated on a blocking solution of PGB at pH 7.4 [PBS, 1% glycine, 1% BSA] for minutes. Following an overnight incubation at 4°C on mouse anti-human insulin (1:20) monoclonal antibody (Biogenex, San Roman, CA, USA) in PGB pH 7.4, the sections were incubated on a goat anti-mouse 10 nm (1:50) gold probe (British Biocell International, UK) in PGB pH 8.2 for 2 hours at room temperature. Primary antibody was omitted from control grids. The sections were stained for 20 min in 2% aqueous uranyl acetate and lead citrate for 20 min before examination in a Phillips CM 10 electron microscope.

Acute stimulation of insulin secretion static stimulation Before stimulation, tissue culture plates were thoroughly washed with basal medium [Phosphate Buffered Saline containing 1 mM CaCI 2 and supplemented with 20 mM HEPES and 2 mg/ml bovine serum albumin, 2.8 mM glucose (unless otherwise stated)] to remove culture medium and FCS. Monolayers were incubated in the basal medium at pH 7.4 for three consecutive 1 hr periods to stabilize the basal secretion of insulin.

Monolayers were then exposed to stimuli for 1 hr. Glucose (20 mM) and 10 mM theophylline (Sigma, St. Louis, MI, USA) were dissolved in basal medium. Calcium mM) was dissolved in basal medium without phosphate. Two controls were used- basal medium alone, and basal medium without phosphate.

To construct the dose response curve to glucose, the basal level of insulin secretion was established as described above and the monolayers were exposed to increasing concentrations of glucose from 0-20 mM over 1 hr periods.

WO 00/62862 PCT/AU00/00318 -26- Insulin synthesis Dose response curve to glucose after trypsinization 10 6 Huh7ins cells were incubated at 37 0 C CD 2 /95% 02) in vials containing 2 ml Krebs-Ringer bicarbonate buffer supplemented with 10 mM HEPES, 2mg/ml BSA (KRB/BSA), 50 pCi/ml L-[4,5- 3

H]

leucine and 20 mM glucose. After 2 hr, the cells were washed with KRB-BSA containing 0.05% nonradioactive leucine and disrupted by sonication in 200 jll distilled water. The amount of labelled insulin was determined by an immunoprecipitation technique as previously described (18).

Statistical analysis Data were expressed as means S.E. Paired sample means were compared with Student's t-test.

(ii) Results Gene transfer into hepatoma cells Huh7 cells were stably transfected with the pRc CMV vector carrying human insulin cDNA and 25 clones of the Huh7ins cells were isolated for further analysis. The transfectants have remained stable for a period of greater than six months in continuous culture.

By Northern analysis, GLUT 2 mRNA was clearly abundant in the RNA from Huh7 and Huh7ins cells, but absent in HEP G2 cells (negative control (Fig. To determine whether the GLUT 2 protein was expressed in the Huh7ins cells, Western blots of membranes were probed with an antiserum raised against human GLUT 2. This antiserum detected a protein of 55 kDa in the Huh7 and Huh7ins cells, having identical electrophoretic mobility to GLUT 2 in pancreatic islets (Fig. This protein was absent WO 00/62862 PCT/AU00/00318 -27from HEP G2 cells, indicating that both the parent Huh7 cell line and the transfected Huh7ins cells retain endogenous expression of the glucose transporter GLUT2.

Glucokinase activity was detected by RT/PCR in human liver tissue (positive control), untransfected Huh7 cells, transfected Huh7ins cells and HEP G2 cells. No glucokinase expression was detected in primary human fibroblasts (negative control) (Fig. 3).

Chronic secretion and storage of insulin The levels of human (pro)insulin secreted into the culture supematant from different clones of Huh7ins cells stably transfected with RcCMP-pC 2 differed 1.8-fold [0.16+ 0.0 to 0.30 0.10 pmoles (pro)insulin/ 106 cells/day]. Examination of acid-ethanol extracts of the cells indicated that the (pro)insulin content of the clones varied 1.6-fold from [4.20 0.60 to 6.93 0.80 pmoles (pro)insulin/10 6 cells]. Huh7 cells transformed with the RcCMV vector alone did not contain or secrete (pro) insulin. The Huh7ins clone 2 which stored the largest amount of (pro)insulin [6.93 0.80 pmoles (pro)insulin/l 06 cells] was used for the experiments detailed in this study. However static incubations and immunohistochemical staining were also carried out on clone 9 and clone 13, which stored 6.3 0.6 and 5.7 0.4 pmoles (pro)insulin/ 06 cells respectively. Identical results to those detailed below for clone 2 were demonstrated by these two. Immunohistochemical analysis using a polyclonal antibody confirmed that (pro)insulin was being stored (Fig. 4b).

Ultrastructural studies of the Huh7 and Huh7ins cells (Fig. 5) revealed aside from the normal structures seen in the cytoplasm of hepatoma cells, a complete secretory apparatus containing rough endoplasmic reticulum, Golgi complex and the secretory granules required for insulin synthesis and storage (Fig. 5. a The granules resembled the characteristic insulin granules seen in a normal P cell (19-21) with an electron dense core surrounded by an electron lucent halo with an outer limiting membrane. In a subpopulation of Huh7ins cells, large vacuoles of approximately (1-4 jpm) diameter were observed (Fig. Similar vacuoles were also described in HEP G2ins/g cells No granules were observed with Huh7 cells, but a smaller number of vacuoles were seen in the untransfected cells.

WO 00/62862 PCT/AU00/00318 -28- Immunostructural observations revealed that insulin was being stored in the granules (Fig These granules containing immunoreactive insulin were present in the cytoplasm of the cell, similar to the pattern seen in a normal 3 cell (19-21) and NIT1 insulinoma cells which were used as positive controls for the immunogold technology. Occasional labelling was seen on the large vacuoles, but nonspecific labeling was seen on cell nuclei or elsewhere in the cell. Control Huh7 cells were negative as were Huh7ins and NIT 1 cells in the absence of primary antibody.

Acute (pro)insulin secretion The effect of three well-known p-cell secretogogues, glucose, theophylline and calcium on the Huh7ins cells was determined. A 3-fold stimulation of (pro)insulin secretion over basal levels (Fig. 6a) was detected when 20 mM glucose was applied as a stimulus to Huh7ins cells for one hour. As would be expected in regulated release, on removal of the stimulus, insulin returned to basal levels. In the same experiment the cells showed a 46fold response to 10 mM theophylline (Fig. 6b) and an eight-fold response to 10 mM calcium over basal levels (Fig. 6c). (Pro)insulin secretion in response to glucose plus theophylline resulted in a significant (P<0.01) increase in (pro)insulin secretion to 2.7+0.3 pmoles (pro)insulin/10 6 cells above that seen for theophylline alone (Fig. 6d).

Similar results to these were seen for clones 9 and 13.

In response to varying concentrations of glucose from 0-20 mM, a dose-response curve for (pro)insulin secretion was generated (Fig. While glucose-responsiveness commenced at a slightly lower concentration than normal islets, a secretion curve approaching normal physiological conditions was generated.

(Pro)insulin synthesis In response to varying concentrations of glucose from 0-20 mM, a dose response curve was generated for (pro)insulin synthesis (Fig. 8) with half maximal synthesis occurring at WO 00/62862 PCT/AU00/00318 -29- 5.4 mM glucose and a Vmax of 42.9+1.4X 03c.p.m./tg DNA not significantly different for adult and fetal islets (22,23).

EXAMPLE 2 REVERSAL OF DIABETES BY TRANSPLANTATION OF AN INSULIN-PRODUCING CELL LINE Research Design and Methods Source of transfected liver cell line HUH7-ins cells were grown as monolayers in DMEM-medium containing 25 mM glucose (Trace Biosciences, Castle Hill, Sydney, Australia) in the presence of 10% fetal calf serum, 23.8 mM sodium bicarbonate and the selective antibiotic geneticin 0.55 mg/mL (Gibco, Grand Island, New York). Culture was in air and 5% CO 2 at 37 0 C. Details of the creation of these cells appear in Example 1. Briefly HUH7 cells were transfected with the human insulin cDNA gene under the control of the constitutive promoter CMV These cells have the capability to synthesize, store and secrete (pro)insulin.

Source of mice Male, inbred SCID mice were obtained from the Walter and Eliza Hall Institute of Medical Research in Melbourne, Victr.ria, Australia. They were housed in sterilized polycarbonate cages covered with autoclavable filter tops, six to a cage, and had unlimited access to sterilized tap water and gamma irradiated mouse food. The temperature of the room in which they were housed varied between 17 0 C and 24 0 C and the relative humidity 53% to 76%.

Induction of diabetes WO 00/62862 PCT/AU00/00318 30 Diabetes was induced in the mice by injection of multi-low dose streptozotocin mg/kg/day for 5 consecutive days This technique was used in preference to a single dose of streptozotocin because of low morbidity. Blood glucose levels were measured on iL blood obtained from a tail vein with the blood being placed on glucose reagent strips (Bayer Diagnostic, Mulgrave, Victoria, Australia) and the blood glucose level read in a glucometer (Bayer Diagnostics). The blood glucose levels of the 18 mice transplanted were 21.4 1.2 mM.

Transplantation of HUH7-ins cells HUH7-ins cells were trypsinized from culture flasks, their concentration determined with a haemocytometer, and aliquots of 1 x 10 7 cells were transplanted into the mice by either direct injection or in a plasma clot.

For the direct injection, the HUH7-ins cells were initially aspirated into polyethylene tubing (internal diameter 0.48 mm; external diameter 0.96 mm) attached to a 23 gauge needle placed on a Hamilton syringe. The end of the tubing was then inserted either beneath the left renal capsule of the mice (10) or into the subcutaneous tissues above the right scapula before the plunger of the syringe was rotated to allow injection of the cells into the mice. Six mice were transplanted in this way, 3 beneath the renal capsule and 3 subcutaneously. In a further 5 diabetic mice the cells were injected subcutaneously using a 1 mL syringe attached to a 213 gauge needle without the polyethylene tubing.

A further 7 mice were subsequently transplanted with cells placed in a plasma clot (11), 5 having grafts placed beneath the renal capsule and 2 subcutaneously.

Blood glucose levels and weight of the mice were monitored every 1-3 days. Insulin was not administered to the mice at any time, as is sometimes done to reduce mortality and morbidity. Grafts were removed from the mice when the blood glucose levels became WO 00/62862 PCT/AU00/00318 -31 low. Blood glucose levels of the mice were monitored thereafter, an increase expected if the graft had been responsible for reversal of the diabetes.

Oral glucose tolerance test An oral glucose tolerance test was performed in 5 mice when blood glucose levels became normal. This was carried out on mice fasted overnight. 16.7 .mol/g body weight of a 1.67 M glucose solution was injected into the stomach of the mice through polyethylene tubing that had been swallowed by the animals Blood glucose levels were measured at 0, 20, 40, 60 and 120 minutes after administration of the glucose.

Human C-peptide measurement Blood was collected from the tail vein of transplanted mice and controls at 0 and minutes during the oral glucose tolerance test for measurement of human C-peptide (Eurodiagnostica, Malm6, Sweden). The antibody used in the assay does not detect human insulin but there is 41% cross reactivity with human proinsulin. C-peptide rather than (pro)insulin was measured because the available antibody to insulin reacted with both human and mouse insulin.

Histological analysis Grafts removed were analysed histologically both with haematoxylin and eosin and immunoperoxidase stains for insulin. Insulin was detected using polyclonal guinea pig anti-insulin as a primary antibody, followed by sequential incubations with rabbit antiguinea pig antibody, biotinylated goat anti-rabbit antibody, and alkaline phosphataselabelled streptavidin. The chromogen 3-amino-9-ethylcarbazole was then added to stain the cytoplasm of insulin-positive cells red and the sections were counterstained with haematoxylin. All reagents were obtained from Dako (Carpintaria, California, USA).

Negative controls were conducted by omitting the primary antibody.

WO 00/62862 PCT/AU00/00318 -32- Ultrastructural analysis Grafts removed were cut into 1 mm 3 cubes which were immersion fixed for 1 hour at 4°C in 2% glutaraldehyde, 1% paraformaldehyde in 0.1M cacodylate buffered at pH 7.4 followed by 1% osmium tetroxide at room temperature for 20 minutes. A block stain was carried out in aqueous 2 uranyl acetate at room temperature for 1 hour. Tissue was dehydrated in solutions of graded alcohol and embedded in Durcupan (Fluka, Switzerland). Ultra-thin sections (80 nm) were cut on a Reichert Ultracut-S and counterstained with Reynolds' lead citrate and examined at 80 kV on a JEOL 100C electron microscope. Images were generated using Adobe Photoshop 4.01.

Liver cell insulin-related peptide content (Pro)insulin was extracted from liver cells with 0.18N HCL in 70% ethanol for 18 hours at 4°C. After evaporation of the ethanol, the samples were lyophylized and reconstituted in 50 iL of 0.1 triflouroacetic acid 0.1 bovine serum albumin (BSA) prior to a high performance liquid chromatographic analysis.

Reverse phase high performance liquid chromatography (RP-HPLC) for separation of insulin-related peptides The HPLC system consisted of a 1050 Hewlett-Packard pump with a Merck (Darinstadt, Germany) LiChrospher 100 RP-18 (5 Vim) 250 x 4 mm column with a guard cartridge.

Cell extract samples (100 piL each) were loaded on the HPLC column and eluted at a rate of 1 mL/min. with the following two buffer system: Buffer A- 3.4 mL. concentrated

H

3

PO

4 14 g NaCIO 4 2.2 g heptanesulfonic acid monohydrate sodium salt brought to pH 3; Buffer B: 90% acetonitrile in H,O. Solution B was held at 38% for 30 min. for insulin elution and then linearly increased to 42% over 65 min. for elution of proinsulinrelated peptides; for column wash solution B was then increased to 60% over 10 min., WO 00/62862 PCT/AU00/00318 33held at 60% for 5 min. and then linearly decreased over 10 min. to 38%. Absorption at 217 nm was monitored with a spectrophotometric detector (Jasco, Tokyo, Japan). One mL fractions were collected in tubes containing 0.1 ml 0.5 M boric acid, 0.1% BSA.

For determination of insulin and its related peptides, the samples were dried in a Speed- Vac apparatus and reconsitituted for radioimmunoanalysis in 0.5 ml phosphate buffered saline 0.1 BSA. Human insulin was determined by standard radioimmunoassay as described Elution positions of human insulin, diarginyl-insulin, split 31,32 proinsulin, split 65,66 proinsulin and intact proinsulin were determined with appropriate standards (kindly provided by Eli Lilly Co, Indianapolis, Indiana, USA).

Tissue culture of grafts Part of the grafts removed from the mice were diced and trypsinized with trypsin/EDTA (Trade Biosciences) for 4 minutes to produce single cells and these were allowed to attach to culture flasks containing collagen and grow. The medium used was the same as that applied to HUH7-ins cells prior to transplantation. Levels of (pro)insulin in conditioned medium was measured by radioimmunoassay. When sufficient cells had grown, they were exposed to abasal medium consisting of phosphate buffered Dulbecco's salt solution supplemented with 20 mM HEPES, 1 mg/mL bovine serum albumin and 2.8 mM glucose for two one hour periods before being exposed to a solution containing mM glucose To act as a control, some cells were exposed to the basal medium alone at this time. Thereafter both groups of cells were exposed for 1 hour to basal medium. Levels of (pro)insulin were measured by radioimmunoassay.

Statistical analysis Data were analysed with the computer statistical package NCSS Statistical significance of differences between groups was tested by Student's paired t test or, if there were more than two groups, by one-way analysis of variance after log WO 00/62862 PCT/AU00/00318 -34transformation of the data. Where the P value for the latter test was <0.05, the differences between individual groups were evaluated with Duncan's multiple range test.

(ii) Results Effect on blood glucose levels The blood glucose levels of all 18 mice that were transplanted became normal, 5.5 0.3 mM, in 5-26 days after the cells were implanted. Animals receiving cells beneath the renal capsule became normoglycaemic faster than those grafted subcutaneously, median of 12 days (range 5-12) vs. 18 days (range 12-26 days) (Figure 9A). Whether cells were injected or transplanted in a plasma clot did not alter the outcome (data not shown).

Blood glucose levels continued to decline reaching subnormal levels, 1-7 0.2 mM, a median of 3 days (range 1-13 days) after blood glucose levels were normalized.

Removal of the grafts at this time resulted in a prompt increase in the blood glucose levels of hyperglycaemic values, 16.7 3.5 mM (Figure 9B).

Oral glucose tolerance test The fasting blood glucose levels of the transplanted mice were lower than those of the controls, 2.2 0.3 mM vs. 6.3 1.1 mM (Figure 10). The levels remained unchanged in the grafted mice for the first 20 minutes, and thereafter fell to 1.2 0.2 mM at minutes, staying at this level for the remainder of the test. In contrast blood glucose levels rose in the control mice, reaching a peak at 20 minutes, 18.0 1.7 declining to 9.5 2.0 by the end of the test (Figure WO 00/62862 PCT/AUOO/00318 Plasma levels of human C-peptide Human C-peptide was present in the blood of transplanted mice but not the untransplanted controls. Fasting levels were 0.38 0.04 nM when the blood glucose levels were 2.2 0.3 mM. Levels 20 minutes after administration of an oral glucose load (oral glucose tolerance test) were unchanged at 0.37 0.01 nM when the blood glucose levels were 2.3 0.5 mM. Blood glucose levels did decline to 1.1 0.1 mM thereafter and this was not associated with clinical features of hypoglycaemia.

Histology Transplanted cells coalesced into a reddish friable mass at the site of grafting and grew rapidly. The weight of the graft removed at the time the animals had subnormal blood glucose levels was 76-505 mg (median 260 mg). Histological analysis showed that the cells had infiltrated into the surrounding tissues, so that they could not be fully removed, especially in the subcutaneously transplanted mice. Thus, the cells originally placed beneath the renal capsule infiltrated the kidney, and those transplanted subcutaneously nearby skeletal muscle. As a result there was regrowth of these remaining cells with blood glucose levels becoming normal and eventually subnormal again. The size of the cells in the grafts was variable and there were numerous mitoses. Immunoperoxidase stain for (pro)insulin showed the presence of this hormone or its precursor molecule in the graft (Figure 11A).

Electron microscopy Spherical electron dense granules surrounded by an outer limiting membrane were found in the grafted HUH7-ins cells. These granules were different from the typical crystalline core observed in adult human P cell granules. In cells that were close to the developing microvasculature of the graft site granules were seen adjacent to prominent Golgi apparatus that is required for the packaging of insulin into granules (Figure 11B). Some WO 00/62862 PCT/AU00/00318 -36cells contained electron dense granules within the rough endoplasmic reticulum (Figure 11C). In the cytoplasm of a third type of cell the granules were found next to vacuoles, of diameter 0.3 0.6 im (Figure 11D).

High performance liquid chromatographic analysis HPLC analysis of transfected liver cell extracts revealed that they contained insulinrelated peptides corresponding mainly to human diarginyl-insulin and two minor peaks detectable in the (pro)insulin radioimmunoassay corresponding to split 31,32 human proinsulin and intact human proinsulin. Only a very small peak was seen to elute at the position of mature human insulin (Figure 12).

(Pro)insulin content and secretion The (pro)insulin content of grafts removed from the mice was 1.47 0.25 pmol/100 mg.

Cells in these grafts retained their ability to store the hormone when cultured, at levels comparable to those of untransplanted cells (Figure 13), 5.05 5.59 pmol/10 6 cells.

They also were able to secrete (pro)insulin chronically in a manner similar to that of the untransplanted cells (Figure 13). Amount of hormone released per day varied from 0.28 to 0.34 pmol per 10 6 cells.

When cells isolated from the grafts were challenged with 20 mM glucose, insulin secretion was enhanced 1.9 2.6 fold (Figure 14). similar degree of glucose responsiveness was observed in untransplanted HUH7-ins cells Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in WO 00/62862 PCT/AU00/00318 -37this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

WO 00/62862 PCT/AU00/00318 -38-

BIBLIOGRAPHY:

1. Bland, B.J. (ed) (1997) International pancreas transplant registry newsletter; 9.

2. Hering, Brendel, Schultz, Schultz, Bretzel, R.G. (1996) International Islet Transplant Registry; 6 3. Permutt, M.A. et al. (1989) Proc. Natl. Acad. Sci. USA 89:8688-8692.

4. Weinhouse, S. (1976) Regulation of glucokinase in liver. In: Current topics in Cellular regulation. Horecker, B.L. Stadtman E.R. (ed) Academic Press: New York, San Francisco, London, pp. 1-50.

Simpson Tuch Swan Tu Marshal G.M. (1995) Gene Therapy 2:223-231.

6. Simpson, Marshall, Tuch, Maxwell, Szymanska, Tu, J., Beynon, Swan, Camacho, M. (1997) Gene Therapy 4:1202-1215.

7. Wilkinson, M. (1988) Nucleic Acids Res. 16:10933.

8. Simpson, Szymanska, Tuch, Marshall, G.M. (1999) Transplant Proc 31:812.

9. Tuch, Chen, J. (1993) Pancreas 8:305-11.

Tuch, Osgerby, Turtle, J.R. (1988) Transplantation 46:608-11.

11. Simeonovic, Brown, Teirrinen, Wilson, J.D. (1992) Preparation and transplantation of fetal proislets. In: Pancreatic Islet Cell Transplantation.

Ricordi C, Ed. Austin, Texas, RG Landes Co, p 238-48.

WO 00/62862 WO 0062862PCT/AUOO/0031 8 39 12. Gross, Leibowitz. Cerasi, Kaiser. N. (1996) Endocrinology 137:~5610- 56 13. Tuch, Maitland, Turtle, J.R. (1984) Culture and perifusion of human fetal pancreas. In: Methods in Diabetes Research Vol 1: Laboratocry methods, Part B. Larner, Pohl, S, Eds. New York, J Wiley Sons, p 153-163.

14. Hintze, J.L. (199 1) Number cruncher statistical system: version 5.0 1. Kaysville, Utah.

Noonan, Beck, Holzmayer, Chin, Wunder, Andrulis, Gazdar, Willman, Griffith, Van Hoff, Roninson, I.B. (1990) Proc Natl A cad Sci USA: 8 7:7160-7164.

16. Tanizawa, Koranyi, Welling, Permutt, M.A. (199 1) Proc Natl Acad Sci USA: 88:7294-7297.

17. Stirling, Graff, P. (1995) JHistochem Cytochem: 43:115-123.

18. Maldonato, Renold, Sharp, Cerasi, E. (1977) Diabetes: 26:538- 545.

19. Saccomanno, Spada, Bassetti, Gil-delAlamo, Faglia, G. (1993) J Histochem Cytochem: 41:1233-1239.

Lukinius, Ericsson, Grimelius, Korsgren, 0. (1992) Dev Bio:1S3:376-385.

21. Solica, E. In: J.V. Johannessen (ed) Electron Microscopy in Human Medicine.

McGraw-Hill International, New York, 1991, pp 189-212.

WO 00/62862 PCT/AUOO/003 18 40 22. Schuit. rN'T Veld, Pipeleers, D.G. (1988) Pro NlaulAcad Sci USA 85:3865-3869.

23. Simpson, Tuch, B.E. (1995) Pancreas 11:48-54.

EDITORIAL NOTE 36490/00 THIS SEQENCE LISTING FORMS A PART OF THE DESCRIPTION AND IS FOLLOWED BY CLAIM PAGES 41 TO 43 .1 1 WO 00/62862 WO 0062862PCT/AUOO/003 18 SEQUENCE LISTING <110> South Eastern Sydney Area Health Service University of Technology, Sydney <120> method of prophylaxis and treatment of diabetes <130> 2284432/TDO <140> <141> International 2000-04-14 <150> 60/129500 <151> 1999-04-15 <160> 2 <170> Patentln Ver. <210> <211> <212> <213> 1

DNA

mammal ian <400> 1 ctacttggaa caaatcgccc <210> 2 <211> <212> DNA <213> mammalian <400> 2 cctcctcctg gacttcttcc

Claims (8)

1. 9. 9 9 9*9 9 *99* 9e .9
2. 21-04-'05 08:35 FROM- T-737 P009/014 F-615 -41- THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS: 1. A method of preventing, reducing or otherwise ameliorating diabetes in a subject, said method comprising introducing into said subject an effective number of insulin producing cells wherein said cells are hepatocytes which have been transfected with a nucleic acid molecule encoding insulin or a derivative, homologue or mimetic thereof. 2. The method according to claim 1 wherein said cells express a high capacity glucose transporter. 3. The method according to claim 2 wherein said transporter is GLUT 2. 4. The method according to claim 3 wherein said hepatocyte is a Huh7 cell. 5. The method according to claim 4 wherein said transfected hepatocyte is Huh7ins. 6. A method for the treatment and/or prophylaxis of a condition characterised by aberrant, unwanted or otherwise inappropriate functional activity or levels of glucose in a subject, said method comprising administering to said subject an effective number of insulin producing cells wherein said cells are hepatocytes which have been transfected with a nucleic acid molecule encoding insulin or a derivative, homologue or mimetic thereof. 7. The method according to claim 6 wherein said condition is diabetes. 8. The method according to claim 7 wherein said cells express a high capacity glucose transporter. 9. The method according to claim 8 wherein said transporter is GLUT 2. COMS ID No: SBMI-01214531 Received by IP Australia: Time 08:41 Date 2005-04-21 21-04-'05 08:35 FROM- T-737 P010/014 F-615 -42- The method according to claim 9 wherein said hepatocyte is a Huh7 cell. 11. The method according to claim 10 wherein said transfected hepatocyte is Huh7ins. 12. A method of up-regulating insulin levels in a subject said method comprising introducing into said subject an effective number of insulin producing cells wherein said cells are hepatocytes which have been transfected with a nucleic acid molecule encoding insulin or a derivative, homologue or mimetic thereof. 13. A method of down-regulating glucose levels in a subject said method comprising introducing into said subject an effective number of insulin producing cells wherein said cells are hepatocytes which have been transfected with a nucleic acid molecule encoding insulin or a derivative, homologuc or mimetic thereof. 9 14. The method according to claim 12 or 13 wherein said cells express a high capacity glucose transporter. 15. The method according to claim 14 wherein said transporter is GLUT 2. 9 9 16. The method according to claim 15 wherein said hepatocyte is a Huh7 cell. 17. The method according to claim 16 wherein said transfected cell is Huh7ins. 18. Use of hepatocytes which produce insulin in the manufacture of a medicament for the treatment of a condition characterised by aberrant, unwanted or otherwise inappropriate functional activity or levels of glucose in a subject, wherein said hepatocytes have been transfected with a nucleic acid molecule encoding insulin or a derivative, homologue or mimetic thereof. 19. Use according to claim 18 wherein said condition is diabetes. COMS ID No: SBMI-01214531 Received by IP Australia: Time 08:41 Date 2005-04-21 21-04-'05 08:35 FROM- T-737 P011/014 F-615 9* 43 Use according to claim 18 or 19 wherein said cells express a high capacity glucose transporter. 21. Use according to claim 20 wherein said transporter if GLUT2.
3. 22. Use according to claim 21 wherein said hepatocyte is a H-uh7 cell.
4. 23. Use according to claixn 22 wherein said transfected hepatocyte is Huh7ins.
5. 24. A cell transfected with a nucleic acid molecule encoding insulin or a derivative, homiologu~e or mimetic thereof wherein said cell is a hepatocyte which produces insulin or a derivative, homologue or mimetic thiereof, which insulin production is regulatable in vivo when used in the method of any one of claims 1 to 17.
6. 25. The cell according to claim 24 wherein said nucleic acid molecule is cDNA encoding insulin.
7. 26. The cell according to claim 24 or 25 wherein said hepatocyte is a Huh7 cell.
8. 27. The cell according to claim 26 wherein said transfected hepatocyte is a Huh7ins cell. DATED this 2 1 day of April 2005 SOUTH EASTERN SYDNEY AREA HEALTH SERVICES and UNIVERSITY OF TECHNOLOGY, SYDNEY by DAVIES COLLISON CAVE Patent Attorneys for the Applicants COMS ID No: SBMI-01214531 Received by IP Australia: Time 08.41 Date 20D5-04-21