+

WO2000062862A1 - Technique de prophylaxie et de traitement des diabetes - Google Patents

Technique de prophylaxie et de traitement des diabetes Download PDF

Info

Publication number
WO2000062862A1
WO2000062862A1 PCT/AU2000/000318 AU0000318W WO0062862A1 WO 2000062862 A1 WO2000062862 A1 WO 2000062862A1 AU 0000318 W AU0000318 W AU 0000318W WO 0062862 A1 WO0062862 A1 WO 0062862A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
insulin
derivative
mimetic
homologue
Prior art date
Application number
PCT/AU2000/000318
Other languages
English (en)
Inventor
Bernard Edward Tuch
Ann Margaret Simpson
Original Assignee
South Eastern Sydney Area Health Service
University Of Technology, Sydney
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by South Eastern Sydney Area Health Service, University Of Technology, Sydney filed Critical South Eastern Sydney Area Health Service
Priority to AU36490/00A priority Critical patent/AU783594B2/en
Publication of WO2000062862A1 publication Critical patent/WO2000062862A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/28—Insulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/067—Hepatocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • 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.
  • Insulin dependant diabetes mellitus also referred to as type 1 diabetes or juvenile onset diabetes due to its appearance in childhood or early adolescence
  • 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 ⁇ cells.
  • 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 ⁇ cells are no longer able to produce.
  • attempts have been made to replace the missing ⁇ cells by transplantation of a whole pancreas, the islets from an adult pancreas or insulin producing fetal pancreatic cells.
  • pancreas While transplantation of whole pancreas is of benefit in reversing diabetes in humans, with 76% one year graft survival (1), 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 (2). 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.
  • 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.
  • nucleotide sequence information prepared using the programme Patentln 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 , ⁇ 210>2, etc).
  • sequence identifier eg. ⁇ 210>1 , ⁇ 210>2, etc.
  • the length, type of sequence (DNA, etc) and source organism for each nucleotide sequence are indicated by information - J -
  • numeric indicator fields ⁇ 211>, ⁇ 212> and ⁇ 213> are 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 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.
  • 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 comprising 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 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.
  • Figure 1 is an image of a Northern blot analysis of GLUT 2 in HEP G2 (lane 1), Huh7 (lane 2), 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 1), Huh7 control cells (lane 2), Huh7ins cells, clone 2 (lane 3), 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 1), Huh 7 cells (lane 2), Huh7ins cells (clone 2, transfected with insulin cDNA, lane 3), Huh7ins cells (clone 9, transfected with insulin cDNA, lane 4), human liver (lane 5) and human skin fibroblasts (lane 6).
  • hGLK human glucokinase
  • Figure 4 is an image of light micrographs of (a) 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 6 is a graphical representation of stimulation by (a) 20 mM glucose, (b) 10 mM theophylline, (c) 10 mM calcium and (d) 20 mM glucose + 10 mM theophylline on the acute regulated release of immunoreactive (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; Bl : 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 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 ⁇ mol 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.
  • Electron dense granules are present in the cytoplasm of the cells in 3 different arrangements.
  • 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; 3- split 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.
  • SCI and SC2 represent two different mice from which grafts were removed.
  • Clone 2 is the HUH7-ins cells prior to transplantation.
  • Figure 14 is a graphical representation of acute (pro)insulin secretion in response to 20 mM glucose from cells isolated from grafts removed diabetic SCID mice, the blood glucose levels of which had been normalized by the grafts.
  • SCI and SC2 represent two different mice from which grafts were removed. Cells were exposed to basal medium both before (“Basal 1 " and "Basal 2") and after ("Basal 3") addition of 20 mM glucose ("Stim"). Note that levels of the hormone rose when glucose was added and declined when glucose was removed.
  • 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.
  • 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.
  • references 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 ⁇ 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.
  • congenital defects in the pancreatic islet cells for example type 1 diabetes/IDDM, gestational diabetes or slowly progressive IDDM which is also referred to as latent autoimmune diabetes in adults
  • 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.
  • the method of the present invention may either reduce the severity of any one or more symptoms or eliminate the existence of any one or more symptoms.
  • 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.
  • 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.
  • 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 ⁇ or ⁇ chain of insulin in isolation or various isoforms of insulin due to the translation of mRNA splice variants.
  • 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 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.
  • derivatives, homologues or mimetics of insulin which exhibit one or more of the functional activities of insulin.
  • 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 least 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.
  • 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.
  • 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.
  • 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 switched on, that is, expression of the gene is induced or even up-regulated where the gene is not expressed in sufficiently high levels.
  • 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.
  • 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.
  • the insulin is secreted.
  • the insulin which is produced may be stored intracellularly for a period of time prior to its release.
  • 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.
  • 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.
  • cells which either naturally or as a result of genetic modification express the high capacity glucose transporter, GLUT 2 (3) and the high capacity glucose phosphorylation enzyme glucokinase (4) 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.
  • the "cells" which are introduced into an individual in accordance with the method of the present invention may be in any suitable form.
  • 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.
  • 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.
  • 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.
  • 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 unrelated individual).
  • 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.
  • 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.
  • the site of transplant may be any suitable site, for example, subcutaneously or, where the donor cells are liver cells, under the renal capsule.
  • 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.
  • the cells are liver cells (hepatocytes) since liver cells express GLUT 2 and glucokinase.
  • 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, wherein said hepatocytes produce insulin or a derivative, homologue or mimetic thereof.
  • said hepatocytes are from the glucose-responsive insulin secreting hepatoma cell line Huh7ins, which has been developed by the inventors.
  • the cells of this cell line have been transfected with insulin cDNA and are capable of storing insulin and expressing and secreting insulin in response to glucose stimulation.
  • 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 (i.e.
  • Huh7ins cells have the ability to package insulin into a form of limiting membrane (i.e. a releasable compartment) until regulated release occurs.
  • 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.
  • other proteinaceous or non- proteinaceous molecules may be coadministered either with the introduction of the insulin- producing cells or during insulin production by the transplanted cells.
  • 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.
  • 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.
  • transplanted cells constituitively release insulin
  • This may be achieved, for example, utilising the tetracycline conditional gene expression system whereby insulin synthesis is inhibited when tetracycline is given to the host when this gene is stimulated.
  • 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.
  • the subject undergoing treatment or prophylaxis may be any human or animal in need of therapeutic or prophylactic treatment.
  • treatment and prophylaxis are to be considered in its broadest context.
  • the term “treatment” does not necessarily imply that a mammal is treated until total recovery.
  • prophylaxis does not necessarily mean that the subject will not eventually contract a disease condition.
  • 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.
  • the subject of the treatment is a mammal and still more preferably a human.
  • 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.
  • 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 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 should be understood as a reference to either permanently or transiently abnormal levels or activities of these molecules or to physiologically normal levels or activities of one or both of these molecules, which levels or activities are nevertheless unwanted or otherwise inappropriate.
  • a molecule which is "directly” modultable by insulin is one which the subject insulin associates or otherwise interacts with to up-regulate, down- regulate 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.
  • 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.
  • said molecule is glucose or insulin or derivative or equivalent thereof.
  • 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.
  • 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.
  • 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.
  • said modulation is up-regulation.
  • 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.
  • said modulation is down-regulation.
  • said cells have been transfected with a nucleic acid molecule encoding insulin or a derivative, homologue or mimetic thereof.
  • said cells are hepatocytes and still more preferably Huh7ins cells.
  • 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.
  • said molecule is insulin and/or glucose or derivative or equivalent thereof.
  • said condition is diabetes.
  • 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.
  • said cells are utilised in the methods hereinbefore defined.
  • 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.
  • DMEM Dulbecco's Modification of Eagles's medium
  • Huh7 cells were grown as monolayers in DMEM supplemented with lu% 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.
  • 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, anti- rabbit immunoglobulin (1 :500)] for 30 min at room temperature.
  • PBS phosphate buffered saline
  • 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. Supematants were prepared by centrifugation at 16,000 g in a refrigerated microfuge.
  • Protein concentration in the supernatant was determined by the method of Bradford (14), and 5 ⁇ g of protein was suspended in an equal volume of 2X sample buffer (100 mM Tris, 4%SDS, 0.2% bromophenol blue, 20% glycerol, 10% ⁇ -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 8.0).
  • 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 (BS A) and bands visualised using the ECL chemiluminescent Western blotting kit (Amersham International, Amersham, Bucks, UK).
  • human GLUT 2 (1 :500) (gift from Prof. M. Permutt, Washington University, St. Louis, MI, USA) in TBST plus 1% bovine serum albumin (BS A) and bands visualised using the ECL chemiluminescent Western blotting kit (Amersham International, Amersham, Bucks, UK).
  • RNA was isolated by the guanidinium thiocyanate method (7).
  • RNA (l ⁇ 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 1 OOng total RNA and 1 unit of Amplitaq polymerase (Perkin/Elmer, Norwalk, IN, USA) in a reaction mix containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl 2 , 0.01% (w/v) gelatin, 250 ⁇ M 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 94°C, a 35 cycle program consisting of 94°C for 45 sec, 61 °C for 1 min and 72°C for 45 sec was performed.
  • This primer pair generated a 155 bp PCR product from bp 244-379 of the published human glucokinase sequence (16).
  • 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.
  • a goal anti guinea pig lOnm (1 :50) primary antibody (Zymed, San Francisco, USA) incubated overnight at 4°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 10 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 CM10 electron microscope.
  • basal medium Phosphate Buffered Saline containing 1 mM CaCl 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 (10 mM) was dissolved in basal medium without phosphate.
  • Two controls were used- basal medium alone, and basal medium without phosphate.
  • 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. Insulin synthesis
  • 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.
  • GLUT 2 mRNA was clearly abundant in the RNA from Huh7 and Huh7ins cells, but absent in HEP G2 cells (negative control (Fig. 1).
  • 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. 2). This protein was absent from 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).
  • 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°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 (8). These cells have the capability to synthesize, store and secrete (pro)insulin.
  • mice Male, inbred SCID mice were obtained from the Walter and Eliza Hall Institute of Medical Research in Melbourne, Victoria, Australia. They were housed ; n 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°C and 24°C and the relative humidity 53 % to 76% .
  • 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.
  • 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.
  • mice were subsequently transplanted with cells placed in a plasma clot (11), 5 having grafts placed beneath the renal capsule and 2 subcutaneously.
  • mice 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 (10). Blood glucose levels were measured at 0, 20, 40, 60 and 120 minutes after administration of the glucose.
  • 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 counter- stained with Reynolds' lead citrate and examined at 80 kV on a JEOL 100C electron microscope. Images were generated using Adobe Photoshop 4.01.
  • the HPLC system consisted of a 1050 Hewlett-Packard pump with a Merck (Darmstadt, Germany) LiChrospher 100 RP-18 (5 ⁇ m) 250 x 4 mm column with a guard cartridge.
  • Cell extract samples (100 ⁇ L 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 NaClO 4 , 2.2 g heptanesulfonic acid monohydrate sodium salt brought to pH 3; Buffer B: 90% acetonitrile in H 2 O. Solution B was held at 38 % for 30 min.
  • 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 (12). 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).
  • 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.
  • 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.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Diabetes (AREA)
  • Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Endocrinology (AREA)
  • Hematology (AREA)
  • Wood Science & Technology (AREA)
  • Obesity (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Gastroenterology & Hepatology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Emergency Medicine (AREA)
  • Genetics & Genomics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Epidemiology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne, de manière générale, une technique de prophylaxie et de traitement des diabètes, et des agents utilisés à ces fins. Elle concerne, plus particulièrement, la transplantation de cellules productrices d'insuline permettant de se protéger contre les symptômes associés aux diabètes ou de les améliorer. Elle concerne, encore plus particulièrement, une technique permettant de prévenir, de réduire, ou d'améliorer les effets des diabètes par transplantation de cellules génétiquement altérées, en particulier de cellules du foie, afin d'exprimer l'insuline.
PCT/AU2000/000318 1999-04-15 2000-04-14 Technique de prophylaxie et de traitement des diabetes WO2000062862A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU36490/00A AU783594B2 (en) 1999-04-15 2000-04-14 Method of prophylaxis and treatment of diabetes

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US12950099P 1999-04-15 1999-04-15
US60/129,500 1999-04-15

Publications (1)

Publication Number Publication Date
WO2000062862A1 true WO2000062862A1 (fr) 2000-10-26

Family

ID=22440274

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/AU2000/000318 WO2000062862A1 (fr) 1999-04-15 2000-04-14 Technique de prophylaxie et de traitement des diabetes

Country Status (2)

Country Link
AU (1) AU783594B2 (fr)
WO (1) WO2000062862A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2185696A4 (fr) * 2007-08-10 2011-04-13 Univ Sydney Tech Cellules génétiquement modifiées pour comprendre la glucokinase d'un îlot pancréatique et leurs utilisations
EP1605965B1 (fr) * 2003-03-26 2012-12-26 DeveloGen Aktiengesellschaft Utilisation de proteines associees a la saposine pour la prevention et le traitement de l'obesite, du diabete et/ou du syndrome metabolique

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994020624A1 (fr) * 1993-03-01 1994-09-15 Genentech, Inc. Cellules transformees par convertase de pro-hormones et synthese de polypeptides
WO1995032740A1 (fr) * 1994-05-31 1995-12-07 Unisearch Limited Lignee de cellules transformees qui proviennent d'un carcinome hepatocellulaire humain et qui sont capables de produire de l'insuline
WO1996040894A1 (fr) * 1995-06-07 1996-12-19 The Regents Of The University Of California Traitement de l'insulinoresistance
WO1997026357A1 (fr) * 1996-01-19 1997-07-24 Board Of Regents, The University Of Texas System Compositions et procedes d'inhibition de l'hexokinase
WO1997029180A1 (fr) * 1996-02-06 1997-08-14 Eli Lilly And Company Therapie des diabetes
WO1998020906A2 (fr) * 1996-11-08 1998-05-22 The Autonomous University Of Barcelona Traitement du diabete avec un gene de facteur de transcription
WO1998031397A1 (fr) * 1997-01-21 1998-07-23 Wisconsin Alumni Research Foundation Traitement du diabete a l'aide de cellules beta de synthese

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994020624A1 (fr) * 1993-03-01 1994-09-15 Genentech, Inc. Cellules transformees par convertase de pro-hormones et synthese de polypeptides
WO1995032740A1 (fr) * 1994-05-31 1995-12-07 Unisearch Limited Lignee de cellules transformees qui proviennent d'un carcinome hepatocellulaire humain et qui sont capables de produire de l'insuline
WO1996040894A1 (fr) * 1995-06-07 1996-12-19 The Regents Of The University Of California Traitement de l'insulinoresistance
WO1997026357A1 (fr) * 1996-01-19 1997-07-24 Board Of Regents, The University Of Texas System Compositions et procedes d'inhibition de l'hexokinase
WO1997029180A1 (fr) * 1996-02-06 1997-08-14 Eli Lilly And Company Therapie des diabetes
WO1998020906A2 (fr) * 1996-11-08 1998-05-22 The Autonomous University Of Barcelona Traitement du diabete avec un gene de facteur de transcription
WO1998031397A1 (fr) * 1997-01-21 1998-07-23 Wisconsin Alumni Research Foundation Traitement du diabete a l'aide de cellules beta de synthese

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CLARK ET AL.: "Novel insulinoma cell lines produced by iterative engineering of GLUT2, glucokinase and human insulin expression", DIABETES, vol. 46, no. 6, 1997, pages 958 - 967 *
EDITORS, BRETZEL ET AL., INTERNATIONAL ISLET TRANSPLANT REGISTRY, NEWSLETTER NO. 7 6/1, 1996, pages 1 - 20 *
SIMPSON ET AL.: "Secretion and storage from a human hepatoma cell line (HUH7-INS)", TRANSPLANT PROC., vol. 31, no. 1-2, February 1999 (1999-02-01) - March 1999 (1999-03-01), pages 812 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1605965B1 (fr) * 2003-03-26 2012-12-26 DeveloGen Aktiengesellschaft Utilisation de proteines associees a la saposine pour la prevention et le traitement de l'obesite, du diabete et/ou du syndrome metabolique
EP2185696A4 (fr) * 2007-08-10 2011-04-13 Univ Sydney Tech Cellules génétiquement modifiées pour comprendre la glucokinase d'un îlot pancréatique et leurs utilisations
US20150030574A1 (en) * 2007-08-10 2015-01-29 University Of Technology, Sydney Cells genetically modified to comprise pancreatic islet glucokinase and uses thereof
US9365829B2 (en) 2007-08-10 2016-06-14 University Of Technology, Sydney Cells genetically modified to comprise pancreatic islet glucokinase and uses thereof
US9732329B2 (en) 2007-08-10 2017-08-15 University Of Technology, Sydney Cells genetically modified to comprise pancreatic islet glucokinase and uses thereof
US10738285B2 (en) 2007-08-10 2020-08-11 University Of Technology, Sydney Cells genetically modified to comprise pancreatic islet glucokinase and uses thereof

Also Published As

Publication number Publication date
AU783594B2 (en) 2005-11-10
AU3649000A (en) 2000-11-02

Similar Documents

Publication Publication Date Title
Korbutt et al. Large scale isolation, growth, and function of porcine neonatal islet cells.
US6703017B1 (en) Reversal of insulin-dependent diabetes by islet-producing stem cells, islet progenitor cells and islet-like structures
US5759536A (en) Use of fas ligand to supress T-lymphocyte-mediated immune responses
KR19990067077A (ko) 기능성 랑게르한스 섬의 시험관내 성장 및 그 생체내 사용방법
US20210040459A1 (en) Cells genetically modified to comprise pancreatic islet glucokinase and uses thereof
CA2246431A1 (fr) Expression recombinee de proteines a partir de lignees cellulaires secretoires
WO2001023528A1 (fr) Inversion de diabetes dependant de l'insuline par des cellules souches insulaires, des cellules insulaires progenitrices et des structures de type insulaire
NO320713B1 (no) Farmasoytisk preparat som omfatter Sertoli-celler og celler som produserer en biologisk faktor, seksjonert sett med beholdere for cellene, produktartikkel som omfatter et embaleringsmateriale med Sertoli-celler og fremgangsmate for okning av utvinning og profilering av celler ex vivo.
Ramesh et al. Pancreatic islet transplantation in type 1 diabetes mellitus: an update on recent developments
Gray et al. Islet cell transplantation for insulin-dependent diabetes mellitus: perspectives from the present and prospects for the future
JPH11505411A (ja) 組換えβ細胞およびその使用
BROWN et al. Pancreas transplantation for diabetes mellitus
Wahoff et al. Islet autotransplantation after total pancreatectomy in a child
CA2477538C (fr) Pancreas chimerique
Rajab et al. Comparison of the portal vein and kidney subcapsule as sites for primate islet autotransplantation
AU783594B2 (en) Method of prophylaxis and treatment of diabetes
Vo et al. Lowering of blood glucose to nondiabetic levels in a hyperglycemic pig by allografting of fetal pig isletlike cell clusters1
Lipes et al. Genetic engineering of insulin expression in nonislet cells: implications for beta-cell replacement therapy for insulin-dependent diabetes mellitus
US7169608B2 (en) Bone marrow cell differentiation
US20050048040A1 (en) Methods for improving pancreatic islet cell transplantation
Karl et al. Transplantation of insulin-secreting tissues
Mullen et al. Islet transplantation for the cure of diabetes
Malone Understanding diabetes in children
Gołebiewska et al. “Old School” Islet Purification Based on the Unit Gravity Sedimentation as a Rescue Technique for Intraportal Islet Transplantation—A Case Report
WO1994015459A1 (fr) Procede d'isolement et de transplantation d'hepatocytes specialises

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DE DK DM DZ EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WWE Wipo information: entry into national phase

Ref document number: 36490/00

Country of ref document: AU

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP

WWG Wipo information: grant in national office

Ref document number: 36490/00

Country of ref document: AU

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载