WO1999054451A1 - Cellules neuroendocrines secretrice d'insuline et ses utilisations - Google Patents

Cellules neuroendocrines secretrice d'insuline et ses utilisations Download PDF

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WO1999054451A1
WO1999054451A1 PCT/US1999/008628 US9908628W WO9954451A1 WO 1999054451 A1 WO1999054451 A1 WO 1999054451A1 US 9908628 W US9908628 W US 9908628W WO 9954451 A1 WO9954451 A1 WO 9954451A1
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cell
insulin
cells
glucose
gene
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PCT/US1999/008628
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English (en)
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Alvin C. Powers
Lan Wu
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Vanderbilt University
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    • 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
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates to methods for treatment of Type I and Type II Diabetes.
  • the invention also relates to insulin-producing cells and methods of using those cells in transplantation or in situ for the treatment of diabetes. More specifically, the invention relates to genetic engineering of non- islet cells to produce insulin in response to physiologic stimuli.
  • the blood glucose concentration is very narrowly controlled (usually 80-90 mg/dl of blood in a fasting person in the morning before breakfast).
  • the concentration rises to 120-140 mg/dl within approximately a n hour following a meal, but feedback systems control the return of glucose concentration back to the control level within approximately two hours after the last absorption of carbohydrates.
  • Glucose is the only nutrient that can be used b y the brain, retina, and germinal epithelium of the gonads for a n adequate supply of energy. Blood glucose concentrations must b e regulated in order to provide the necessary nutrition to these organs.
  • the role of insulin is crucial to this process. Within seconds after insulin binds to its receptors on the cell membrane , many cell types in the body become highly permeable to glucose, allowing rapid entry of glucose into the cells.
  • Type I diabetes is caused by an autoimmune destruction of the pancreatic islet cells which produce insulin. This insulin deficiency results in total dependence of the individual on insulin administration by either multiple insulin injections each day or an insulin infusion device.
  • Type 1 1 Diabetes there is a resistance to the action of insulin and, in addition, insulin deficiency.
  • the molecular pathogenesis of Type II Diabetes is not known, but it does involve autoimmune destruction of the islets.
  • Type II Diabetes in its later stages, is also treated with multiple insulin injections and sometimes insulin infusion devices. In both types of diabetes, the currently available methods for administering insulin are inadequate. Insulin administration does not mimic the normal output of insulin from the pancreas.
  • Glucose-regulated insulin secretion correlates with the level of glucose utilization and the rate-limiting step in this process is phosphorylation of glucose by glucokinase.
  • Glucose metabolism is thought to alter the intracellular ratio of ATP/ADP and NAD(P)H, which influence the activity of the ATP-sensitive K+ ion channel.
  • the heteromeric, ATP-sensitive K + channel, the site of sulfonylurea binding, is crucial for initiating and terminating insulin secretion, since homozygous mutations in the sulfonylurea receptor cause insulin release at subphysiologic glucose concentrations.
  • glucose phosphorylation by glucokinase is of great importance in the control of insulin secretion, additional events such as the glycerol phosphate shuttle, generation of acetyl-CoA/malonyl-CoA, increases in intracellular pH, cAMP and G proteins, phosphoinositol metabolites, phospholipase A2/C, and cytosolic enzymes (pyruvate kinase, mitochondrial glycerophosphate dehydrogenase, lactate dehydrogenase) are also important.
  • the final event in insulin secretion is thought to be activation of a voltage-sensitive Ca +2 ion channel with a subsequent increase in intracellular Ca +2 .
  • Insulin secretagogues other than glucose bypass the glucose phosphorylation step and enter the signal transduction pathways at more distal steps. Fatty acids and neural input also modulate insulin secretion.
  • GLP-1 and GIP are secreted by neuroendocrine cells within the small and large intestine in response to oral glucose and other nutrients and their contribution to insulin secretion has been termed the "entero- insular-axis" .
  • Each peptide interacts with a distinct beta cell receptor and appears to stimulate insulin secretion independently .
  • the GLP-1 receptor belongs to the VIP/PACAP receptor family of G-coupled receptors and interacts with signal transduction pathways for cAMP, intracellular Ca +2 , Na + and Ca +2 channels, an d protein phosphorylation.
  • the GIP receptor also influences several signal transduction pathways including MAP kinase, cAMP and Ca +2 , and the protein kinase C pathway.
  • GLP-1 and GIP were originally thought to act independently of the GLUT2/glucokinase/ATP-sensitive K+ channel pathway, recent evidence suggests that the signal transduction pathway for both peptides may interact with the ATP-sensitive K+ channel pathway .
  • the beta cell response to GLP-I requires the presence of glucose, which also suggests that these two signal transduction p athway s are interrelated.
  • GLP-1 has potential as a therapeutic agent in Type II diabetes as it is able to stimulate insulin release from th e beta cell when glucose is not present.
  • Neuroendocrine cell types contain secretory granules which not only serve as a reservoir for the hormone o r neurotransmitter, but also interact with signal transduction systems during the final steps of exocytosis.
  • any groups have identified the molecular components of the secretory granules in neurons and neuroendocrine cells, and a recurring theme is that the same molecules are involved in hormone secretion by islet cells, neuroendocrine cells, and neurons (synaptophysin, synaptotagmin, snares, etc.).
  • the final signal for exocytosis is a common feature of regulated hormone or neurotransmitter secretion from a variety of cell types.
  • GLUT 2 and glucokinase are expressed in liver (the hepatic glucokinase isoform differs slightly in th e amino terminus), but the liver does not have secretory vesicles.
  • GLUT2 nor glucokinase is expressed in most neuroendocrine cell types though some glucose-responsive neurons in the brain may express these proteins.
  • the metabolic steps following glucose phosphorylation in the beta cell involve a series of enzymes and mitochondrial events but these are not beta cell specific.
  • the GLP-I receptor and the GIP receptor have a limited distribution, but are found in other tissues as well.
  • the distal events in insulin secretion in the beta cell utilize a set of proteins o r infrastructure that are neuron or neuroendocrine cell specific, bu t not beta cell specific. Utilization of non-islet cells as the foundation cell into which proteins important for physiologically-regulated insulin secretion are introduced would overcome the obstacle of a recurrence of the autoimmune process of Type I diabetes. Despite the ability of many cell types to express an introduced insulin gene, however, physiologic regulation of insulin secretion still presents a number of challenges.
  • Non-islet cells can b e engineered to express insulin, but these cells (eg. hepatocytes, myocytes, and fibroblasts) lack the ability to store and properly process insulin because they do not contain secretory granules with their accompanying hormone processing enzymes. Furthermore, these cells secrete insulin via the constitutive secretory pathway and thus will secrete some insulin regardless of the extracellular glucose concentration.
  • these cells eg. hepatocytes, myocytes, and fibroblasts
  • these cells secrete insulin via the constitutive secretory pathway and thus will secrete some insulin regardless of the extracellular glucose concentration.
  • Non-islet cells Insulin secretion independent of the glucose concentration is a serious limitation of the use of non-islet cells. Thus, most non-islet cells must be engineered to not only sense the extracellular glucose concentration, but also to express a large number of proteins required for regulated secretion via secretory vesicles. The ability of pituitary cells to correctly process insulin reflects expression of similar hormone processing enzymes in a variety of neuroendocrine cells. Non-islet, neuroendocrine cells (pituitary and adrenomedullary cells) express most, but not all, beta-cell proteins required for glucose-regulated insulin secretion. What is needed is a method of stimulating these neuroendocrine cells to secrete insulin in vitro and in vivo in response to physiologically relevant stimuli.
  • the prior art is deficient in genetically engineered cells that express non-glucose insulin secretagogue receptors and produce insulin.
  • the present invention fulfills this need in the art.
  • the present invention is directed to an engineered cell comprising a gene encoding a non-glucose insulin secretagogue receptor and an insulin gene, wherein at least one of said genes is a recombinant gene and the cell secretes insulin in response to glucose wherein at least one of said genes has been introduced into the cell by means of a recombinant vector.
  • non-glucose insulin secretagogue receptors include
  • the recombinant gene may be a cDNA.
  • the cell is derived from a cell capable of forming secretory granules and is a neuroendocrine such as a pituitary and a thyroid cell, or neuroendocrine-like cell.
  • the present invention is also directed to a method of producing insulin, comprising: (a) culturing the engineered cell of described herein; (b) stimulating said cell to secrete insulin; and (c) collecting the secreted insulin.
  • the cell is stimulated to secrete insulin by contacting said cell with a non- glucose compound.
  • the cell may be grown in contact with a solid support such as gelatin beads.
  • the cell may also be contained within a column.
  • the insulin produced is human insulin.
  • Representative examples of non-glucose compound include forskolin, dibutyryl cAMP or isobutylmethylxanthine.
  • the present invention is also directed to an artificial beta cell device, comprising a population of engineered cells as described herein, wherein at least one of said genes is a recombinant gene, wherein the cell population is positioned into a selectively permeable membrane.
  • the selectively permeable membrane is a biocompatible coating.
  • the cell population is encapsulated by the biocompatible coating such as a semipermeable capsule.
  • the cells are microencapsulated, e.g., encapsulated in a hydrogel coating or encapsulated in an alginate coating.
  • the device may b e further defined as a semipermeable fiber, with the cells seeded into the fiber.
  • the device has a cell population which comprises from about 1 ,000 to about 10,000 engineered cells.
  • the present invention is also directed to a method of producing insulin in an individual in need of such treatment, comprising the step of: removing non-islet, neuroendocrine cells from said individual; transforming said cells with a gene encoding a non-glucose insulin secretagogue receptor and gene encoding insulin; and returning said cells to said individual so as to produce insulin.
  • the non-glucose insulin secretagogue receptor is selected from the group consisting of glucagon-like peptide 1 , glucose-dependent insulin releasing polypeptide, cholecystokinin, gastrin, secretin, and gastric inhibitory peptide.
  • Figure 1 shows that normal rat pituitary does not express GLP-1 receptor.
  • Total RNA form normal rat islets, rat hypothalamus, and rat pituitary was used to synthesize cDNA using MMLV reverse transcriptase.
  • the amount of cDNA equivalent to 10 ng of total RNA was used in a RT-PCR reaction with 32p_dc ⁇ p and primers specific for the rat GLP-1 receptor ( 30
  • the expected sized band is 207 bp and is marked with a n arrow.
  • One-third of the RT-PCR product was separated on a 8% non-denaturing poly aery lamide gel. The gel was dried an d exposed for autoradiography (18 hour exposure). Molecular weight standards are shown in lane 1. Both islets and hypothalamus are known to express the GLP-1 receptor.
  • FIG. 2 shows that normal rat pituitary has SURl and Kir 6.2.
  • Membranes were prepared from normal r at pituitaries or alpha TC cells (islet cell line) and photolabeled with 10 nM l ⁇ Si-azido-giibenclamide, solubilized in SDS samples buffer, and separated on 7.5 % polyacrylamide gels.
  • Lanes A and B alpha TC cell membranes plus (B) or minus (A) l ⁇ M unlabeled glibenclamide;
  • Lanes C and D pituitary cell membranes plus (D) or minus (C) l ⁇ M unlabeled glibenclamide.
  • the 140 kDa and 1 50 kDa SURl protein (reflecting differences in glycosylation) is marked with arrows.
  • the lower molecular weight Kir 6.2 is not shown in this figure.
  • Figure 3 shows that glibenclamide stimulates growth hormone secretion by normal rat pituitary cells. Rat pituitary cells were cultured for 24 hours after isolation and then stimulated with 1 and 10 nM glibenclamide. Growth hormone in the culture media was quantified by radioimmunoassay. The mean +_ SEM is shown.
  • Figure 4 shows that pituitary cells infected with a recombinant insulin-adenovirus co-secrete insulin and pituitary hormones.
  • Rat pituitary cells were infected with AdhlNS (expressing the human insulin cDNA under the control of the CMV promoter) at a MOI of 10 for 48 hours.
  • AdhlNS expressing the human insulin cDNA under the control of the CMV promoter
  • Figure 5 shows that pituitary cells infected with both a recombinant GLP-I receptor adenovirus and a recombinant insulin adenovirus secrete insulin at physiologic GLP-1 levels.
  • the pituitary cells were then evaluated in a cell perifusion system a s the GLP-1 concentration was raised from 20 pM to 30 nM (perifusate contained 25 mM glucose). Human insulin was quantified by radioimmunoassay.
  • FIG. 6 shows that glucose does not regulate hormone secretion by pituitary cells.
  • Normal rat pituitary cells, infected were infected with a recombinant GLP-I receptor adenovirus, or wild type pituitary cells were evaluated in a cell perifusion system where the glucose concentration was raised from 2.5 mM to 20 mM.
  • the GLP-1 stimulation w as preceded by perifusion at 20 mM glucose and there was no increase in GH secretion.
  • the cells were stimulated with 10 nM GLP-I or 10 nM GHRH. Growth hormone in the perifusate was quantified b y radioimmunoassay. If CRH was substituted for GHRH and ACTH was measured, GLP-I and CRH stimulated ACTH secretion with a similar profile. Likewise, if TRH was substituted for GHRH and
  • prolactin was measured, GLP-I and TRH stimulated prolactin secretion with a similar profile. Neither ACTH nor prolactin secretion was altered by changes in the serum glucose.
  • Figure 7 shows that engineered pituitary cells secrete insulin in response to GLP-I independent of extracellular glucose concentration.
  • Normal rat pituitary cells infected with both a recombinant GLP-I receptor-adenovirus and a recombinant insulin-adenovirus, were evaluated in a cell perifusion system. The cells were stimulated with 10 nM GLP-I at a range of glucose concentrations (2.5 mM, 5 mM, 10 mM, or 20 mM).
  • Panel A shows GH secretion and Panel B show insulin secretion in the s ame sample of the perifusate.
  • Wild type pituitary cells, cells expressing only the GLP-1 receptor, and cells expressing both the GLP-1 receptor and human insulin are shown.
  • Figure 8 shows expression of human GLUT3 in r at pituitary cells. Rat pituitary cells were infected with AdhGLUT3 at a MOI of 10 for 48 hours. The infected cells were incubated with a rabbit anti-human GLUT3 antibody, followed by a cy3 - conjugated donkey anti-rabbit antibody. Similarly, expression of human GLUT2 in rat pituitary cells with the AdhGLUT2 virus has been demonstrated with GLUT2 specific antibody produced in our laboratory.
  • Figure 9 shows that the expression of human GLUT2 in rat pituitary cells increases glucose transport.
  • Rat pituitary cells were infected with AdLacZ and AdhGLUT2 at a MOI of 10 for 48 hours. The infected cells were cultured in a 48-well plate at a density of 1 x 105 cells/well. The uptake of ( 1 4c)-2-deoxyglucose during a 15 minute incubation at room temperature w as
  • Figure 10 shows that glucokinase adenovirus expresses protein with expected function.
  • Figure 10 A RIN 1046-38 cells were infected with an adenovirus expressing human GLUT2 (GLUT2) or an adenoviruses expressing both human GLUT2 and human islet glucokinase (GLUT2 + GK). Uninfected RIN cells (wild type) served as a control. Twenty-four hours after infection, extracts of the cells were prepared and assayed for hexokinase activity as previously described (234,235). The ability of glucose 6-phosphate to inhibit hexokinase, but not glucokinase, at 3 mM glucose is shown.
  • FIG 10B Normal rat pituitary cells were infected with an adenovirus expressing either lacZ (upper panel) or human islet glucokinase (lower panel). Twenty four hours later cells w ere permeabilized, fixed with 4% paraformaldehyde, and stained with a polyclonal antiserum to GK A secondary antibody coupled to Cy-2 was added and the cells were analyzed by fluorescent activated cell sorting (FACS). Cells expressing for GK are shown in the lower panel by a shift of the curve to the right (compare upper and lower panel).
  • FACS fluorescent activated cell sorting
  • Figure 11 shows that two transgenes can b e expressed in one cell.
  • Figure 11 A Rat pituitary cells w ere simultaneously infected with AdhlNS (expressing the human insulin cDNA under the control of the CMV promoter) an d adenovirus expressing islet glucokinase (AdhGK). The infected cells were then stained with an antiserum against insulin and CK and analyzed by confocal microscopy. The insulin antiserum w as
  • FIG 11B RIN 1046-38 cells (rat islet cell line) were simultaneously infected with AdhGLUT2 (expressing the human GLUT2 cDNA under the control of the CMV promoter) and adenovirus expressing islet glucokinase (AdhGK).
  • AdhGLUT2 expressing the human GLUT2 cDNA under the control of the CMV promoter
  • AdhGK adenovirus expressing islet glucokinase
  • the infected cells were then stained with an antiserum specific for human GLUT2 (raised in our laboratory against the carboxyterminal tail of GLUT2; This antiserum does not cross-react with rat GLUT2.) and GK and analyzed by confocal microscopy.
  • This clone of RIN cells has little GK staining before infection with AdhGK.
  • the filter active for which antibody/antigen complex is shown above each panel. Note that GLUT2 staining is cell surface and GK staining is cytoplasmic.
  • the present invention provides such artificial beta cells that can be employed in the clinical treatment of IDDM.
  • the invention relates generally to a n engineered cell that includes a gene, preferably a recombinant gene, encoding a functional non-glucose insulin secretagogue receptors, wherein the engineered cells secrete insulin in response to glucose.
  • engineered cell is intended to refer to a cell into which a recombinant gene has been introduced. Therefore, engineered cells are distinguishable from naturally occurring cells, which do not contain a recombinantly introduced gene.
  • Engineered cells are thus cells having a gene or genes introduced through the hand of man.
  • Recombinantly introduced genes will either be in the form of a cDNA gene (i.e., they will not contain introns), a copy of a genomic gene, or will include genes positioned adjacent to a promoter not naturally associated with the particular introduced gene.
  • a cDNA version of the gene it will be more convenient to employ as the recombinant gene a cDNA version of the gene. It is believed that the use of a cDNA version will provide advantages in that the size of the gene will generally be much smaller and more readily employed to transfect the targeted cell than will a genomic gene, which will typically be up to an order of magnitude larger than the cDNA gene. However, this does not exclude th e possibility of employing a genomic version of a particular gene where desired.
  • Engineered cells of the present invention will generally be derived from a cell line comprised of cells capable of forming secretory granules.
  • Secretory granules are generally confined to mammalian cells whose main function is the synthesis and secretion of peptides. Generally speaking, secretory granules are found in endocrine cells. Secretory granules are formed b y budding of intracellular membranous structures known as th e Golgi apparatus. Polypeptide hormones are usually synthesized a s
  • the initial protein product of the insulin gene in beta cells is preproinsulin.
  • This precursor differs from mature insulin in that it has a so-called "signal sequence" at its N-terminus, consisting of a stretch of hydrophobic amino acids that guide the polypeptide to the rough endoplasmic reticulum.
  • I t also has a connecting peptide between the A and B chains that comprise the mature insulin molecule; this connector is known a s the "C-peptide”.
  • the preproinsulin molecule enters the lumen of the endoplasmic reticulum, in the process having its hydrophobic N-terminal "pre” region proteolytically removed.
  • the processed, correctly folded proinsulin molecule (still containing the C- peptide) is then transported to the Golgi apparatus. As th e precursor is transported through the Golgi apparatus, enzymatic removal of the C-peptide connector begins.
  • Secretory granules are derived from Golgi membranes by a process of budding off and eventual separation.
  • the resulting granule envelops the mixture of unprocessed proinsulin and the small amount of mature insulin.
  • Most of the processing of proinsulin to insulin occurs shortly after formation of the secretory granules by virtue of the fact that the enzymes responsible for this processing are found at highest concentration within the granules.
  • the granules are transported to the plasma membrane surface of the cell in response to secretory stimuli such as glucose; whereupon they fuse with the plasma membrane an d release their stores of the mature hormone.
  • the present invention proposal outlines a n experimental approach based on the following criteria for th e "ideal" foundation cell: the foundation cell must be non-malignant and non-transformed so that the malignant potential is zero; th e foundation cell must be non-islet in origin so that it will not destroyed by a recurrence of the autoimmune process which causes Type I diabetes; the foundation cell must possess th e regulated secretory pathway and secretory vesicles; th e foundation should be closely related to beta cells in protein expression to minimize the number of introduced genes required to endow the foundation cell with physiologically-regulated insulin secretion; the foundation cell must be capable of expressing components of the glucose signal transduction pathway found in beta cells or ideally would already express most of these proteins; the foundation cell should be capable of expressing the signal transduction pathways for non-glucose insulin secretogogues such as GLP-1 and GIP.
  • cells used in this aspect will preferably be derived from an endocrine cell, such as a pituitary or thyroid cell.
  • an endocrine cell such as a pituitary or thyroid cell.
  • the type of engineering that will be required in order to achieve a cell that secretes insulin in response to glucose will depend on the property of the starting cell. I n general, in addition to the ability to form secretory granules, the ability to functionally express certain genes is important.
  • the functional genes that are required include an insulin gene and a GLP gene. In the practice of the invention, one or more of these genes will be a recombinant gene.
  • constitutive promoters are generally viral in origin, and include the cytomegalovirus (CMV) promoter, the Rous sarcoma long-terminal repeat (LTR) sequence, and the SV40 early gene promoter. The use of these constitutive promoters will ensure a high, constant level of expression of the introduced genes.
  • CMV cytomegalovirus
  • LTR Rous sarcoma long-terminal repeat
  • the level of expression from the introduced gene(s) of interest can vary in different clones, probably as a function of the site of insertion of the recombinant gene in the chromosomal DNA.
  • the level of expression of a particular recombinant gene can be chosen by evaluating different clones derived from each
  • the constitutive promoter ensures that the desired level of expression is permanently maintained. It may also be possible to use promoters that are specific for cell type used for engineering, such as the insulin promoter in insulinoma cell lines, or the prolactin or growth hormone promoters in anterior pituitary cell lines.
  • the present invention is directed to a method of providing a physiologically-responsive insulin-secreting capability to a mammal in need of such capability.
  • the method includes generally implanting engineered cells that secrete insulin in response to physiologic stimuli into such a mammal.
  • Techniques presently in use for the implantation of islets will be applicable to implantation of cells engineered in accordance with the present invention.
  • One method involves the encapsulation of engineered cells in a biocompatible coating. I n this approach, cells are entrapped in a capsular coating that protects the encapsulated cells from immunological responses, and also serves to prevent uncontrolled proliferation of clonal engineered cells.
  • a preferred encapsulation technique involves encapsulation with alginate-polylysine-alginate. Capsules made employing this technique generally contain several hundred cells and have a diameter of approximately 1 mm. If the engineered cells are from the same person, then this would not be necessary.
  • An alternative approach is to seed Amicon fibers with engineered cells.
  • the cells become enmeshed in the fibers, which are semipermeable, and are thus protected in a manner similar to the micro encapsulates.
  • the cells After successful encapsulation or fiber seeding, the cells, generally approximately 1,000- 10,000, may b e
  • non-islet neuroendocrine cells are genetically-engineered to express receptors for non-glucose insulin secretagogues, along with hu man insulin.
  • neuroendocrine cells for example, from the pituitary or adrenal medulla
  • a non-glucose secretagogue is GLP-1
  • cells are genetically engineered to respond to GLP-1 by the introduction of human GLP-1 receptor cDNA into the cell for expression.
  • non-glucose secretagogues especially cholecystokinin (CKK), gastrin, secretin, or gastric inhibitory peptide (GIP), which are released in the gastrointestinal tract in the postprandial state
  • CKK cholecystokinin
  • GIP gastric inhibitory peptide
  • m ay be used as factors to stimulate insulin secretion in neuroendocrine cells, in conjunction with co-infection of a first recombinant adenovirus expressing human insulin and a second recombinant adenovirus expressing the appropriate receptor for the factor of choice.
  • CKK cholecystokinin
  • GIP gastric inhibitory peptide
  • cells may be co-infected with a first recombinant virus expressing human insulin and a second recombinant virus expressing a sulfonylurea receptor.
  • Stimulation with amino acids, especially lysine and arginine produces stimulation of insulin secretion following the digestion of foods and the release of amino acids (and glucose) into the system.
  • DNA may b e introduced into the cell by any suitable vector which is known to those of skill in the art of genetic engineering.
  • suitable vectors for example, and methods for their construction, are known to those of skill in the art. Mol. Med. Today, 1(9): 410-417 ( 1995) . Ferrari, S., Gene Ther. 4(10): 1 100- 1 106 (1997). Gorman, CM., Gene Ther. 4(9): 983-992 (1997).
  • human glucagon-like peptide- 1 receptor cDNA GenBank accession no. U10037, Wei and Mojsov, FEBS Letters 385 :219-224 (1995) is used to construct a recombinant adenovirus that expresses human glucagon-like peptide- 1 receptor.
  • Methods for construction of recombinant adenovirus are well known to those of skill in the relevant art. A basic method is described in Graham FL, Prevec L. "Manipulation of adenovirus vectors.” In: Murray EJ (Ed). Methods in Molecular Biology, Vol. 7: Gene Transfer and Expression Protocols. The Human Press Inc.: Clifton, NJ, 1991 , p. 109-127. Production of recombinant adenovirus is also described
  • human GLP-1 receptor DNA used to construct recombinant adenovirus was supplied as a n insert in the pcDNA3 vector.
  • the glucagon-like peptide- 1 cDNA construct supplied by Svetlana Mojsov of The Rockefeller University, consisted of glucagon-like peptide- 1 cDNA inserted into the pcDNA3 vector at the EcoRl site, with the 5'-end of th e glucagon-like peptide- 1 receptor cDNA next to the T7 promoter of the pcDNA3 vector.
  • AdhlNS a recombinant adenovirus expressing th e human insulin cDNA under the control of the CMV promoter
  • the recombinant virus expressing a human insulin cDNA and th e recombinant virus expressing the receptor for a non-glucose secretagogue is a retrovirus or other virus appropriate as a vector for gene transfer. Techniques for production of these recombinant viruses and infection of the cells to produce protein expression are known to those of skill in the art.
  • cells are co-infected with a first recombinant adenovirus
  • recombinant adenovirus co-infection of cells results in th e expression of glucagon-like peptide- 1 receptor on the cell surface.
  • Stimulation with glucagon-like peptide- 1 (7-36) amide, a derivative of glucagon-like peptide 1 (GLP-1) stimulates the co- secretion of both insulin and pituitary hormones in these co- infected cells.
  • GLP-1 is normally released from the intestine in the postprandial state.
  • Stimulation of insulin and pituitary hormone secretion by GLP-1 occurs in a dose-dependent fashion, starting at 30 pM and reaching a maximum at 3 nM.
  • the effect of glucagon-like peptide- 1 in pituitary cells, in contrast to beta cells, is independent of the extracellular glucose concentration.
  • cells are removed from an individual, co-infected with a first recombinant adenovirus expressing GLP-1 and a second recombinant adenovirus expressing human insulin, and transplanted back into the same individual, where glucagon-like peptide 1 released from the intestine in the postprandial state stimulates the secretion of insulin from the transplanted cells in a dose-dependent fashion.
  • cells are removed from an individual, co-infected with a first recombinant virus expressing human insulin and a second recombinant virus expressing the receptor for another non- glucose secretagogue, such as the sulfonylurea receptor, an d transplanted back into the same individual, where the non-glucose secretagogue released following ingestion of food stimulates th e production and secretion of insulin in the transplanted cell.
  • a first recombinant virus expressing human insulin
  • a second recombinant virus expressing the receptor for another non- glucose secretagogue, such as the sulfonylurea receptor
  • DNA encoding the appropriate receptor (GLP-1 , sulfonylurea, GIP, for example) and DNA encoding the insulin gene may b e introduced into cells in vivo or in situ. Methods for in vivo gene delivery are known to those of skill in the art of gene therapy . Oudrhiri, N., Proc. Nat'l Acad. Sci. USA 94: 1651-1656 (1997).
  • Rat pituitary cells are isolated by collagenase digestion of the pituitary.
  • Rat adrenomedullary cells are isolated b y dissecting the medulla from the cortex following collagenase digestion. Islets are isolated and for fractionation into different pituitary cell types, biotinylated hypothalamic releasing hormones and a commercial electromagnetic microbead system (MAC cell sorting system from Miltenyi Biotec). Originally optimized to separate lymphocyte subsets, the electromagnetic microbead system allows cells to be separated on the basis of a biotinylated protein, which binds the surface of the cell of interest (via a n antibody or ligand). The biotinylated releasing hormones are
  • Cells can be either positively or negatively selected and we have utilized this system to isolate pituitary cell type (under sterile conditions). The purity of the cell preparation is tested by immunocytochemistry for each pituitary hormone and by cell perifusion and stimulation with releasing hormones that were not used to select the cell population. Cell Perifusion is performed in neuroendocrine cells and islet cells a s previously reported.
  • membranes are photolabeled with 10 nM ⁇ I-azido-glibenclamide, solubilized in
  • °°Rb efflux are performed as reported. Neuroendocrine cells are incubated with °"RbCl for 12-24 hours, rinsed, and then
  • glibenclamide 26 incubated in media, media with oligomycin and 2 deoxy-glucose (metabolically inhibited), or media with oligomycin and 2 deoxy- glucose plus 1 ⁇ M glibenclamide (K ⁇ y/p channels inhibited). At designated time points, aliquots of media are removed for counting. The % glibenclamide-inhibitable efflux are calculated a s the difference between %°°Rb efflux-metabolically inhibited and
  • Patch clamping are performed as previously reported .
  • Single cell recordings are obtained from neuroendocrine cells in the inside-out patch orientation.
  • Current-voltage relationships are constructed by measuring single channel current amplitudes a t different membrane potentials as previously reported.
  • ATP-dose response cures (concentrations of 1-1000 ⁇ M) will be obtained for each inside-out patch with the current measured as a fraction of the mean current obtained in a control solution of 1 ⁇ M ATP.
  • recombinant adenovirus or AAV vector which expresses GIP Receptor, SURl and Kir 6.2, and adenoviruses which bicistronically express the transgene and green fluorescent protein (GFP)
  • recombinant adenoviruses that express islet proteins of interest are created using homologous recombination.
  • AAV vectors are created using established methods .
  • adenoviruses have been created and utilized to introduce the cDNAs for a variety of proteins into primary rat pituitary cells and adrenomedullary cells.
  • Adenoviruses infect a wide number of cell types with high efficiency and adenoviral infection usually does not interfere with
  • Table 1 summarizes information from multiple laboratories concerning expression of key "islet” proteins in pituitary and adrenal tissue.
  • Sulfonylurea receptor expressed in normal rat pituitary influences hormone secretion
  • Isolated pituitary cells actually represent 5 distinct neuroendocrine cell types that secrete different hormones (ACTH, GH, TSH, prolactin, and LH/FSH) and can be stimulated with their natural secretogogues (CRH, GHRH, TRH, somatostatin, and GnRH).
  • Glibenclamide (1 nM) increases growth hormone secretion b y normal rat pituitary cells ( Figure 3).
  • Co-expression of the GLP-1 receptor and insulin in pituitary cells endows these cells with capacity to secrete insulin at physiologic levels of GLP-1
  • a recombinant adenovirus that expresses the human glucagon-like peptide-I receptor has been created and expres sed in rat pituitary cells (the human cDNA was from Dr. Svetlana Mojsov, Rockefeller Institute).
  • the human glucagon-like peptide-I receptor has been co-expressed with the human insulin cDNA b y infecting cells with both recombinant adenoviruses simultaneously.
  • glucagon-like peptide-I stimulates insulin secretion (indicating that a single cell has been infected by both adenoviruses) and secretion of the endogenous hormone (growth hormone, prolactin, ACTH, and TSH) ( Figures 4, 5, 6).
  • growth hormone, prolactin, ACTH, and TSH the endogenous hormone
  • Figures 4, 5, 6 This demonstrates that the glucagon-like peptide- 1 receptor in pituitary cells has a similar dose response as the islet glucagon-like peptide- 1 receptor and that the remainder of the glucagon-like peptide- 1 signal transduction pathway is present in all pituitary cell phenotypes.
  • the hypothalamic releasing hormones stimulate the release of their respective pituitary hormone and also human insulin ( Figures 4, 5, 6).
  • GLP-1 stimulated hormone secretion from normal rat pituitary cells is not dependent on the extracellular glucose concentration
  • Figure 7 A shows GH secretion and Figure 7B shows insulin secretion in the same sample of the perifusate. Wild type pituitary cells, cells expressing only the GLP-1 receptor, and cells expressing both the GLP-1 receptor and human insulin (H.insulin/GLP-1 R) are shown.
  • GLUT2 AdhGLUT2
  • AdhGLUT3 human GLUT3
  • Figures 7 and 8 demonstrate the presence and function of human GLUT proteins.
  • An adenovirus expressing islet glucokinase (AdhGK) expresses a protein that has the expected properties for glucokinase-i.e. its glucose phosphorylating activity is not inhibited by glucose 6 - phosphate and its enzymatic activity is maximal at 4-10 m M glucose concentrations (Figure 10).
  • Two transgenes can be co-expressed in rat pituitary cells b y adenovirus-mediated gene transfer
  • FIG. 11 shows that insulin and GK can be co- expressed while Figure 11B shows the co-expression of GLUT2 an d GK in RIN cells.
  • Figure 5 demonstrates that this approach is also capable of co-expressing insulin and the GLP- 1 receptor.
  • Figure 11A also demonstrates that pituitary cells can synthesize GK.
  • Pituitary cells are known to express GK mRNA, but not GK protein, -presumably as the result of alternative RNA splicing. The GK adenovirus used the GK cDNA and thus circumvented this problem.
  • two different transgenes can be expressed in a single pituitary cell. Insulin and glucokinase can be co-expressed. Rat pituitary cells were simultaneously infected with AdhlNS (expressing the human insulin cDNA under the control of the CMV promoter) and adenovirus expressing islet glucokinase (AdhGK). The infected cells were then stained with an antiserum against insulin, against glucokinase, or both and analyzed by confocal microscopy. Co- expression of GLUT2 and glucokinase in RIN cells was shown. RIN 1046-38 (rat islet cell line) cells were simultaneously infected with AdhGLUT2 (expressing the human GLUT2 cDNA under th e control of the CMV promoter) and adenovirus expressing islet
  • glucokinase (AdhGK).
  • the infected cells were then stained with a n antiserum specific for human GLUT2 (which does not cross-react with rat GLUT2), with antiserum specific for glucokinase or both and analyzed by confocal microscopy.
  • GLUT2 staining was cell- surface and glucokinase staining was cytoplasmic.
  • Pituitary cells can synthesize glucokinase.
  • Pituitary cells are known to express glucokinase mRNA, but not glucokinase protein—presumably a s the result of alternative RNA splicing.
  • the recombinant adenovirus used the glucokinase cDNA and thus circumvented this problem.
  • Transplanted primary neuroendocrine cells (pituitary cells and adrenomedullary cells') modified to secrete insulin
  • the secretion of GLP-1 and GIP in response to oral glucose is primarily responsible for the greater insulin secretion stimulated by oral compared to intravenous glucose. Since GLP-1 and GIP are physiologic stimuli of insulin secretion, this experiments shows that: 1) primary neuroendocrine cells, engineered to secrete human insulin in response to GLP-1 in vitro, secretes insulin in vivo in response to GLP-1 or GIP released b y oral glucose; and 2) GLP-1 and GIP-induced insulin secretion b y the engineered neuroendocrine cells impacts glucose homeostasis in normal physiology and in experimental diabetes.
  • the insulin- secreting neuroendocrine cells will be transplanted into the liver or beneath the kidney capsule of normal rats to examine their ability to secrete human insulin in response to GLP-I in vivo.
  • catheters are implanted using microsurgical techniques into the portal vein, carotid artery , jugular vein, and duodenum of normal Sprague-Dawley rats. The catheters are tunneled subcutaneously so that they can b e accessed 1-2 weeks later while the animal is conscious and unrestrained. This is a standard surgical procedure. Modified neuroendocrine cells are infused into the portal vein and allowed to embolize into the liver as has been reported for pancreatic islets.
  • modified neuroendocrine cells will b e transplanted beneath the renal capsule.
  • Transplantation of insulin-secreting neuroendocrine cells into the liver will expose cells to maximal levels of endogenous GLP-I (released from the GI tract into the portal circulation), but the renal capsule has been widely used as a transplantation site and would exposed to th e same level of GLP-1 as islets in their native pancreatic location.
  • oral glucose tolerance testing will b e performed weekly using the surgically-implanted duodenal catheter. As an example, the results of insulin secretion in a normal rat or mouse given an duodenal glucose load is shown.
  • an unanesthetized, unrestrained rats are given a duodenal glucose load and samples were taken from the jugular catheter every 15-30 minutes. Samples are assayed for glucose or rat insulin.
  • Initial experiments establish optimal transplantation parameters. The optimal number of neuroendocrine cells, th e effect of such cells on animal health (growth, weight), the length of function and survival of the transplanted cells (using serial oral glucose tolerance testing and infusion of releasing hormones directly into the portal vein) is determined. Oral glucose tolerance testing is compared to intravenous glucose tolerance testing (using the carotid and jugular catheters).
  • Immunocytochemical analysis of hepatic or renal histologic sections is utilized to monitor neuroendocrine cell survival and expression of the insulin and GLP-I receptor transgenes.
  • Controls include wild type neuroendocrine cells, neuroendocrine cells which have been infected with an adenovirus expressing lac Z, and neuroendocrine cells infected with the insulin adenovirus alone.
  • Adenovirus and some herpes viral vectors are examples of th e former, while adeno-associated virus (AAV), lentivirus, and HIV are types of defective viral vectors.
  • AAV adeno-associated virus
  • lentivirus lentivirus
  • HIV HIV
  • defective viral vectors do not have native viral genes and require that these viral genes be provided by the cell line in which the defective viral vector is produced.
  • GLP-1 stimulated insulin secretion from neuroendocrine cells is independent of th e glucose concentration (in contrast to GLP-1 stimulated insulin secretion from beta cells) and this raises the possibility of insulin secretion at sub-physiologic glucose levels.
  • One experimental approach (response to non-glucose stimuli and response a t different in vivo concentrations) examines whether this occurs in animals transplanted with the engineered neuroendocrine cells.
  • Transplanted primary neuroendocrine cells pituitary cells and adrenomedullary cells modified to secrete insulin in response to GIP
  • Rat pituitary cells and adrenal medulla do not express the GIP receptor nor respond to GIP in a cell perifusion system.
  • the human GIP receptor is co-expressed with human insulin in primary neuroendocrine cells. After GIP-regulated insulin secretion is demonstrated in the cell perifusion system, these cells are transplanted into the liver or beneath the renal capsule and the ability of oral glucose to regulate insulin secretion will be examined.
  • cells are engineered to express both the GLP-1 and GIP receptor to determine whether the responses to GLP-I and GIP are additive.
  • Recombinant adenovirus-mediated gene transfer o r AAV-mediated gene transfer is utilized.
  • An alternative approach is to use a single recombinant adenovirus that encodes both GLUT2 and GK (either using a separate promoter and 3' untranslated region for each transgene or a bicistronic mRNA under the control of a single promoter) (257-259).
  • Viruses w ere created with a separate promoter and 3' untranslated region for each transgene and this will be used to used to infect pituitary cells and adrenomedullary cells. In this way, one can be certain that each cell is infected with the same virus and that the level of expression will be relatively uniform in all cells infected.
  • RXR retinoid X receptor
  • the insect hormone ecdysone does not bind nuclear receptors from mammalian systems, but in concert with a modified nuclear receptor, regulates transcription through a hybrid ecdysone response element.
  • both the hybrid nuclear receptor and the promoter controlling the gene of interest must be expressed in the same cell.
  • This system is transcriptionally-inactive in mammalian cells in the basal state, but in the presence of ecdysone or an ecdysone analogue (ponasterone A) transcription can increase up to 1 000 fold. This will allow the effect of different levels of islet protein expression on hormone secretion and intracellular events in glucose stimulatory pathway to be examined.
  • a similar approach using tetracycline-regulated expression of glucokinase has been used by Iynedjian and co-workers. In fact, by combining th e
  • a recombinant adenovirus may be created using one of two two adenoviruses-one of which carries the VP16-RXR receptor and another one which carries the regulatable promoter and the gene of interest. Conversely, one could create one recombinant adenovirus to carry the cDNA's for both the nuclear receptor and the promoter. In this approach, the expression of th e VP16-RXR receptor would be controlled by a constitutive promoter/enhancer and the regulated promoter/enhancer would control the expression of the gene of interest. The capacity of th e recombinant adenovirus is sufficient to carry the cDNA for VP 16- RXR, the regulated promoter/enhancer, and the gene of interest.
  • GK or GK with a glucose transporter endows neuroendocrine cells with glucose-regulated insulin secretion
  • the islet form of GK alone, GK and GLUT2, or GK an d GLUT3 is expressed in primary rat pituitary cells o r adrenomedullary cells using adenovirus-mediated gene transfer and the ability of changes in the extracellular glucose (2.5 mM to 25 mM) to alter hormone secretion examined. Since human insulin expressed in neuroendocrine cells is co-secreted with th e endogenous pituitary hormones or catecholamines, the endogenous pituitary hormones or neurotransmitters initially serve as the marker of hormone secretion. Neither neuroendocrine cell type alters the secretion of their native
  • adrenomedullary cells will be stimulated with acetylcholine ( 50 ⁇ M) or l , l -dimethyl-4-phenylpiperazinium (3-50 ⁇ M) (252) .
  • Neuroendocrine cell secretion profiles will be compared to insulin secretion of isolated pancreatic islets and dispersed islet cells when stimulated with increased extracellular glucose (2.5 mM to 25 mM) as previously reported (272), in wild-type pituitary cells or adrenomedullary cells, and in pituitary cells o r adrenomedullary cells infected with a control adenovirus (AdLacZ).

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Abstract

L'invention porte sur une cellule obtenue par génie génétique comportant un gène codant pour un récepteur non glucosique sécrétagogue d'insuline et sur un gène d'insuline, l'un au moins de ces gènes étant un gène de recombinaison, et la cellule sécrétant de l'insuline en réponse au glucose lorsque l'un au moins desdits gènes a été introduit dans la cellule à l'aide d'un vecteur de recombinaison. L'invention porte également sur un procédé de production d'insuline consistant: (a) à cultiver la cellule de la revendication 1; (b) à stimuler ladite cellule pour qu'elle secrète de l'insuline; puis (c) à recueillir l'insuline ainsi produite.
PCT/US1999/008628 1998-04-20 1999-04-20 Cellules neuroendocrines secretrice d'insuline et ses utilisations WO1999054451A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5744327A (en) * 1990-02-20 1998-04-28 Board Of Regents, The University Of Texas System Methods for producing insulin in response to non-glucose secretagogues
US5747325A (en) * 1991-06-03 1998-05-05 Board Of Regents, The University Of Texas System Devices comprising genetically engineered βcells

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5744327A (en) * 1990-02-20 1998-04-28 Board Of Regents, The University Of Texas System Methods for producing insulin in response to non-glucose secretagogues
US5747325A (en) * 1991-06-03 1998-05-05 Board Of Regents, The University Of Texas System Devices comprising genetically engineered βcells

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
HUGHES S D, ET AL.: "ENGINEERING OF GLUCOSE-STIMULATED INSULIN SECRETION AND BIOSYNTHESIS IN NON-ISLET CELLS", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, NATIONAL ACADEMY OF SCIENCES, US, vol. 89, 1 January 1992 (1992-01-01), US, pages 688 - 692, XP002921739, ISSN: 0027-8424, DOI: 10.1073/pnas.89.2.688 *
IRMINGER J C, ET AL.: "HUMAN PROINSULIN CONVERSION IN THE REGULATED AND THE CONSTITUTIVE PATHWAYS OF TRANSFECTED ATT20 CELLS", JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY, US, vol. 269, no. 03, 1 January 1994 (1994-01-01), US, pages 1756 - 1762, XP002921743, ISSN: 0021-9258 *
OKA Y, ET AL.: "EFFECTS OF LONG-TERM INCUBATION WITH NON-GLUCOSE INSULIN SECRETAGOGUE ON GLUCOSE-INDUCED INSULIN SECRETION AND EXPRESSION OFGLUCOSE TRANSPORTER AND GLUCOKINAS IN INSULIN-SECRETING CELL LINE, MIN6 CELLS", DIABETES, AMERICAN DIABETES ASSOCIATION, US, vol. 43, 1 May 1994 (1994-05-01), US, pages 179A, XP002921740, ISSN: 0012-1797 *
PASMANTIER R, ET AL.: "P19, A HORMONALLY REGULATED PHOSPHOPROTEIN OF PEPTIDE HORMONE-PRODUCING CELLS: SECRETAGOGUE-INDUCED PHOSPHORYLATION IN ATT-20 MOUSE PITUITARY TUMOR CELLS AND IN RAT AND HAMSTER INSULINOMA CELLS", ENDOCRINOLOGY, THE ENDOCRINE SOCIETY, US, vol. 119, no. 03, 1 September 1986 (1986-09-01), US, pages 1229 - 1238, XP002921741, ISSN: 0013-7227 *
SCHMIDT W K, MORRE H-P H: "SYNTHESIS AND TARGETING OF INSULIN-LIKE GROWTH FACTOR-I TO THE HORMONE STORAGE GRANULES IN AN ENDOCRINE CELL LINE", JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY, US, vol. 269, no. 43, 1 October 1994 (1994-10-01), US, pages 27115 - 27124, XP002921742, ISSN: 0021-9258 *

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