US20120195864A1 - Cells genetically modified to comprise pancreatic islet glucokinase and uses thereof - Google Patents

Cells genetically modified to comprise pancreatic islet glucokinase and uses thereof Download PDF

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US20120195864A1
US20120195864A1 US12/672,832 US67283208A US2012195864A1 US 20120195864 A1 US20120195864 A1 US 20120195864A1 US 67283208 A US67283208 A US 67283208A US 2012195864 A1 US2012195864 A1 US 2012195864A1
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cells
insulin
cell
genetically modified
hepatocyte
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Ann Margaret Simpson
Chang Tao
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University of Technology Sydney
Merck Sharp and Dohme LLC
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University of Technology Sydney
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/37—Digestive system
    • A61K35/407—Liver; Hepatocytes
    • 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
    • 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
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/45—Transferases (2)
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/62—Insulins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/33—Insulin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/20—Vector systems having a special element relevant for transcription transcription of more than one cistron
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/01—Phosphotransferases with an alcohol group as acceptor (2.7.1)
    • C12Y207/01002—Glucokinase (2.7.1.2)

Definitions

  • the present invention relates generally to a population of cells genetically modified to produce insulin in a glucose responsive manner and uses thereof. More particularly, the present invention relates to a population of cells genetically modified to produce insulin in response to physiologically relevant levels of glucose and uses thereof.
  • the cells of the present invention are useful in a wide variety of applications, in particular in the context of therapeutic and prophylactic regimes directed to the treatment of diabetes and/or the amelioration of symptoms associated with diabetes, based on the transplantation of the cells of the present invention into mammals requiring treatment. Also facilitated is the design of in vitro based screening systems for testing the therapeutic effectiveness and/or toxicity of potential adjunctive treatment regimes.
  • Diabetes mellitus is characterised by an abnormality of carbohydrate metabolism resulting in elevated glucose levels in both the blood and the urine.
  • the failure of the human body to properly metabolise the glucose is caused by defects in insulin secretion or use of insulin.
  • Insulin is produced by ⁇ -cells in the islets of the pancreas and permits the body to utilise glucose as a source of energy. When this process cannot occur, the body compensates by utilising alternative sources of energy such as stored fats. However, this leads to rapidly rising levels of glucose and the accumulation of ketones in the bloodstream due to the occurrence of extensive fat metabolism.
  • Type 1 diabetes is broadly classified into two groups termed Type 1 diabetes and Type 2 diabetes.
  • Type 1 diabetes (often referred to as 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 ⁇ -cells in the islets of the pancreas. Its onset is abrupt and occurs typically prior to the age of 20 years.
  • Type 1 diabetes is increasingly presenting in adults. This disease is characterised by lack of n-cell function and no insulin production, and therefore insulin therapy is required.
  • Type 2 diabetes is characterised by insulin resistance, a condition in which the body fails to properly use insulin, which is often accompanied by obesity and other metabolic disorders. There are frequently no overt symptoms observed. Insulin secretory defects are evident very early in disease in both Type 1 and Type 2 diabetes, despite their differing aetiology.
  • diabetes In the absence of treatment, 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 diabetes and also severe symptoms of type ⁇ diabetes is generally by daily insulin injection to replace the insulin which the damaged cells are no longer able to produce.
  • complications and side effects are common.
  • diabetic vascular complications affecting both micro- and macro-blood vessels, represent major causes of disability and death in the patients with type 1 and type 2 diabetes.
  • diabetes is now recognized as a potent and independent risk factor for the development of coronary, cerebrovascular and peripheral atherosclerotic disease (Beckman et al., 2002 , JAMA 287:2570-2581).
  • Treatment of diabetes can also be effected via the transplantation of insulin-secreting tissue.
  • this latter strategy relies on the use of scarce human tissue as a source, it seems unlikely that there will ever be sufficient numbers of organs available to assist more than a selected number of insulin-dependent diabetics. Furthermore, these patients would have to undergo a long term regimen of immunosuppressive drugs.
  • the term “derived from” shall be taken to indicate that a particular integer or group of integers has originated from the species specified, but has not necessarily been obtained directly from the specified source. Further, as used herein the singular forms of “a”, “and” and “the” include plural referents unless the context clearly dictates otherwise.
  • nucleotide sequence information prepared using the programme PatentIn Version 3.1, presented herein after the bibliography.
  • Each nucleotide sequence is identified in the sequence listing by the numeric indicator ⁇ 210> followed by the sequence identifier (eg. ⁇ 210>1, ⁇ 210>2, etc).
  • the length, type of sequence (DNA, etc) and source organism for each nucleotide sequence is indicated by information provided in the numeric indicator fields ⁇ 211>, ⁇ 212> and ⁇ 213>, respectively.
  • Nucleotide sequences referred to in the specification are identified by the indicator SEQ ID NO: followed by the sequence identifier (eg. SEQ ID NO:1, SEQ ID NO:2, etc.).
  • sequence identifier referred to in the specification correlates to the information provided in numeric indicator field ⁇ 400> in the sequence listing, which is followed by the sequence identifier (eg. ⁇ 400>1, ⁇ 400>2, etc). That is SEQ ID NO:1 as detailed in the specification correlates to the sequence indicated as ⁇ 400>1 in the sequence listing.
  • One aspect of the present invention is directed to a genetically modified mammalian cell, which cell is capable of secreting insulin, said genetic modification comprising the transfection of said cell with a nucleic acid molecule encoding pancreatic islet glucokinase.
  • a genetically modified mammalian hepatocyte which hepatocyte is capable of secreting insulin, said genetic modification comprising the transfection of said hepatocyte with a nucleic acid molecule encoding pancreatic islet glucokinase.
  • Yet another aspect of the present invention is directed to a genetically modified mammalian hepatocyte, which hepatocyte is capable of secreting insulin, said genetic modification comprising the transfection of said hepatocyte with a nucleic acid molecule encoding pancreatic islet glucokinase and wherein said cell is responsive to glucose in a physiologically relevant manner.
  • Still another aspect of the present invention provides a genetically modified human hepatocyte, which hepatocyte is capable of secreting insulin, said genetic modification comprising the transfection of said hepatocyte with a nucleic acid molecule encoding pancreatic islet glucokinase.
  • Yet still another aspect of the present invention provides a genetically modified human hepatocyte, which hepatocyte is capable of secreting insulin, said genetic modification comprising the transfection of said cell with a vector, which vector comprises a nucleic acid molecule encoding pancreatic islet glucokinase.
  • Still yet another aspect of the present invention provides a genetically modified Huh7ins cell, said genetic modification comprising the transfection of said Huh7ins cell with a vector, which vector comprises a nucleic acid molecule encoding pancreatic islet glucokinase.
  • a further aspect of the present invention is directed to a method of therapeutically and/or prophylactically treating a condition in a mammal, which condition is characterised by the aberrant production of functional insulin, said method comprising introducing into said mammal an effective number of the genetically modified cells hereinbefore defined.
  • Another further aspect of the present invention provides a method of therapeutically and/or prophylactically treating diabetes in a mammal, said method comprising introducing into said mammal an effective number of the genetically modified cells hereinbefore defined.
  • Still another aspect of the present invention contemplates a method of modulating insulin levels in a mammal said method comprising introducing into said subject an effective number of the genetically modified cells hereinbefore defined.
  • Yet another aspect of the present invention contemplates a method of modulating glucose levels in a mammal said method comprising introducing into said subject an effective number of the genetically modified cells hereinbefore defined.
  • Still another aspect of the present invention is directed to the use of genetically modified cells hereinbefore defined in the manufacture of a medicament for the treatment of a condition in a mammal, which condition is characterised by the aberrant production of functional insulin.
  • a method of assessing the effect of a treatment or culture regime on the phenotypic state of the genetically modified cells as hereinbefore defined comprising subjecting said cells to said treatment regime and screening for an altered phenotypic state.
  • FIG. 1 is a schematic representation of pIRESpuro3.
  • the human islet glucokinase cDNA was cut out of the vector pBluescriptSK at the Eco R I site and subsequently cloned into the EcoRI site of the multi-cloning site of the pIRESpuro3 vectors.
  • FIG. 2 is a schematic representation of the pIERSpuro3 vector sequence of the pIERSpuro3 vector (5157 base pairs sequence SEQ ID NO:1) with insert human islet glucokinase cDNA (2733 base pairs SEQ ID NO:2) highlighted in enlarged text.
  • the nucleotides in bold text represent the remnant nucleotides of the pBluescriptSK cloning vector.
  • SEQ ID NO:3 represents the vector sequence incorporating the islet glucokinase cDNA sequence.
  • FIG. 3 is an image of the RT/PCR expression of human islet glucokinase, 220 bp product.
  • DNA marker (lane 1), Melligen cells clone 6 (lane 2), Huh7ins cells with pIRISpuro3 vector only (lane 3), Huh7ins cells (lane 4).
  • FIG. 4 is an image of a Western blot analysis for human glucokinase in Huh7ins (lane 1), Huh7ins cells with pIRISpuro3 vector only (lane 2), Melligen cells (lane 3).
  • (b) Immuno-electron micrographs showing localization of insulin in Melligen cells (bar 460 nm).
  • FIG. 11 is a graphical representation of the growth kinetics of MIN 6 cells and the three liver cell lines used.
  • Cells were initially seeded at a density of 1 ⁇ 10 4 cells/mL, for liver cell lines, and 2 ⁇ 10 4 cells/mL, for MIN-6 cells, into six well plates.
  • FIG. 15 is an image of Annexin/PI staining of Huh7ins at both 24 h and 48 h which shows no early or late apoptotic cell death. Only a necrotic population was detected and the difference between control and treated cells was not significant p>0.05. These figures are representative of four independent experiments.
  • FIG. 16 is an image showing that at both 24 h and 48 h Melligen cells showed no early or late apoptotic cell death. Only a necrotic population was detected and the difference between control and treated cells was not significant p>0.05. These figures are representative of four independent experiments.
  • FIG. 17 is an image of Huh7ins PI only staining showing that there was no sub-G1 peak evident in treated and control cells at 24 h and 48 h. These figures are representative of four independent experiments.
  • FIG. 18 is an image of Melligen cells PI only staining showing that there was no sub-G1 peak evident in treated and control cells at 24 h and 48 h. These figures are representative of four independent experiments.
  • FIG. 19 is an image of the cell morphology of untreated MIN-6 cells following a) 1 day, b) 6 days, and c) 12 days of incubation without cytokines.
  • the cell morphology of MIN-6 cells following cytokine treatment with IFN- ⁇ , TNF- ⁇ and IL-1 ⁇ can be seen in d) 1 day, e) 6 days and f) 12 days. (100 ⁇ magnification).
  • FIG. 20 is an image of the cell morphology of untreated Huh7ins cells following a) 1 day, b) 6 days, and c) 12 days.
  • the cell morphology of Huh7ins cells incubated with the cytokines IFN- ⁇ , TNF- ⁇ and IL-1 ⁇ following the same time points d) 1 day, e) 6 days, and f) 12 days. (100 ⁇ magnification). Morphology of the Melligen cells was the same as the Huh7ins cells.
  • FIG. 21 is an image showing RT-PCR performed using primers for IFNR1, IFNR2, IL1R1, IL1R2, TNFR1 and TNFR2 cytokine receptors.
  • Lanes contain cDNA for: 1-Pancreatic Islet Cells (positive control) 2-Huh7 Cells 3-Huh7ins Cells 4-TAO Cells 5-Negative control. Results show bands for IFNR1, IFNR2, IL1R1, IL1R2 and TNFR1 but not TNFR2 cytokine receptor.
  • the TNFR2 Cytokine Receptor was not detected at the Molecular Level in the liver cell lines.
  • FIG. 22 is an image of RT-PCR performed using primers for I ⁇ B ⁇ , I ⁇ B ⁇ and I ⁇ B ⁇ . Lanes contain cDNA for: control and treated pancreatic islet cells (positive control), control and treated Huh7 Cells, control and treated Huh7ins Cells, and control and treated Melligen Cells. Results show bands for I ⁇ B ⁇ , I ⁇ B ⁇ , I ⁇ B ⁇ and iNOS but not MCP-1.
  • FIG. 23 is a graphical representation of real time PCR results showing that inhibitors of NF ⁇ B are down-regulated in the Melligen cells. Down-stream effector molecule, Fas, is also down-regulated. These trends in gene expression were also seen in Huh7 and Huh7ins cells.
  • FIG. 28 is a graphical representation showing a 10-day cytokine treatment did not affect (a) Huh7ins and (b) Melligen cell's responsiveness to glucose in the millimolar range. Melligen cells secrete insulin in response to 4.25 mM glucose (the physiological range) and Huh7ins cells to 2.5 mM glucose.
  • FIG. 29 is an image of ⁇ -cell transcription factors, pancreatic hormones, proinsulin convertase, and factors of the glucose sensing apparatus expressed in transfected liver cell lines.
  • RT-PCR analysis for ⁇ -cell transcription factors [PDX-1, NEUROG3, NEUROD1, NKX2-2, NKX6-1, Pax6]; pancreatic hormones [glucagon, somatostatin (SST) and pancreatic polypeptide (PP)]; GLUT2 and Glucokinase (GK) [islet and liver form]; proinsulin convertases [PC1/3 and PC2] in Huh7 cells (lane 1), Huh7ins cells (lane 2), Melligen cells (lane 3), human islet (lane 4, the positive control), and water (lane 5, the negative control).
  • FIG. 30 is an image of real-time PCR analysis of (a) Human islet glucokinase in lane 2 human islet cells, lane 3 Melligen cells, lane 4 Huh7ins with vector only, lane 5 Huh7ins, lane 6 Huh7 cells, lane 7 dH 2 O and (b) human liver glucokinase in lane 2: human islet cells, lane 3: Melligen cells, lane 4: Huh7ins with vector only, lane 5: dH 2 O. Lane 1: DNA marker in both cases.
  • FIG. 31 is a graphical representation of real-time PCR expression of liver GK in a) Melligen cells and Huh7ins with empty vector, and b) Huh7ins cells and Huh7ins cells with empty vector.
  • FIG. 34 is a graphical representation of Real-time PCR expression of the glucose transporter GLUT2: in a) Melligen cells and Huh7 cells, and b) Melligen cells and Huh7ins cells.
  • FIG. 35 is an image of qualitative western blot analysis for the expression of PDX-1 in: Huh7 (lane 1), Huh7ins (lane 2), Melligen (lane 3), and the positive control, human islet cells (lane 4).
  • the present invention is predicated, in part, on the determination that glucose responsive insulin secretion by genetically engineered cells which are not pancreatic ⁇ cells can be more appropriately designed to mimic normal physiological events by engineering the cell to express pancreatic islet glucokinase, as opposed to other forms of glucokinase. It has been determined that in the absence of the production of this enzyme, hypersensitive responsiveness to extracellular glucose levels can occur, leading to the induction of a hypoglycaemic state in individuals treated with such cells, due to the fact that insulin production is upregulated even where systemic levels of glucose are below the lower physiological threshold required to stimulate insulin production by normal pancreatic ⁇ cells. This determination, and the generation of cells based thereon, has now facilitated the improvement of therapeutic and prophylactic treatment regimes directed to treating diabetes and/or the symptoms associated with diabetes.
  • one aspect of the present invention is directed to a genetically modified mammalian cell, which cell is capable of secreting insulin, said genetic modification comprising the transfection of said cell with a nucleic acid molecule encoding pancreatic islet glucokinase.
  • Reference to a “cell capable of secreting insulin” should be understood as a reference to a cell which either does or has the capacity to produce insulin.
  • Reference to “produce” is a reference to the expression (being transcription and translation) of an insulin encoding nucleic acid molecule and secretion of the insulin expressed thereby. It should be understood, however, that although the cell may be any type of eukaryotic cell, the cell is not a functionally normal pancreatic ⁇ cell. The cell may be one which, even in the absence of the genetic modification of the present invention can nevertheless produce insulin either constitutively or in response to a stimulus or it is one which although not producing insulin prior to incorporation of the genetic modification of the present invention, will be able to do so thereafter.
  • the capacity of a normal pancreatic ⁇ cell to produce insulin in a physiologically relevant glucose responsive manner is due both to its capacity to express the insulin gene and to the functionality of a “glucose sensing system” which regulates insulin release in response to small external nutrient changes.
  • the glucose sensing system essentially comprises a high capacity glucose transporter, such as GLUT 2, and a glucose phosphorylation enzyme, such as glucokinase.
  • GLUT 2 high capacity glucose transporter
  • glucokinase a glucose phosphorylation enzyme
  • insulin 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 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 therefore constitutively express insulin but effects its secretion only upon receipt of an appropriate stimulus.
  • the cell which is the subject of the genetic modification of the present invention includes, but is not limited to:
  • the subject cells may have been freshly isolated from an individual (such as an individual who may be the subject of treatment) or they may have been sourced from a non-fresh source, such as from a culture (for example, where cell numbers were expanded) or a frozen stock of cells (for example, an established stem cell line such as the Huh7ins cell line), which had been isolated at some earlier time point either from an individual or from another source.
  • a non-fresh source such as from a culture (for example, where cell numbers were expanded) or a frozen stock of cells (for example, an established stem cell line such as the Huh7ins cell line), which had been isolated at some earlier time point either from an individual or from another source.
  • the subject cells prior to undergoing the genetic manipulation of the present invention, may have undergone some other form of treatment or manipulation, such as but not limited to enrichment or purification, modification of cell cycle status or the formation of a cell line.
  • the subject cell may be a primary cell or a secondary cell.
  • a primary cell is one which has been isolated from an
  • the subject cell is a hepatocyte.
  • hepatocytes are known to play a crucial role in intermediary metabolism, synthesis and storage of proteins in the liver.
  • liver cells inherently express the high capacity glucose transporter GLUT 2, this being one of the key elements of the glucose sensing system which regulates insulin release from pancreatic ⁇ cells in response to small external nutrient changes.
  • GLUT 2 high capacity glucose transporter
  • hepatocytes are used, other than introducing the genetic modification of the present invention (being the incorporation of a gene expressing pancreatic islet glucokinase) they need generally only otherwise be manipulated to introduce the capacity to express the insulin gene.
  • the present invention therefore more preferably provides a genetically modified mammalian hepatocyte, which hepatocyte is capable of secreting insulin, said genetic modification comprising the transfection of said hepatocyte with a nucleic acid molecule encoding pancreatic islet glucokinase.
  • glucokinase per se, although enabling cellular glucose responsiveness, may not necessarily result in glucose responsiveness which mimics the physiological events associated with normal pancreatic functioning.
  • the glucokinase enzyme which is endogenously expressed by hepatocytes although ideally acceptable in the context of normal hepatic functioning, is not ideal in the context of pancreatic functioning since it is effectively “hyperresponsive” in that such cells, if transfected with an insulin encoding gene, will be stimulated to express and secrete insulin at extracellular glucose concentrations which are below those at which normal pancreatic islet ⁇ cells would produce insulin.
  • pancreatic islet ⁇ cells will produce insulin in response to extracellular concentrations of glucose of the order of 4-5 mM while some hepatocytes, if genetically engineered to produce insulin, are responsive to glucose levels well below 4 mM, thereby exhibiting the potential to induce hypoglycaemia if not appropriately managed.
  • a genetically modified cell which is responsive to glucose in a “physiologically relevant manner” should be understood as a reference to a cell which produces insulin in response to substantially the same glucose concentration range to which the pancreatic islet ⁇ cells of the mammal in issue would normally respond. That is, the subject glucose responsiveness is not such that a physiologically unacceptable state of hypoglycaemia would be induced to occur. It would be appreciated, therefore, that this concentration range may vary from one mammal to another. In the context of the human, the relevant glucose concentration range is about 4-5 mM of glucose.
  • the present invention is directed to a genetically modified mammalian hepatocyte, which hepatocyte is capable of secreting insulin, said genetic modification comprising the transfection of said hepatocyte with a nucleic acid molecule encoding pancreatic islet glucokinase and wherein said cell is responsive to glucose in a physiologically relevant manner.
  • pancreatic islet glucokinase should be understood as a reference to a form of glucokinase which is expressed by pancreatic islet cells.
  • proteins which are expressed by the cells of the present invention such as “insulin”, “glucose transporter”, “GLUT 2”, “glucokinase” and “pancreatic islet glucokinase” should be understood as a reference to all forms of these proteins and to functional derivatives and homologues thereof. This includes, for example, any isoforms which arise from alternative splicing of the mRNA encoding these molecules or functional mutants or polymorphic variants of these proteins.
  • 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.
  • the pancreatic islet glucokinase is the human form of this molecule and, even more preferably, the form encoded by SEQ ID NO:2.
  • mammal should be understood to include reference to a mammal such as but not limited to human, primate, livestock (animal (eg. sheep, cow, horse, donkey, pig), companion animal (eg. dog, cat), laboratory test animal (eg. mouse, rabbit, rat, guinea pig, hamster), captive wild animal (eg. fox, deer).
  • animal eg. sheep, cow, horse, donkey, pig
  • companion animal eg. dog, cat
  • laboratory test animal eg. mouse, rabbit, rat, guinea pig, hamster
  • captive wild animal eg. fox, deer
  • the present invention more preferably provides a genetically modified human hepatocyte, which hepatocyte is capable of secreting insulin, said genetic modification comprising the transfection of said hepatocyte with a nucleic acid molecule encoding pancreatic islet glucokinase.
  • pancreatic islet glucokinase is human pancreatic islet glucokinase and, even more preferably, the form encoded by SEQ ID NO:2.
  • “Derivatives” of the molecules herein described include functional fragments, parts, portions or variants. Derivatives may be derived from insertion, deletion or substitution 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 in the protein although random insertion is also possible with suitable screening of the functionality 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 a sequence has been removed and a different residue inserted in its place. Additions to amino acid sequences include fusions with other peptides, polypeptides or proteins, as detailed above.
  • Derivatives also include fragments having particular regions of the entire protein fused to peptides, polypeptides or other proteinaceous or non-proteinaceous molecules. For example, insulin or derivative thereof may be fused to another molecule in order to agonise its activity. In another example, it may be desirable to facilitate co-expression of both pro-insulin and a cleavage enzyme.
  • Derivatives of nucleic acid sequences which may be utilised in accordance with the method of the present invention may similarly be derived from single or multiple nucleotide substitutions, deletions and/or additions including fusion with other nucleic acid molecules. Derivatives of nucleic acid sequences also include degenerate variants.
  • a “variant” should be understood to mean a molecule which exhibits at least some of the functional activity of the form of molecule of which it is a variant.
  • a variation may take any form and may be naturally or non-naturally occurring.
  • homologue is meant that the molecule is derived from a species other than that which is being treated in accordance with the method of treatment aspects of the present invention. This may occur, for example, where it is determined that a species other than that which is being treated produces a form of the subject molecule which exhibits suitable functionality.
  • insulin for example, one might utilise the insulin gene of a non-human mammal, even where the cells of the invention are proposed to be utilised in a human context.
  • the cells of the present invention are genetically modified. This genetic modification may occur in one or both of two contexts.
  • the cell may have been genetically modified in order to render it “capable of secreting insulin”.
  • the subject cell is also transfected with a nucleic acid molecule encoding pancreatic islet glucokinase. Accordingly, by “genetically modified” is meant that the subject cell has undergone some form of molecular manipulation relative to that which is observed in the context of the majority of a corresponding unmodified population. Such modifications include but are not limited to:
  • nucleic acid should be understood as a reference to both deoxyribonucleic acid and ribonucleic acid thereof.
  • the subject nucleic acid molecule may be any suitable form of nucleic acid molecule including, for example, a genomic, cDNA or ribonucleic acid molecule.
  • expression refers to the transcription and translation of DNA or the translation of RNA resulting in the synthesis of a peptide, polypeptide or protein.
  • a DNA construct corresponds to the construct which one may seek to transfect into a cell for subsequent expression while an example of an RNA construct is the RNA molecule transcribed from a DNA construct, which RNA construct merely requires translation to generate the protein of interest.
  • expression product is a reference to the product produced from the transcription and translation of a nucleic acid molecule.
  • protein should be understood to encompass peptides, polypeptides and proteins. It should also be understood that these terms are used interchangeably herein.
  • the protein may be glycosylated or unglycosylated and/or may contain a range of other molecules fused, linked, bound or otherwise associated to the protein such as lipids, carbohydrates or other peptides, polypeptides or proteins (such as would occur where the protein of interest is produced as a fusion protein with another molecule, for example GST or EGFP).
  • Reference hereinafter to a “protein” includes a protein comprising a sequence of amino acids as well as a protein associated with other molecules such as amino acids, lipids, carbohydrates or other peptides, polypeptides or proteins.
  • genetic material is generally conveniently introduced to cells via the use of an expression construct.
  • a cell type which either naturally or as a result of earlier random or directed genetic manipulation is characterised by one or more of the genetic modifications of interest (for example, one may seek to introduce the pancreatic islet glucokinase modification into a cell which has previously been modified in terms of rendering it “capable of secreting insulin”.
  • the modification of a cell line such as Huh7ins, as discussed in more detail hereinafter, which is a hepatic cell line transfected with insulin encoding DNA is one such example).
  • said genetic modification is the transfection of a cell capable of secreting insulin with an expression construct comprising one or more DNA regions comprising a promoter operably linked to a sequence encoding a pancreatic islet glucokinase and, optionally, a second DNA region encoding a selectable marker and, optionally, a third DNA region encoding a suicide protein.
  • the construct may also comprise DNA encoding insulin and/or a glucose transporter where the subject cell has not previously been rendered “capable of secreting insulin”.
  • the subject promoter may be constitutive or inducible. Where the subject construct expresses more than one protein of interest, these may be under the control of separate promoters or they may be under the control of a single promoter, such as occurs in the context of a bicistronic vector which makes use of an IBES sequence to facilitate the translation of more than one protein product, in an unfused form, from a single RNA transcript.
  • the subject construct may additionally be designed to facilitate use of the Cre recombinase mediated splicing inducible gene expression system.
  • nucleic acid “expression construct” should be understood as a reference to a nucleic acid molecule which is transmissible to a cell and designed to undergo transcription. The RNA molecule is then transcribed therefrom.
  • expression constructs are also referred to by a number of alternative terms, which terms are widely utilised interchangeably, including “expression cassette” and “vector”.
  • the expression construct of the present invention may be generated by any suitable method including recombinant or synthetic techniques.
  • the subject construct may be constructed from first principles, as would occur where an entirely synthetic approach is utilised, or it may be constructed by appropriately modifying an existing vector. Where one adopts the latter approach, the range of vectors which could be utilised as a starting point are extensive and include, but are not limited to:
  • an appropriate vector for modification to the extent that one chooses to do this rather than synthetically generate a construct, will depend on a number of factors including the ultimate use to which the genetically modified cell will be put. For example, where the cell is to be administered in vivo into a human, it may be less desirable to utilise certain types of vectors, such as viral vectors. Further, it is necessary to consider the amount of DNA which is sought to be introduced to the construct. It is generally understood that certain vectors are more readily transfected into certain cell types. For example, the range of cell types which can act as a host for a given plasmid may vary from one plasmid type to another.
  • the size of the inserted DNA can vary depending on factors such as the size of the DNA sequence encoding the protein of interest, the number of proteins which are sought to be expressed, the number of selection markers which are utilised and the incorporation of features such as linearisation polylinker regions and the like.
  • the expression construct which is used in the present invention may be of any form including circular or linear.
  • a “circular” nucleotide sequence should be understood as a reference to the circular nucleotide sequence portion of any nucleotide molecule.
  • the nucleotide sequence may be completely circular, such as a plasmid, or it may be partly circular, such as the circular portion of a nucleotide molecule generated during rolling circle replication (this may be relevant, for example, where a construct is being initially replicated, prior to its introduction to a cell population, by this type of method rather than via a cellular based cloning system).
  • the “circular” nucleotide sequence corresponds to the circular portion of this molecule.
  • a “linear” nucleotide sequence should be understood as a reference to any nucleotide sequence which is in essentially linear form.
  • the linear sequence may be a linear nucleotide molecule or it may be a linear portion of a nucleotide molecule which also comprises a non-linear portion such as a circular portion.
  • An example of a linear nucleotide sequence includes, but is not limited to, a plasmid derived construct which has been linearised in order to facilitate its integration into the chromosomes of a host cell or a construct which has been synthetically generated in linear form. To this end, it should also be understood that the configuration of the construct of the present invention may or may not remain constant.
  • a circular plasmid-derived construct may be transfected into a cell where it remains a stable circular episome which undergoes replication and transcription in this form.
  • the subject construct may be one which is transfected into a cell in circular form but undergoes intracellular linearisation prior to chromosomal integration. This is not necessarily an ideal situation since such linearisation may occur in a random fashion and potentially cleave the construct in a crucial region thereby rendering it ineffective.
  • the nucleic acid molecules which are utilised in the method of the present invention are derivable from any human or non-human source.
  • Non-human sources contemplated by the present invention include primates, livestock animals (eg. sheep, pigs, cows, goats, horses, donkeys), laboratory test animal (eg. mice, hamsters, rabbits, rats, guinea pigs), domestic companion animal (eg. dogs, cats), birds (eg. chicken, geese, ducks and other poultry birds, game birds, emus, ostriches) captive wild or tamed animals (eg. foxes, kangaroos, dingoes), reptiles, fish, insects, prokaryotic organisms or synthetic nucleic acids.
  • livestock animals eg. sheep, pigs, cows, goats, horses, donkeys
  • laboratory test animal eg. mice, hamsters, rabbits, rats, guinea pigs
  • domestic companion animal eg.
  • the constructs of the present invention may comprise nucleic acid material from more than one source.
  • the construct may originate from a bacterial plasmid, in modifying that plasmid to introduce the features defined herein nucleic acid material from non-bacterial sources may be introduced.
  • sources may include, for example, viral DNA (e.g. IRES DNA), mammalian DNA (e.g. the DNA encoding the pancreatic islet glucokinase) or synthetic DNA (e.g. to introduce specific restriction endonuclease sites).
  • the cell type in which it is proposed to express the subject construct may be different again in that it does not correspond to the same organism as all or part of the nucleic acid material of the construct.
  • a construct consisting of essentially bacterial and viral derived DNA may nevertheless be expressed in the mammalian stem cells contemplated herein.
  • the present invention is exemplified in the context of a pancreatic islet glucokinase expressing bicistronic vector which is transfected into cells which are already “capable of secreting insulin” in the context of the earlier definition. Specifically, cDNA encoding pancreatic islet glucokinase is transfected into the multicloning site of pIRESpuro3.
  • the pIRESpuro3 bicistronic vector exemplified herein contains the internal ribosome entry site of the encephalomyocarditis virus, which permits the translation of two open reading frames from one messenger RNA (Jackson et al., 1990 , Trends Biochem. Sci. 15:477-483; Jang et al., 1988 , J. Virol. 62:2636-2643; Rees et al., 1996 , BioTechniques 20:102-104).
  • pancreatic islet glucokinase After selection with puromycin, most surviving colonies are likely to stably express the pancreatic islet glucokinase, thus decreasing the need to screen large numbers of colonies to find functional clones (this being a particular advantage of bicistronic vectors).
  • the selective pressure for antibiotic resistance is increased due to the positioning of the puromycin resistance gene downstream to a less optimal position for translation as directed by the IRES sequence (Rees et al., 1996, supra).
  • the selective pressure on the entire expression cassette is increased, resulting in selection for cells that express the entire transcript, including the pancreatic islet glucokinase, at high levels.
  • the expression cassette of pIRESpuro3 contains the human cytomegalovirus major immediate early promoter/enhancer followed by a multiple cloning site that precedes stop codons in all three reading frames, a synthetic intron known to enhance the stability of the mRNA (Huang and Gorman, 1990 , Nucleic Acids Res. 18:937-947), the ECMV IRES followed by the gene encoding puromycin resistance (puromycin-N-acetyl-transferase; de la Luna, et al., 1988 , Gene 62:121-128), and the polyadenylation signal from SV40.
  • Ribosomes can enter the bicistronic mRNA at the 5′ end to translate the gene of interest and at the ECMV IRES to translate the antibiotic resistance marker. It should be understood that the expression vector exemplified herein is provided solely by way of example and is in no way intended to limit the range and design of vectors which could be used to achieve the object of the present invention.
  • the present invention therefore more preferably provides a genetically modified human hepatocyte, which hepatocyte is capable of secreting insulin, said genetic modification comprising the transfection of said cell with a vector, which vector comprises a nucleic acid molecule encoding pancreatic islet glucokinase.
  • said vector is a bicistronic vector and said pancreatic islet glucokinase is the form encoded by SEQ ID NO:2.
  • said bicistronic vector is pIRESpuro3 and most preferably defined by SEQ ID NO:3.
  • said genetically modified cell is responsive to glucose in a physiologically relevant manner and, most preferably, to extracellular glucose levels in the range of 3-8 mM, preferably 3.5-7 mM, more preferably 4-6 mM and most preferably 4-5 mM.
  • the generation of the cells of the present invention may require the application of a screening and selection step to identify and isolate cells which have successfully incorporated the genetic modification of interest. Identification methods would be well known to the person of skill in the art and include, but are not limited to:
  • the modified cell of the present invention is preferably a hepatocyte, this being a cell type which arguably requires less modification to render it capable of secreting insulin than other cell types due to the fact that it inherently expresses the high capacity glucose transporter, GLUT 2.
  • hepatocytes also inherently express glucokinase, in the context of insulin responsiveness, this molecule ideally functions in a hypersensitive manner in that it results in the expression of a transfected insulin gene at extracellular glucose levels of as low as 2.5 mM, this being below the physiological range that pancreatic islet cells generally produce insulin in the human. Accordingly, the developments of the present invention are a significant step forward in that they overcome this problem.
  • the subject hepatocyte may be freshly harvested or it may be derived from a cell line. Still further, it may be one which is required to be made capable of secreting insulin via transfection of the DNA encoding insulin (this occurring either separately to or concomitantly with the pancreatic islet glucokinase genetic modification herein described) or it may already have been rendered so.
  • said hepatocyte is a Huh7ins cell, this being a cell line which has been modified to express a nucleic acid molecule encoding insulin.
  • Huh7ins is a genetically engineered liver cell line which stores and secretes insulin in response to glucose.
  • these cells are hyperresponsive in the physiological sense in that they can commence secreting insulin at sub-physiological levels of glucose (eg. 2.5 mM), as compared to between 4-5 mM of glucose for normal human pancreatic ⁇ cell functioning, thereby potentially leading to the onset of hypoglycaemia. It is thought that these cells exhibit an imbalance in the glucokinase:hexokinase ratio in favour of hexokinases which results in enhanced glycolytic flux at low glucose levels and consequently increased sensitivity of the glucose-stimulated insulin secretion response.
  • glucose eg. 2.5 mM
  • the present invention therefore most preferably provides a genetically modified Huh7ins cell, said genetic modification comprising the transfection of said Huh7ins cell with a vector, which vector comprises a nucleic acid molecule encoding pancreatic islet glucokinase.
  • said vector is a bicistronic vector and said pancreatic islet glucokinase is the form encoded by SEQ ID NO:2.
  • said bicistronic vector is pIRESpuro3 and most preferably is SEQ ID NO:3.
  • said genetically modified Huh7ins cell is a Melligen cell.
  • the development of the method of the present invention has now facilitated the development of means for therapeutically or prophylactically treating disease conditions characterised by aberrant, preferably insufficient or inadequate, production of functional insulin.
  • This problem may be due to any one of a number of causes including, but not limited to, pancreatic ⁇ cell destruction, the aberrant functioning of the ⁇ cell glucose sensing system, defects in insulin gene expression or defects in the functionality of the insulin expression product itself.
  • reference to a disease condition “characterised by aberrant production of functional insulin” should be understood as a reference to any condition, a symptom or cause of which is insufficient or inadequate levels of functionally effective insulin. Accordingly, and as detailed above, this may be due to defects in the ⁇ cell itself, the glucose sensing system or the expression levels or functionality of the insulin expression product.
  • another aspect of the present invention is directed to a method of therapeutically and/or prophylactically treating a condition in a mammal, which condition is characterised by the aberrant production of functional insulin, said method comprising introducing into said mammal an effective number of the genetically modified cells hereinbefore defined.
  • said condition is diabetes.
  • the present invention therefore more particularly provides a method of therapeutically and/or prophylactically treating diabetes in a mammal, said method comprising introducing into said mammal an effective number of the genetically modified cells hereinbefore defined.
  • 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
  • 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 the onset of a particular condition. “Treatment” may also reduce the severity of an existing condition.
  • the present invention should therefore be understood to encompass preventing, reducing or otherwise ameliorating diabetes in a mammal. This 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.
  • 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.
  • the subject cells are preferably autologous cells which are isolated and genetically modified ex vivo and transplanted back into the individual from which they were originally harvested.
  • the present invention nevertheless extends to the use of cells derived from any other suitable source where the subject cells exhibit the same major histocompatability profile as the individual who is the subject of treatment. Accordingly, such cells are effectively autologous in that they would not result in the histocompatability problems which are normally associated with the transplanting of cells exhibiting a foreign MHC profile.
  • Such cells should be understood as falling within the definition of “autologous”.
  • the subject cells are isolated from a genetically identical twin, or from an embryo generated using gametes derived from the subject individual or cloned from the subject individual (in this case the cells are likely to correspond to stein cells which have undergone directed differentiation to an appropriate somatic cell type).
  • the cells may also have been engineered to exhibit the desired major histocompatability profile. The use of such cells overcomes the difficulties which are inherently encountered in the context of tissue and organ transplants.
  • allogeneic cells are those which are isolated from the same species as the subject being treated but which exhibit a different MHC profile. Although the use of such cells in the context of therapeutics would likely necessitate the use of immunosuppression treatment, this problem can nevertheless be minimised by use of cells which exhibit an MHC profile exhibiting similarity to that of the subject being treated, such as a cell population which has been isolated/generated from a relative such as a sibling, parent or child. Also contemplated herein is the use of established cell lines such as Huh7ins or the Melligen cells which have been derived therefrom. The present invention should also be understood to extend to xenogeneic transplantation. That is, the cells which are genetically modified in accordance with the method of the invention and introduced into a patient are isolated from a species other than the species of the subject being treated.
  • an “effective number” means that number of cells necessary to at least partly attain the desired effect, or to delay the onset of, inhibit the progression of, or halt altogether the onset or progression of the particular condition being treated. Such amounts will depend, of course, on the particular condition being treated, the severity of the condition and individual patient parameters including age, physical conditions, size, weight, physiological status, concurrent treatment, medical history and parameters related to the disorder in issue.
  • One skilled in the art would be able to determine the number of cells of the present invention that would constitute an effective dose, and the optimal mode of administration thereof without undue experimentation, this latter issue being further discussed hereinafter. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is preferred generally that a maximal cell number be used, that is, the highest safe number according to sound medical judgement. It will be understood by those of ordinary skill in the art, however, that a lower cell number may be administered for medical reasons, psychological reasons or for any other reasons.
  • the method of the present invention is predicated on the introduction of genetically modified cells to an individual suffering a condition as herein defined, it may not necessarily be the case that every cell of the population introduced to the individual will have acquired or will maintain the subject modification. For example, where a transfected and expanded cell population is administered in total (i.e. the successfully modified cells are not enriched for), there may exist a proportion of cells which have not acquired or retained the genetic modification. The present invention is therefore achieved provided the relevant portion of the cells thereby introduced constitute the “effective number” as defined above.
  • the population of cells which have undergone differentiation will be subjected to the identification of successfully modified cells, their isolation (for example by EGFP based FACS sorting or GST selection) and testing for a functional genetic modification and introduction to the subject individual.
  • This provides a means for selecting a specific subpopulation of cells for administration, such as cells expressing appropriate levels of the insulin and glucose sensing molecules in issue.
  • the subject cells require introduction into the subject individual.
  • the cells may be introduced 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.
  • the cells may also be encapsulated prior to transplantation. Encapsulation is a technique which is useful for preventing the dissemination of cells which may continue to proliferate (i.e. exhibit characteristics of immortality), although this is not expected to be a significant problem where a pure population of terminally differentiated cells are administered (but may be an issue if the cell population is derived from an immortalised cell line) or for minimising tissue incompatibility rejection issues.
  • the cells 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 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.
  • the cells which are administered to the patient can be administered as single or multiple doses by any suitable route.
  • a single administration is utilised.
  • Administration via injection can be directed to various regions of a tissue or organ, depending on the type of treatment required.
  • 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.
  • Other examples of circumstances in which such co-administration may be required include, but are not limited to:
  • the method of the present invention can either be performed in isolation to treat the condition in issue or it can be performed together with one or more additional techniques designed to facilitate or augment the subject treatment.
  • additional techniques may take the form of the co-administration of other proteinaceous or non-proteinaceous molecules, as detailed hereinbefore.
  • 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 treatment 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” modulatable 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 contemplates a method of modulating insulin levels in a mammal said method comprising introducing into said subject an effective number of the genetically modified cells hereinbefore defined.
  • Yet another aspect of the present invention contemplates a method of modulating glucose levels in a mammal said method comprising introducing into said subject an effective number of the genetically modified cells hereinbefore defined.
  • Still another aspect of the present invention is directed to the use of genetically modified cells hereinbefore defined in the manufacture of a medicament for the treatment of a condition in a mammal, which condition is characterised by the aberrant production of functional insulin.
  • said condition is diabetes.
  • a method of assessing the effect of a treatment or culture regime on the phenotypic state of the genetically modified cells as hereinbefore defined comprising subjecting said cells to said treatment regime and screening for an altered phenotypic state.
  • altered is meant that one or more of the phenotypic or functional parameters which are the subject of analysis are changed relative to untreated cells. This may be a desirable outcome where the treatment regime in issue is designed to improve or assist cellular functioning. However, where the treatment regime is associated with a detrimental outcome, this may be indicative of toxicity and therefore the unsuitability for use of the treatment regime. It is now well known that the differences which are observed in terms of the responsiveness of an individual to a particular drug are often linked to the unique genetic makeup of that individual. Accordingly, the method of the present invention provides a valuable means of testing either an existing or a new treatment regime which may be used concurrently with the administration of the cells of the invention.
  • This provides a unique means for evaluating the likely effectiveness of a drug, such as a drug which is proposed to be co-administered with the cells of the invention, prior to administering the drug and the cells in vivo.
  • a drug such as a drug which is proposed to be co-administered with the cells of the invention, prior to administering the drug and the cells in vivo.
  • this aspect of the present invention provides a means of optimising a treatment regime.
  • the method of the present invention can be used to screen and/or test drugs, other treatment regimes or culture conditions.
  • this aspect of the present invention can be utilized to monitor for changes to the gene expression profiles of the subject cells and tissues.
  • the method according to this aspect of the present invention can be used to determine, for example, gene expression pattern changes in response to a proposed concomitant treatment regime, such as a treatment regime which is required to be maintained in order to treat an unrelated condition from which the patient also suffers.
  • the treatment to which the cells or tissues of the present invention are subjected is the exposure to a compound.
  • the compound is a drug or a physiological ion.
  • the compound can be a growth factor or differentiation factor.
  • the present invention is further defined by the following non-limiting Examples.
  • Human islet glucokinase cDNA contained in the pBluescript commercial vector was a gift from Dr M. Alan Permutt, the University of Washington (St Louis, USA).
  • the human islet glucokinase cDNA was cut out of the pBrescript SK+ by restriction enzyme E COR I.
  • the 2733 base pair fragment containing the human islet glucokinase cDNA was inserted into the pIERSpuro3 expression vector (Clontech, USA) at the E COR I site in the multi cloning site (971-972 bp) ( FIG. 1 ).
  • Huh7ins cells were cultured as monolayers in Dulbecco's Modification of Eagles's Medium containing 10% fetal calf serum (FCS) in 5% CO 2 in air plus 0.55 mg/ml G418 as described (Tuch et al., 2003 , Gene Therapy 10:490-503).
  • FCS fetal calf serum
  • Huh7ins cells were transfected with the pIRESpuro3-glucokinase construct or the pIRESpuro3 vector alone using Effectenen transfection reagent (Qiagen Germmay). Twenty four hours after transfection the eukaryocidal antibiotic puromycin (1.1 ⁇ g/ml) was added to the cultures. Medium plus drugs were changed every 2-3 days. After 14 days of selection, colonies were picked up and expanded into mass cultures. The cells containing the pIRESpuro3-glucokinase construct will hereafter be referred to as Melligen cells.
  • the primers designed for human islet glucokinase cDNA were as follows:
  • Amplification was undertaken for 35 cycles at denaturing temperature 95° C. for 45 seconds, annealing temperature 56° C. for 30 seconds and extension temperature 72° C. for 35 seconds.
  • the PCR product was separated on 2% agarose gel with TBE buffer ( FIG. 3 ).
  • Protein samples from the three different cell types (15 ⁇ g/30 ⁇ l) were run on 10% polyacrylamide gels for Western blot analysis at 100 v and then transferred to a nitrocellulose membrane (Millipore Corporation, USA).
  • the nitrocellulose membrane was blocked in phosphate buffered saline (PBS) with 5% skim milk overnight at 4° C. to avoid any non-specific binding. After washing three times (10 min) with PBS containing 0.05% Tween 20 .
  • PBS phosphate buffered saline
  • the nitrocellulose membrane was incubated with primary antibody—rabbit anti-human glucokinase antibody (1/1000 dilution) (Santa Cruz Biot USA) for 2 hours at room temperature, then washed again three times with PBS (0.05% Tween n ), the nitrocellulose membrane was incubated with second antibody—a polyclonal (donkey) anti-rabbit horseradish peroxidase IgG conjugate (1/800 dilution) (Sigma) for 1 hour at room temperature. After washing three times with PBS (0.05% Tween 20 ), glucokinase protein expression in the nitrocellulose membrane was detected using 3,3′-Diaminobenzidine (peroxidase substrate) (Sigma).
  • the primary human glucokinase was raised in rabbits against a recombinant protein corresponding to amino acids 318-405 mapping near the carboxy terminus of glucokinase of human origin, conjugated to a monoclonal anti-rabbit IgG antibody and detects a protein of 52 kD ( FIG. 4 ).
  • Glucose phosphorylation was measured in cell homogenates by following the conversion of [U- 14 C] glucose to [U- 14 C] glucose-6-phosphate as described (Kuwajima et al., 1996 , J. Biol. Chem. 261: 8849-53). Glucokinase and hexokinase activities were discriminated by performing the assay in the presence or absence of 10 mmol/L glucose-6-phosphate, an inhibitor of low K m hexokinase activity (Wilson, 1984, Regulation of carbohydrate metabolism, p. 45-85).
  • Huh7, Huh7ins and Huh7ins empty vector cells contain 16 ⁇ 1.2, 24.2 ⁇ 2.3, 25.4 ⁇ 2.6 U/g protein of glucose phosphorylating activity, respectively when assayed at 20 mM glucose in the absence of glucose-6-phosphate, but this activity is reduced to 3.0 ⁇ 0.5, 4.8 ⁇ 0.4 and 4.7 ⁇ 0.5 U/g protein respectively when the assay is conducted in the presence of 10 mmol/1 glucose-6-phosphate ( FIG. 5 ).
  • Huh7ins with the vector only and Melligen cells were washed with PBS, trypsinised and removed from tissue culture flasks. The suspended cells were centrifuged at 1000 rpm for minutes and supernatant was aspirated. The cells were resuspended in the desired volume of fresh medium, a cell count was performed and the cells were distributed into 1.5 ml tubes at a density of 5 ⁇ 10 6 cells per tube. The tubes were centrifuged at 1800 rpm for 5 minutes and the supernatant was discarded. The cells were resuspended in 300 ⁇ l of 0.18N HCL in 70% ethanol for 48 hours at 4° C., to allow sufficient time for lysis of cells and release of stored insulin. For measurement of insulin content, samples were diluted in 1:10 before being placed in the radioimmunoassay (RIA).
  • RIA radioimmunoassay
  • a post-embedding immunogold procedure was used to confirm the localization of intracellular insulin.
  • Single cell suspensions of Melligen and MIN-6 cells (mouse insulinoma cell line, used as a positive control) were analyzed. Briefly, the sections were incubated in 50% sodium metaperiodate at 50° C. for 3.5 minutes followed by 0.01M sodium citrate buffer pH 6.0 at 95° C. for 10 minutes and a 15 minute cooling period. Non specific binding was blocked using 1% goat serum for 30 minutes at room temperature.
  • a goat anti guinea pig 10 nm (1:50) gold probe (Aurion, Wageningen, The Netherlands) incubated for 2 hours at room temperature was directed against a polyclonal guinea pig anti human insulin (1:20) 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 ( FIG. 8 b ).
  • FIG. 2 shows the sequence of the pIERSpuro3 vector (5157 base pairs sequence) with insert human islet glucokinase cDNA (2733 base pairs), which was cloned into the vector at the 972 bp site. Both junctions of the vector and insert have been sequenced (results not shown).
  • Melligen cells (10 7 cells) are transplanted subscapularly into non-autoimmune non-obese diabetic severe combined immunodeficiency (NOD.scid) mice. Groups of eight animals are required for these experiments. Diabetes (blood glucose levels exceeding 14 mM on two separate occasions and serum insulin concentration below 0.15 ng/mL) is induced by a single high dose of STZ (250 mg/kg body weight). After transplantation, body weight and blood glucose is monitored three times each week and then daily if blood glucose concentrations decrease below 4 mM.
  • NOD.scid non-autoimmune non-obese diabetic severe combined immunodeficiency mice. Groups of eight animals are required for these experiments. Diabetes (blood glucose levels exceeding 14 mM on two separate occasions and serum insulin concentration below 0.15 ng/mL) is induced by a single high dose of STZ (250 mg/kg body weight). After transplantation, body weight and blood glucose is monitored three times each week and then daily if blood glucose concentrations decrease below 4 mM.
  • Transplantation is considered successful if the non-fasting blood glucose concentration returns to normal (less than 8.4 mM) within 5 days after surgery. Animals are maintained on exogenous insulin immediately after transplantation if necessary. Transplants are considered unsuccessful if the blood glucose concentration increases to more than 20 mM on more than two occasions. After transplantation and when blood glucose levels are between 5 and 10 mM, glucose tolerance tests are performed and blood glucose and serum insulin levels assayed. If blood glucose levels fall to less than 2 mM for more than 24 h the animal is sacrificed and the transplant removed. If the normoglycaemic state is retained, then explants will be harvested at 2, 6 and 12 weeks after transplantation from a cohort to verify that hyperglycaemia subsequently returns.
  • pancreas is prepared for insulin immunohistochemistry. After resection, the graft is weighed and the proliferative characteristics of Melligen cells quantified by MTT assay over a period of 72 h. The insulin content of the graft is measured. Explants are also examined for gene expression (of glucokinase, GLUT2 glucose transporter, and insulin by PCR), histologically (for cellular integrity, insulin secretory granules, and vascularisation) and immunohistochemically (for insulin).
  • gene expression of glucokinase, GLUT2 glucose transporter, and insulin by PCR
  • histologically for cellular integrity, insulin secretory granules, and vascularisation
  • immunohistochemically for insulin
  • results indicate that that unlike pancreatic ⁇ cells, insulin-expressing liver cells may not be susceptible to autoimmune destruction. However, inflammatory reactions may be generated if antigenic products from encapsulated dying cells diffuse through the microcapsule. Therefore, an analysis is performed to determine if microcapsules prevent communication between Melligen cells and immune cells that might cause lymphocyte activation. This is achieved using a conventional splenocyte co-culture system.
  • IgG deposition is also assessed on the microcapsules and the Melligen cells contained in the microcapsules (after incubation of microcapsules with FITC-labelled anti-mouse IgG antibody) at 6 and 12 weeks after transplantation, to determine if antibodies are induced by either antigens shed from the cell surface, proteins secreted by live cells, or liberated after cell death that may diffuse through the capsule.
  • MIN-6 and Huh7 cells are of murine pancreatic and human liver origin, respectively, each cell line exhibited distinct growth kinetics. These cell growth characteristics were used to determine the time course for experiments in which cytokines were co-incubated with cell lines. Since MIN-6 cells reached the log growth phase at a later time point, this cell line was plated at higher seeding densities when run in parallel experiments with Huh7 and Huh7ins to ensure that the cells were in log phase when used in the cytokine toxicity experiments.
  • MTT assay was to be used to determine cell viability in both the presence and absence of cytokines, preliminary experiments were performed to determine cell viability of untreated Huh7ins cells.
  • Huh7ins cells were seeded at an initial density of 1 ⁇ 10 3 cells/well into a 96-well plate and cultured for 8 days.
  • MTT is metabolised by mitochondrial dehydrogenase within the viable mitochondria of a cell to produce a purple coloured crystal, formazan. When dissolved in DMSO the absorbance of the formazan solution can be read at 570 nm.
  • the MTT assay is a rapid and reproducible method for determining cellular response to cytotoxic agents.
  • the results obtained from this preliminary experiment showed that the seeding density chosen approached an absorbance value of 1.0 nm over the 8-day period.
  • the initial seeding density used was optimal since subsequent experiments to assess the cytotoxic effect of cytokines were to be conducted for between six and twelve days. These results also showed that the exponential growth of the cells was reflected by the mitochondrial activity of the cells. From the growth kinetic results ( FIG. 11 ) it was also determined that MIN-6 cells would be plated at twice the seeding density as the liver cell lines.
  • results from the MTT assay revealed that co-incubation of MIN-6 cells with the concentrations of single cytokines at the concentrations listed in Table 1 did not reduce the viability of MIN-6 cells as compared to untreated cells even after 14 days of co-incubation (data not shown). Consequently, the cytokines were used in combination and at higher cytokine concentrations.
  • the concentrations employed were similar to those used by Tabiin et al. (2001) in studies investigating the cytokine treatment of both the rodent insulinoma cell line, NIT-1, and the insulin secreting human hepatocyte cell line, HEPG2 ins/g.
  • the triple cytokine combination concentration were further titrated by adding the cytokines at twice IFN- ⁇ (768 ng/mL), TNF- ⁇ (20 ng/mL) and IL-1 ⁇ (4000 pg/mL) and half the triple cytokine concentrations IFN- ⁇ (192 ng/mL), TNF- ⁇ (5 ng/mL) and IL-1 ⁇ (1000 pg/mL) previously used.
  • MIN-6 and Huh7ins cells that were treated with cytokines had viabilities of 72 ⁇ 5%.
  • Untreated MIN-6 cells remained exponentially viable (100 ⁇ 1%) over the 10 days of the experiment.
  • Huh7, Huh7ins and Melligen cells When exposed to the cytokine combination for 10 days, Huh7, Huh7ins and Melligen cells showed no significant decrease in cell viability compared to the untreated control cells of each cell line ( FIG. 14 b - d ).
  • Annexin V is a calcium dependent phospholipid-binding protein with high affinity for phosphatidylserine.
  • Propidium iodide (PI) is a standard cytometric viability probe that is excluded by cells with intact membrane.
  • PI staining is performed simultaneously with the annexin V staining to differentiate apoptotic cells (single annexin V-positive) from necrotic cells (double annexin V-PI-positive), as necrotic cells also expose phosphatidylserine to annexin V because of the loss of membrane integrity (Vermes et al., 1995). Student's t-test was used and P values less than 0.05 were considered to be statistically significant.
  • annexin V binding was performed at 24 and 48 h with and without cytokine treatment. Staining Melligen cells at 24 h time-point showed close to 0% annexin V-single positive cells (apoptotic) and 0% annexin V PI-double positive cells (late apoptotic) and 20% staining PI-only (necrotic). At 48 h, the percentage of necrotic cells in the cytokine-treated group did not differ significantly from that recorded in the untreated samples (19%).
  • the cells were incubated with the cytokine cocktail for 48 h.
  • the viable cell number was not decreased in the insulin-secreting hepatoma cells by the cytokine cocktail treatment at 24 h and 48 h according to the MTT assay ( FIG. 14 ).
  • the DNA content in these cells were analysed using flow cytometry after propidium iodide staining.
  • FIGS. 19 a , 19 b and 19 c the untreated MIN-6 cells appeared to have intact cell membranes and remained attached to the plate in colonies growing as monolayers.
  • MIN-6 cells were confluent.
  • cytokine-treated MIN-6 cells started to degenerate with ruptured membranes causing cells to detach after 6 days ( FIG. 19 e ).
  • Complete degeneration with cell debris scattered between remaining cells was seen on day 12 ( FIG. 19 f ).
  • RT-PCR was performed using cDNA generated from RNA isolated from human primary islet (positive control), Huh7 (parent cell line), Huh7ins (Huh7 transfected with the insulin gene) and Melligen (further modified Huh7ins) cells with primers for IFNR1, IFNR2, IL1R1, IL1R2, TNFR1 and TNFR2.
  • Molecular expression of cytokine receptors IFNR1, IFNR2, IL1R1, IL1R2 and TNFR1 was confirmed in all cells ( FIG. 21 ). Therefore, the reduced susceptibility to cytokine-induced killing displayed by Melligen cells cannot be attributable to the absence of receptors for the cytokines.
  • RT-PCR was performed using primers for IFNR1, IFNR2, IL1R1, IL1R2, TNFR1 and TNFR2 cytokine receptors.
  • Lanes contain cDNA for: 1-Pancreatic Islet Cells (positive control) 2-Huh7 Cells 3-Huh7ins Cells 4-Melligen Cells 5-Negative control. Results show bands for IFNR1, IFNR2, IL1R1, IL1R2 and TNFR1 but not TNFR2 cytokine receptor.
  • NF- ⁇ B Downstream of TNF receptor (TNFR) 1 associated death domain protein (TRADD), receptor-interaction protein and TNF receptor associated factor (TRAF) 2 activate the NF- ⁇ B pathway.
  • TNFR TNF receptor 1 associated death domain protein
  • TRADF receptor-interaction protein
  • TNF receptor associated factor 2 activates the NF- ⁇ B pathway.
  • NF- ⁇ B has been reported to initiate the expression of various genes associated with anti-apoptosis, cell growth, and immune response in liver cells. No significant difference in the cytokine-induced activation of NF- ⁇ B was observed between the treated Huh7, Huh7ins and Melligen cells ( FIG. 23 ). These results indicate that the cytokine cocktail induces NF- ⁇ B activation irrespective of the presence of the insulin gene, and the anti-apoptotic mechanism in these liver cell lines seems to be independent of NF- ⁇ B activation.
  • cytokine-induced gene expression in Huh7, Huh7ins and Melligen cells was evaluated.
  • Gene expression of Fas and iNOS was switched off after the addition of cytokines to the liver cell lines ( FIG. 22 ).
  • the relative abundance of iNOS and Fas mRNAs was, respectively about 2-fold lower in control cells not treated with the cytokine cocktail expression of the housekeeping gene GAPDH was not affected by exposure to cytokines.
  • the iNOS enzymatic activity was estimated by measurements of medium nitrite (a stable product of nitric oxide (NO) oxidation) accumulation by the modified Griess reaction during a 48 h exposure to cytokines, IFN- ⁇ (384 ng/mL), TNF- ⁇ (10 ng/mL) and IL-1 ⁇ (2000 pg/mL).
  • medium nitrite a stable product of nitric oxide (NO) oxidation
  • IFN- ⁇ 384 ng/mL
  • TNF- ⁇ 10 ng/mL
  • IL-1 ⁇ 2000 pg/mL
  • nitric oxide production by MIN-6, Huh7, Huh7ins and Melligen cells was measured as nitrite accumulation in conditioned medium and determined by the modified Griess reaction.
  • 50 ⁇ L of cell free medium were mixed with an equal volume of 1% sulphanilamide (Sigma, USA) in 5% phosphoric acid.
  • NED solution (0.1% N-1-naphthylethylenediamine dihydrochloride in water) (Sigma, USA), 50 ⁇ L per well, was then added to all wells and the plate was again incubated for 5-10 minutes at room temperature, protected from light.
  • the nitrite concentration was determined in triplicate within a concentration range that corresponded to the linear part of the standard curve. Absorbance was measured at 540 nm in a microplate reader (BioTek, USA).
  • MIN-6 The glucose-responsive insulin secreting pancreatic ⁇ -cell line
  • IFN- ⁇ 384 ng/mL
  • TNF- ⁇ 10 ng/mL
  • IL-1 ⁇ 2000 pg/mL
  • Insulin levels for cytokine-treated MIN-6 cells at day 10 represented the total amount of insulin secreted over the entire 10 days.
  • Huh7ins cells co-incubated with cytokines secreted amounts of insulin that were not significantly different to those secreted by the untreated Huh7ins and Melligen cells over the 10-day period ( FIG. 25 b ).
  • MIN-6, Huh7ins and Melligen cells were treated over 10 days with cytokine cocktail IFN- ⁇ ; TNF- ⁇ and IL-1 ⁇ . Stored insulin was extracted using acid ethanol method and amounts of insulin determined by RIA on days 1, 2, 3, 5, 8, and 10. MIN-6 cells were significantly affected by the cytokine treatment at day 2 (P ⁇ 0.05) in contrast to this Huh7ins and Melligen cells retained their ability to store insulin over the entire 10 days without significant difference between the treated and untreated cells (P>0.05).
  • the effect of the cytokines on glucose-responsiveness was also determined for the Huh7ins and Melligen cells.
  • Untreated Huh7ins and Melligen cells gave a 5-fold increase in insulin secretion when stimulated with 20 mM glucose, with return to basal levels of insulin secretion (0.13 ⁇ 0.014 pmol/well/h) upon removal of the glucose stimulus ( FIG. 27 b and c ).
  • Huh7ins and Melligen cells incubated with cytokines for 10 days showed a 4.5-fold increase in insulin secretion upon the 20 mM glucose stimulus and a return to basal levels of secretion (0.06 ⁇ 0.006 pmol/well/h) within 1 h after stimulation ( FIG. 27 b and c ).
  • the amount of insulin secreted by the treated Huh7ins and Melligen cells during the stimulus was not significantly different to the result obtained for the untreated Huh7ins and Melligen cells respectively. This indicates that Huh7ins and Melligen cells retain the ability to respond to a glucose stimulus even after 10 days of cytokine treatment.
  • Huh7ins and Melligen cells were cultured for 10 days with and without cytokines. At day 10, the cells were stimulated with increasing concentrations of glucose in basal medium. Huh7ins cells secreted increased amounts of insulin in response to 2.5 mM glucose and Melligen cells at 4.25 mM glucose. In both cell lines there was no significant difference observed between cytokine treated and untreated cells at any glucose concentration (P>0.05) ( FIG. 28 ).
  • pancreatic transdifferentiation that has occurred in Huh7ins and Melligen cells was detected.
  • RNA obtained from the cell lines was used to reverse transcribe complimentary DNA (cDNA) by using the reverse transcription reagents (Promega, U.S.A), which were made up to a 40 ⁇ L reaction mixture, containing RT buffer, Random primers, RNase inhibitor, dNTP mixture, Reverse transcriptase, RNase-free dH 2 O.
  • the volume for each reagent is listed in Table 2. All reagents were spun down to mix and incubated at 37° C. for 1 hour; this was followed by a 99° C. heat shock for 1 minute. The tubes were immediately transferred to ice and were ready to use for further amplification.
  • RNA contamination in total isolated RNA was excluded, due to the addition of 1.5 ⁇ L of DNase (1 unit/ ⁇ L) to 60 ⁇ L of mRNA preparation.
  • the sample was also examined following electrophoresis in a 1.5% agarose gel.
  • primer sequences are listed in Table 3, including: PDX1, NEUROG3, NEUROD1, NKX2-2, NKX6-1, PAX6, PC1/3, PC2, liver GK, islet GK, GLUT2, glucagon, somatostatin (SST), and pancreatic polypeptide (PP). These primers were diluted to 1 ⁇ g/ ⁇ L, and further diluted 1:8 for PCR reactions.
  • Real-time PCR was performed to determine the level of expression of some factors that were detected by RT-PCR in different cell lines, and to evaluate the effect of the overexpression of human islet GK in Melligen cells ( FIG. 30 ).
  • Quantitative real time PCR was performed by using a Prism 7500 (ABI). Platinum SYBR Green qPCR supermix-UDG kit (Invitrogen) was used as amplification reagents. The primers are listed in Table 3.
  • Amplification conditions included initiation 50° C. for 2 min and denatured at 96° C. for 10 min, followed by 40 cycles and each cycle included denaturation at 96° C. 35 seconds, annealing at 58° C. (for human islet glucokinase cDNA) or 66.5° C. (for liver glucokinase gene) 35 seconds and extension at 72° C. 35 seconds.
  • Relative quantitative analysis was performed according to the comparative C T value by using the arithmetic formula 2 ⁇ ( ⁇ Ct).
  • the cDNA levels were normalized to house keeping gene (human GAPDH).
  • the specific PDX-1 protein was revealed by western blotting analysis in Huh7, Huh7ins, and Melligen cells was detected at 35 kDa ( FIG. 35 ).

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US9459203B2 (en) 2014-09-29 2016-10-04 Zyomed, Corp. Systems and methods for generating and using projector curve sets for universal calibration for noninvasive blood glucose and other measurements
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