WO1995032740A1 - Lignee de cellules transformees qui proviennent d'un carcinome hepatocellulaire humain et qui sont capables de produire de l'insuline - Google Patents

Lignee de cellules transformees qui proviennent d'un carcinome hepatocellulaire humain et qui sont capables de produire de l'insuline Download PDF

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WO1995032740A1
WO1995032740A1 PCT/AU1995/000327 AU9500327W WO9532740A1 WO 1995032740 A1 WO1995032740 A1 WO 1995032740A1 AU 9500327 W AU9500327 W AU 9500327W WO 9532740 A1 WO9532740 A1 WO 9532740A1
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insulin
cells
glucose
hep
hepatocyte
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PCT/AU1995/000327
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Bernard Edward Tuch
Ann Margaret Simpson
Glenn Mark Marshall
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Unisearch Limited
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    • 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
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C12N2510/02—Cells for production

Definitions

  • the present invention relates to a method of treating Type I diabetes using a transformed hepatocyte.
  • the present invention further relates to a transformed hepatocyte which secretes insulin.
  • a suitable cell type needs to be determined.
  • the cell type of choice for gene therapy of diabetes is not the beta cell.
  • Islet cells are either greatly reduced or absent in patients suffering from Type I diabetes because of autoimmune destruction (Eisenbarth et al, 1986).
  • the donor cell type must be accessible and capable of being engineered to synthesise, process, store and secrete insulin.
  • it is essential that the insulin output of such a modified cell type must be regulated appropriately. To achieve this insulin gene expression must be under the control of a suitable promoter.
  • trans aryotic mice bearing an intact human insulin gene inserted into mouse Ltk fibroblast cells pioneered the field of somatic gene therapy in diabetes by proving that transfected fibroblasts can supply enough insulin in diabetic mice to initially produce healthy animals .
  • the fibroblasts had no facility to store insulin and no developed regulatory pathways to control its secretion the mice died of hypoglycaemia. It would appear that cell types worth foremost consideration are those already specifically adapted to protein secretion, which have well developed regulatory pathways capable of processing prohorraones .
  • the AtT-20 cell line derived from the mouse anterior pituitary when transfected with cDNA for human proinsulin has been shown to be capable of processing proinsulin to insulin (Moore et al, 1983).
  • the islet isoform of glucokinase is naturally expressed in AtT-20ins cells, but the cells lack ability to express the glucose transporter GLUT-2 and fail to respond to glucose (Hughes et al, 1991).
  • the AtT-20ins cells Upon stable transfection with rat GLUT-2, the AtT-20ins cells exhibit increased intracellular storage of insulin, glucose potentiation of non-glucose secretogogues and a direct stimulation of insulin release by glucose, although the maximal effect was seen at non-physiological concentrations of glucose (Hughes et al, 1992).
  • Work in this laboratory has generated stable transformants of the AtT-20 cell line containing insulin cDNA attached to an inducible promoter (Simpson et al, 1993). These clones secreted
  • AtT-20 immortal cell line provides a useful place to start in developing a model system for gene therapy of diabetes, their primary equivalent is unlikely to be available from humans; therefore accessible primary cells must be considered.
  • the present inventors considered hepatocytes as possible target cells .
  • Hepatocytes are known to play a crucial role in intermediary metabolism, synthesis and storage of proteins in the liver, exhibit glucokinase expression (similar to pancreatic islets) and are accessible. Introduction and stable expression of foreign genes into primary mammalian hepatocytes has been demonstrated recently using human liver tissue (Grossman et al, 1991).
  • liver cells which are readily accessible by biopsy may be engineered to secrete insulin acutely in response to glucose and possibly other physiological secretogogues.
  • the present invention consists in a method of treating Type I diabetes in a subject comprising transfecting an hepatocyte with a gene encoding insulin, the gene being under the control of an appropriate promoter, and introducing the transformed hepatocyte into the subject.
  • the present invention consists in a transformed hepatocyte for use in treatment of Type I diabetes, the hepatocyte including a gene encoding insulin, the gene being under the control of an appropriate promoter.
  • the hepatocyte used in the invention is a primary hepatocyte isolated from the subject and, following transfection with an insulin gene under the control of an appropriate promoter, is re-introduced to the subject.
  • Such primary hepatocytes naturally express the glucose transporter GLUT-2, however, it may be desirable to increase the expression of GLUT-2 in the transfected hepatocyte by further transfecting with an homogeneous or heterogeneous GLUT-2 gene under the control of an appropriate promoter.
  • HEP G2 HEP G2
  • HEP G2 HEP G2
  • HEP G2 HEP G2
  • HEP G2 HEP G2
  • the hepatocyte is further transfected with either an homogeneous or heterogeneous gene encoding the high capacity glucose phosphorylation enzyme glucokinase.
  • the hepatocyte includes either homogeneous or heterogeneous genes encoding the glucose transporter GLUT
  • glucose phosphorylation enzyme glucokinase 2 and the high capacity glucose phosphorylation enzyme glucokinase.
  • Figure 1 provides light micrographs of a) HEP G2 cells
  • HEP G2ins cells immunochemically stained for insulin and counterstained with Harris heamotoxylin and Scott bluing. Granular positive staining in cytoplasm of HEP G2ins cells is marked with arrows (X 495).
  • Figure 2a provides a transmission electron micrograph of part of the cytoplasm of a HEPG2ins cell. There are many membrane-bound vacuoles (v) in the cytoplasm, each containing electron dense material. Scale 1 ⁇ m.
  • Figure 2b provides a transmission electron micrograph of part of the nucleus and cytoplasm of a HEP G2 control cell. There is an abundance of rough endoplasmic reticulum (r) , mitochondria (m) and a Golgi complex (G) .
  • Figure 3 shows insulin synthesis of HEP G2ins cells in response to varying concentrations of glucose (0-20 mM) .
  • Figure 4 shows the effect of 20 mM glucose and 5 mM 8-Br- cAMP on a) the chronic release of insulin, and b) the insulin content of HEP G2ins cells. Values are expressed as means + S.E. for 3 observations.
  • Figure 5 shows Northern blot analysis of Glut 2 in HEP G2 (untransfected cells, lane 1), HEP G2 control cells (transfected with CMV and Rep 4 vectors alone, lane 2) HEP G2ins cells (transfected with insulin cDNA alone, lane 3) and HEP G2ins/ g cells (lane 4.
  • Figure 6 shows stimulation by 20 mM glucose on the acute regulated release of immunoreactive insulin and proinsulin.
  • HEP G2ins/ g cells were incubated in the basal medium for 2 consecutive 1 hr periods to stabilise the basal secretion of insulin. Monolayers were exposed to the stimulus for 1 hr.
  • n number of experiments, B: basal, S: stimulus. Values are express as means + S.E.
  • Figure 7 shows stimulation by (a) 5 mM 8 Br cAMP; (b) 10 mM theophylline; (c) 20 mM arginine on the acute regulated release of immunoreactive (pro)insulin.
  • HEP G2ins/ g cells were incubated in the basal medium for 2 consecutive 1 hr periods to stabilise the basal secretion of insulin. Monolayers were exposed to the stimulus for 1 hr. n: number of experiments, B: basal, S: stimulus. Values are expressed as means + S.E.
  • Figure 9 shows perifusion of HEP G2ins/ g cells with 20 mM glucose.
  • EXAMPLE 1 METHODS
  • Insulin cDNA pC2 was kindly provided by Dr. M. Walker, Weizmann Institute, Israel.
  • the expression vector pRcCMV was purchased from Invitrogen (San Diego, Cal, USA) and pSKII "1" ' from Statagene (La Jolla, Cal, USA).
  • Eagles Minimal Essential Medium (MEM) and G418 antibiotic were purchased from Gibco Laboratories, Grand Island, NY, USA.
  • Fetal calf serum (FCS) was supplied by Cytosystems Pty Ltd, Sydney, Australia. Restriction enzymes came from Boehringer Mannheim, Germany.
  • Biosynthetic human proinsulin (hPI) and a polyclonal antibody to this peptide were kindly supplied by Lilly Research Laboratories, Indianapolis, USA.
  • the radioimmunoassay for insulin was carried out with a human insulin kindly provided by Novo Nordisk, Sydney, Australia and guinea pig insulin antibody donated by D. Yue and ⁇ l25 by J. Bryson, University of Sydney, Australia.
  • ⁇ H leucine was purchased from New England Biolabs, Ontario, Canada.
  • Guinea pig anti-insulin antibody was supplied by Dako Corp. Ca, USA. 8-Br-cAMP was purchased from Sigma, St. Louis, Mo, USA.
  • Millipore filters were purchased from Millipore, Bedford, MA, USA.
  • Biorad Protein Assay Kit was obtained from Biorad, Richmond, CA, USA and pansorbin from Calbiochem, Behring Diagnostics, La Jolla, USA. Cell Culture; HEP G2 cells were grown in monolayers in Minimal
  • the full-length 0.6 Kb human insulin cDNA pC2 was ligated into the multi cloning site of the mammalian expression vector pSKII + (EcoRl/BamHl) .
  • the Xbal/Hindlll 0.6 Kb fragment was subsequently cloned into the multi- cloning site of pRcCMV which expresses resistance to the antibiotic neomycin/G418.
  • HEP G2 cells were transfected with 20 ⁇ g of the recombinant plasmid and vector alone (these clones are used throughout the experiments as "control" cells) by electroporation at 200V and 960 ⁇ F in a Biorad gene pulser at a cell concentration of 5 x 10 ⁇ cells per cuvette, in MEM medium containing no FCS. 2.5 x 10 ⁇ cells were subsequently plated into culture dishes in MEM medium containing 10% FCS. To obtain stable transfectants of HEP G2 cells containing insulin cDNA, 48 hr later 1 mg/ml of G418 antibiotic was added to the culture medium. The antibiotic G418 had a purity of 46-49% (active drug, 460-490 ⁇ g/mg) .
  • the concentration referred to above is the crude compound and not the actual drug.
  • Medium plus drug was changed every 2-3 days.
  • After 3-4 weeks of selection colonies were picked using cloning rings and screened for production of insulin by radioimmunoassay (RIA) .
  • RIA radioimmunoassay
  • the packed cells were embedded in Spurr's resin, sectioned grey on a MT-1 ultramicrotome, stained with uranyl acetate and lead citrate, and examined in a Jeol JEM-1010 transmission electron microscope at 80 kv.
  • Static Stimulation of Insulin Secretion Before stimulation, tissue culture plates were thoroughly washed with basal medium (Dulbecco's Phosphate Buffered Saline containing 1 mM CaCl2 and supplemented with 20 mM HEPES and 2 mg/ml bovine serum albumin (BSA) to remove FCS. Monolayers were incubated in the basal medium at pH 7.4 for three consecutive 1 hr periods to stabilise the basal secretion of insulin.
  • basal medium Dulbecco's Phosphate Buffered Saline containing 1 mM CaCl2 and supplemented with 20 mM HEPES and 2 mg/ml bovine serum albumin (BSA)
  • HEP G2ins cells was incubated at 37°C (5% C0 /95% 0 2 ) in vials containing 2 ml Krebs-Ringer bicarbonate buffer (Krebs, 1932) supplemented with 10 mM HEPES, 2 mg/ml BSA (KRB-BSA), 50 ⁇ Ci/ l L-[4,5,- 3 H]leucine and 0-20 mM glucose. After 2 hr, the cells were washed in nonradioactive leucine solution and disrupted by sonication in 200 ⁇ l distilled water.
  • the amount of labelled insulin was determined by an immunoprecipitation technique (Halban et al, 1980) with guinea pig anti- insulin antibody; guinea pig serum was used as a control.
  • the antibody was precipitated by pansorbin. The total binding capacity of this system was 400 ng.
  • the antibody bound and trichloroacetic acid precipitable radioactivity was estimated by liquid scintillation counting and a sample of the aqueous homogenate was analysed for DNA content (Labarea et al, 1980) . Specificity of the antibody was established by blocking measurement of ⁇ E- insulin with an excess of cold insulin (7 mM) , a similar concentration having no effect. ii .
  • Pulse labelling of the cells was stopped by washing in ice cold KRB-BSA and incubated in 2 ml KRB-BSA (containing 2.8, 20 mM glucose or 5 mM 8-Br-cAMP) for 175 min postlabel (chase) incubation at 37°C.
  • the period of pulse labelling was considered as time zero to 5 min, and the chase period a further 175 min, for a total of 180 min.
  • the chase period was ended and the cells centrifuged to form a pellet. The supernatant was removed for analysis and the pelleted cells were sonicated and the total immunoreactive insulin in the chase medium and the cell sonicates analysed by the immunoprecipitation technique outlined above.
  • the glucose phosphorylation activity was determined by measuring the rate of glucose-6-phosphate formation in a modification of the method of Trus et al (1981). HEP G2 (control) and HEP G2ins cells were harvested with trypsin- EDTA, washed with pre-chilled Phosphate Buffered Saline twice to remove glucose and homogenized in ice-cold buffer (pH 7.7) containing 20 mM K2HPO4, 1 mM EDTA, 100 mM KCl and 5 mM dithiotreitol . The homogenate was centrifuged at 12,000 g for 10 min at 4°C, the supernatant was retained to measure glucose phosphorylation activity and protein. Protein was estimated by the Biorad protein assay kit.
  • the assay volume contained 4 ⁇ l of supernatant (containing 7-18.7 ⁇ g protein) in 100 ⁇ l of HEPES HCI 50 mM (pH 7.7), 100 mM KCl, 7.4 mM MgCl 2. 15 mM ⁇ -mercaptoethanol, 0.5 mM ⁇ -NAD + , 0.05% BSA, 2.5 mg/ml glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides, 5 mM ATP and glucose concentrations ranging from 0.03 to 100 mM. Glucose was added at 0.03, 0.06, 0.12, 0.25 and 0.5 mM to measure glucokinase activity.
  • the HEP G2ins clone 11 was finally selected from 25 different clones isolated with cloning rings and five mixtures of clones to be used for further experimentation.
  • Electron microscopy revealed large membrane-bound vacuoles, approximately 2.6 ⁇ m in diameter, containing electron-dense material in the HEP G2ins cells (Fig. 2a), while these were not seen in the control HEP G2 cells (Fig. 2b).
  • hexokinase activity of the transformed cells was significantly (P ⁇ 0.05) lower.
  • DISCUSSION The data presented in this example demonstrates that similar to what has been reported for other non-islet types transfected with the insulin gene (Laub & Rutter, 1993; Lomedico, 1982; Moore et al, 1983), the chronic insulin release of HEP G2ins cells was constitutive and 84% of the material secreted into the medium was proinsulin. Regulated release is that induced by exposure to a secretogogue, in this case 8-Br-cAMP. Following acute stimulation for periods of one hour 86% of the material secreted into the medium was mature insulin not proinsulin.
  • HEP G2ins cells have the ability to package insulin into some form of limiting membrane until regulated release occurs .
  • the cells also appear to be functioning normally following transfection, as with regards to albumin secretion which was unaltered from untransfected cells.
  • Electron microscopy revealed large membrane-bound vacuoles containing electron-dense material in HEP G2ins cells, which were not seen in control cells. This provides morphological evidence that storage of insulin could occur here, but this will have to be determined in future work by immunoelectron microscopy.
  • HEP G2ins cells exhibit enhancement of insulin synthesis in response to both glucose and elevated levels of cAMP, similar to that seen in a pancreatic ⁇ cell (Maldonato et al, 1977).
  • insulin biosynthesis is primarily stimulated by glucose.
  • the short term effects of glucose on preproinsulin synthesis are restricted to a stimulation of translation and this occurs within minutes of raising ambient glucose (Welsh et al, 1986). Over a longer period, glucose is thought to stimulate transcription and stabilise mRNA.
  • a dose response curve for (pro)insulin biosynthesis of HEP G2ins cells in response to increasing concentrations of glucose was generated with a half V max of 4.9 mM glucose, not significantly different to that recorded for adult rat islets (Shuit et al, 1988) and human fetal islets (Simpson, abstract, 1991).
  • the sigmoidal appearance of the curve is also similar to the dose response curves generated by these tissues.
  • Newly synthesised (pro)insulin was only released from HEP G2ins cells after a 60 minute lag period. This is most likely a result of the time required from synthesis in the rough endoplasmic reticulum to passage in the Golgi complex, and thence exocytosis. A lag period of 45 minutes before the appearance of newly synthesised insulin was seen in a similar experiment using rat islets (Rhodes & Halban, 1987). The fact that insulin was released in response to elevated levels of glucose and 8-Br-cAMP could imply release via a regulated pathway or simply the fact that translation is stimulated.
  • HEP G2ins cells Like transfected AtT-20 cells (Moore et al, 1983, Simpson et al, 1993) HEP G2ins cells differ from pancreatic beta cells in at least one important way - they do not respond to an acute glucose stimulus . As there is no defect in signal tranduction concerning insulin synthesis it would appear that the glucose insensitivity of HEP G2ins cells is confined to the secretory process.
  • glucose sensing system which regulates insulin release from pancreatic ⁇ cells in response to small external nutrient changes
  • high capacity glucose transporter GLUT 2 Kerati et al, 1990; Thorens, et al, 1990; Newgard et al, 1990; Johnson et al, 1990
  • high capacity glucose phosphorylation enzyme glucokinase Weinhouse, 1976. Both of these are known to function similarly to pancreatic ⁇ cells and liver cells.
  • HEP G2ins hepatoma cell lines lack of responsiveness to glucose is probably linked at least in part to failure to express the high K m islet-liver transporter GLUT 2.
  • the parent HEP G2 cells express the erythrocyte/brain glucose transporter (Permutt et al, 1989) which has 55% sequence homology to GLUT 2 (thorens, 1988), and it is reasonable assumption that the HEP G2ins cells do likewise.
  • Human GLUT 2 may be of paramount importance in augmenting the glucose- sensitive insulin release from any given cell type, as illustrated in work by Ferber et al (1993), who have stably transfected rat GLUT 2 and glucokinase into
  • results show that the introduction of insulin cDNA into a liver cell line results in synthesis, storage and acute regulated insulin release. However, chronic insulin release was constitutive and the cells did not secrete insulin in response to glucose.
  • the transfected liver cell described in this example in the treatment of diabetic humans it appears to be necessary to include an additional gene encoding for glucose transport. This is in order to render it responsive to the same physiological stimuli as the normal beta cell.
  • utilisation of these cells in humans could only be carried out if they were placed inside immunoprotective capsules. Rejection of these cells would occur without this.
  • the liver cells were taken from the patient with diabetes, infected with the insulin gene (using, for example, a retrovirus), and these beta cytes then transplanted back into the patient. Rejection should not be a problem as the recipient and donor are the same.
  • HEP G2ins cells were grown in monolayers in Eagles minimal essential medium (MEM) supplemented with 10% fetal calf serum (FCS) and 1 mg/ml G418 antibiotic (presence of G418 maintains presence of insulin cDNA/RcCMV construct through subculture) in air at 37 C.
  • MEM Eagles minimal essential medium
  • FCS fetal calf serum
  • G418 antibiotic presence of G418 maintains presence of insulin cDNA/RcCMV construct through subculture
  • the full length human GLUT 2 cDNA was ligated into the multi cloning site of the vector pREP4 (Groger et al . , 1989) which expresses resistance to the eucaryocidal antibiotic hygromycin.
  • HEP G2ins cells were transfected with 40 ⁇ g of the recombinant plasmid and vector. Cells transfected with the vector alone have been used as controls. Transfection was accomplished by electroporation at 200 V and 960 ⁇ F in a Biorad gene pulser at a cell concentration of 5 x 10 cells per cuvette, in MEM medium containing no FCS.
  • Immunohistochemical analysis was carried out on formalin-fixed samples of cultures that had been trypsinised, using Dako LSAB kit and guinea pig anti- insulin (first antibody), a second incubation with anti- rabbit antibody and a final incubation with rabbit anti- guinea pig antibody and the streptavidin-diaminobenzidine chromogen complex. Static simulation of insulin secretion;
  • tissue culture plates were thoroughly washed with basal medium [Dulbecco's phosphate buffered saline (PBS) containing 1 mM CaCl 2 and supplemented with 20 mM HEPES and 2 mg/ml bovine serum albumin (BSA)] to remove culture medium and FCS.
  • Basal medium Dulbecco's phosphate buffered saline (PBS) containing 1 mM CaCl 2 and supplemented with 20 mM HEPES and 2 mg/ml bovine serum albumin (BSA)] to remove culture medium and FCS.
  • Basal medium Dulbecco's phosphate buffered saline (PBS) containing 1 mM CaCl 2 and supplemented with 20 mM HEPES and 2 mg/ml bovine serum albumin (BSA)] to remove culture medium and FCS.
  • Monolayers were incubated in the basal medium at pH 7.4 for three consecutive 1 hr periods to stabilise
  • Glucose (20 mM) , 5 mM 8-Br-cAMP and 10 mM theophylline were dissolved in basal medium, 12-0- tetradecanolyphorbol-13-acetate (TPA: 1.3 ⁇ m) was dissolved in DMSO and diluted in basal medium (DMSO final concentration 0.8%). Calcium (10 mM) was dissolved in basal medium without phosphate. Three controls were used - basal medium alone, medium with 0.8% DMSO and basal medium without phosphate. The dose response curve to insulin was measured in a similar fashion. The basal level of insulin was established and monolayers were exposed to increasing concentrations of glucose from 0-20 mM.
  • HEP G2ins/g cells were incubated at 37°C (5% C0 2 /95% 0 2 ) in vials containing 2 ml Krebs-Ringer bicarbonate buffer supplemented with 10 mM HEPES, 2 g ml BSA (KRB/BSA) ,
  • the immunoreactive material secreted into the medium was proinsulin and/or its split products .
  • insulin and proinsulin assays were carried out. It can be seen from Table III, that three times more proinsulin than insulin was released daily by the transformed HEP G2ins/g cells.
  • the parental HEP G2ins cell line did not possess GLUT 2 chronically secreted a greater proportion of proinsulin (6X) compared to mature insulin.
  • HEP G2 ins cells with the GLUT 2 cDNA also resulted in a 6-fold increase in intracellular insulin content (Table III) compared to ells lacking GLUT 2.
  • the proportion of insulin : proinsulin was also greater (14.6 : 1) in the HEP G2ins/g cells compared to 3.6 : 1 in the HEP G2ins line.
  • Fig. 9 It can be seen from this figure that there is a definite increase in insulin secretion at 30 min on the addition of 20 mM glucose, which returns to basal levels on removal of the stimulus .
  • DISCUSSION This example examined whether transfection of GLUT 2 cDNA, which is absent from HEP G2ins cells, will allow glucose to exert an effect on insulin secretion. It was found that the cell line established from this transfection (HEP G2ins/g cells) were capable, following synthesis, to process proinsulin to insulin and store it until regulated insulin release occurs .
  • the HEP G2ins/g cells also secrete and store significantly greater amounts of (pro)insulin than the parental line (HEP G2ins) .
  • liver cells which naturally express GLUT2, may serve as useful vehicles for the gene therapy of diabetes.
  • HEP G2ins and control HEP G2 cells Results expressed as mean ⁇ S.E. for five observations.
  • Immunoreactive insulin and proinsulin from culture supematants was compared with that isolated from the cell extracts in transfected HEP G2ins/ g, Hep G2ins and HEP G2 (control: transfected with CMV and REP 4 vectors alone) cells.

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Abstract

L'invention concerne une méthode pour traiter le diabète du type (I) chez un sujet, consistant à transfecter des cellules hépatiques avec un gène codant pour l'insuline, ce gène étant contrôlé par un promoteur approprié et à introduire les cellules hépatiques transformées dans le sujet.
PCT/AU1995/000327 1994-05-31 1995-05-31 Lignee de cellules transformees qui proviennent d'un carcinome hepatocellulaire humain et qui sont capables de produire de l'insuline WO1995032740A1 (fr)

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Cited By (6)

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WO1997026334A1 (fr) * 1996-01-19 1997-07-24 Board Of Regents, The University Of Texas System Expression par recombinaison de proteines issues de lignees cellulaires secretoires
WO1998031397A1 (fr) * 1997-01-21 1998-07-23 Wisconsin Alumni Research Foundation Traitement du diabete a l'aide de cellules beta de synthese
WO2000062862A1 (fr) * 1999-04-15 2000-10-26 South Eastern Sydney Area Health Service Technique de prophylaxie et de traitement des diabetes
WO2001070940A1 (fr) * 2000-03-24 2001-09-27 National Cancer Centre Of Singapore Pte Ltd Constructions genetiques pour l'expression regulee de l'insuline
US6352857B1 (en) 1997-01-21 2002-03-05 Wisconsin Alumni Research Foundation Treatment of diabetes with synthetic beta cells
WO2009021276A1 (fr) * 2007-08-10 2009-02-19 University Of Technology, Sydney Cellules génétiquement modifiées pour comprendre la glucokinase d'un îlot pancréatique et leurs utilisations

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US6194176B1 (en) 1996-01-19 2001-02-27 Board Of Regents, The University Of Texas System Recombinant expression of proteins from secretory cell lines
WO1998031397A1 (fr) * 1997-01-21 1998-07-23 Wisconsin Alumni Research Foundation Traitement du diabete a l'aide de cellules beta de synthese
US6352857B1 (en) 1997-01-21 2002-03-05 Wisconsin Alumni Research Foundation Treatment of diabetes with synthetic beta cells
US6933133B2 (en) 1997-01-21 2005-08-23 Wisconsin Alumni Research Foundation Treatment of diabetes with synthetic beta cells
WO2000062862A1 (fr) * 1999-04-15 2000-10-26 South Eastern Sydney Area Health Service Technique de prophylaxie et de traitement des diabetes
WO2001070940A1 (fr) * 2000-03-24 2001-09-27 National Cancer Centre Of Singapore Pte Ltd Constructions genetiques pour l'expression regulee de l'insuline
WO2009021276A1 (fr) * 2007-08-10 2009-02-19 University Of Technology, Sydney Cellules génétiquement modifiées pour comprendre la glucokinase d'un îlot pancréatique et leurs utilisations
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

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