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  • C12N15/09—Recombinant DNA-technology
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  • C12N2710/10011—Adenoviridae
  • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
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  • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination

Definitions

• the present invention relates generally to the preparation, culture and use of engineered cells having the ability to secrete insulin in response to glucose, to gene transfer protocols, to methods for the detection of diabetes-associated antigens, and to methods employing engineered cells in the production of human insulin.
• the present invention relates to recombinant adenoviruses containing genes involved in glucose sensing, such as GLUT-2, the gene encoding glucokinase, and antisense versions of the hexokinase gene, and to the use of adenovirus-mediated gene therapy to direct the expression of recombinant genes in pancreatic islet cells, islet-derived cells or other neuroendocrine cell lines capable of insulin secretion.
• IDDM Insulin-dependent diabetes mellitus
• NIDDM non-insulin dependent diabetes
• IDDM involves specific destruction of the insulin producing ⁇ cells of the islets of Langerhans in the pancreas.
• the destruction of ⁇ cells in IDDM appears to be a result of specific autoimmune attack, in which the patient's own immune system recognizes and destroys the ⁇ cells, but not the surrounding ⁇ (glucagon producing) or ⁇ (somatostatin producing) cells that comprise the islet.
• IDDM islet ⁇ cell destruction is ultimately the result of cellular mechanisms, in which "killer T-cells” destroy ⁇ cells which are erroneously perceived as foreign or harmful.
• the humoral component of the immune system comprised of the antibody-producing ⁇ cells, is also inappropriately active in IDDM patients, who have serum antibodies against various ⁇ cell proteins. Antibodies directed against intracellular proteins probably arise as a consequence of ⁇ cell damage which releases proteins previously "unseen” by the immune system.
• a progressive loss of ⁇ cell function is observed in the early stages of NIDDM and IDDM, even prior to the autoimmune ⁇ cell destruction in IDDM.
• the specific function of glucose-stimulated insulin release is lost in islets of diabetic patients, despite the fact that such islets continue to respond to non-glucose secretagogues such as amino acids and isoproterenol (Srikanta et al . , 1983) .
• pancreatic islets of Langerhans in fuel homeostasis is mediated in large part by their ability to respond to changes in circulating levels of key metabolic fuels by secreting peptide hormones. Accordingly, insulin secretion from islet ⁇ cells is stimulated by amino acids, three-carbon sugars such as glyceraldehyde, and most prominently, by glucose.
• the capacity of normal islet ⁇ cells to "sense" a rise in blood glucose concentration, and to respond to elevated levels of glucose (as occurs following ingestion of a carbohydrate containing meal) by secreting insulin is critical to control of blood glucose levels. Increased insulin secretion in response to a glucose load prevents chronic hyperglycemia in normal individuals by stimulating glucose uptake into peripheral tissues, particularly muscle and adipose tissue.
• Mature insulin consists of two polypeptide chains, A and B, joined in a specific manner.
• the initial protein product of the insulin gene in ⁇ cells is not insulin, but preproinsulin.
• This precursor differs from mature insulin in two ways. Firstly, it has a so-called N-terminal "signal" or "pre” sequence which directs the polypeptide to the rough endoplasmic reticulu , where it is proteolytically processed.
• the product, proinsulin still contains an additional connecting peptide between the A and B chains, known as the C-peptide, which permits correct folding of the whole molecule.
• Proinsulin is then transported to the Golgi apparatus, where enzymatic removal of the C-peptide begins. The processing is completed in the so-called secretory granules, which bud off from the Golgi, travel to, and fuse with, the plasma membrane thus releasing the mature hormone.
• Glucose stimulates de novo insulin biosynthesis by increasing transcription, mRNA stability, translation, and protein processing. Glucose also rapidly stimulates the release of pre-stored insulin. While glucose and non-glucose secretagogues may ultimately work through a final common pathway involving alterations in K + and Ca ++ channel activity and increases in intracellular Ca ++ (Prentki et al . , 1987; Turk et al . , 1987), the biochemical events leading from changes in the levels of a particular fuel to insulin secretion are initially diverse.
• GLUT-2 glucose phosphorylating enzyme
• glucokinase glucose phosphorylating enzyme
• Both proteins are members of gene families; GLUT-2 is unique among the five-member family of glucose transporter proteins in that it has a distinctly higher Km and V ax for glucose.
• Glucokinase is the high Km and high Vmax counterpart of GLUT-2 among the family of hexokinases (Weinhouse, 1976) .
• both proteins have affinities for glucose that allow dramatic changes in their activities over the physiological range of glucose.
• RINm5F clonal insulinoma cells derived from islet ⁇ cells express GLUT-1, a transporter with a substantially lower Km and Vmax for glucose, as their predominant glucose transporter instead of GLUT-2. This may explain the finding that the clonal cells fail to respond to glucose as an insulin secretagogue (Thorens et al . , 1988).
• ICA-cyt cytoplasmic islet cell antibodies
• Gleichmann et al . , 1987 is a diagnostic procedure for detecting the onset of diabetes, which involves binding of patients' antibodies to cryostat sections of fresh human or primate pancreas.
• One evident disadvantage of this is the requirement for fresh human or primate tissue.
• Further difficulties are: false negatives (40%) ; subjective interpretation; poor reproducibility; and the inability to detect cell surface-directed antibodies which are known to specifically damage ⁇ cells (Doberson et al . , 1980) .
• Recombinant methods generally include the expression of recombinant proinsulin in bacteria or yeast, followed by chemical treatment of the proinsulin to ensure correct disulfide bond linkages between the A and B chains of the mature insulin molecule.
• the proinsulin produced by microorganisms is processed to insulin by the addition of proteolytic enzymes. Thereafter, the mature insulin peptide must be purified away from the bacterial or yeast proteins, as well from the added proteases.
• the bacterial procedure involves 40 distinct steps.
• the non-recombinant methods typically include the purification of pig insulin from freshly isolated porcine pancreas or pancreatic islets.
• Each of the above methods suffer from the drawbacks of being technically difficult and laborious.
• the latter method is further complicated by the fact that the pancreas is a complex proteinaceous tissue with high levels of active proteases that can degrade insulin and render it inactive as a hormone.
• the present invention is intended to address such disadvantages present in the prior art.
• the invention is based on the discovery that recombinant DNA technology and cell culture methods may be employed to engineer an "artificial ⁇ cell" that secretes insulin in response to glucose.
• the present invention provides a means of preparing artificial ⁇ cells that it is proposed can be employed in a variety of applications, such as, e.g., in the detection of diabetes-associated antigens, in the clinical treatment of IDDM and even in the large- scale production of correctly-folded insulin.
• the current invention provides methods for growing artificial ⁇ cells in liquid culture on gelatin beads and for the increased production of human insulin by perfusion of such recombinant cells with glucose- containing buffers.
• this aspect of the invention relates generally to an engineered cell that incudes a gene, preferably a recombinant gene, encoding a functional glucose transporter protein, wherein the engineered cells secrete insulin in response to glucose.
• This aspect of the invention is based generally on the finding that where a cell is competent to secrete insulin generally, it may be converted to a glucose-responsive cell through the introduction of a gene encoding a functional glucose transporter protein, such as a GLUT gene.
• GLUT-2 For most purposes leading up to the ultimate treatment of the diabetic condition, one will desire to employ GLUT-2 as the recombinant glucose transporter gene. This is because the GLUT-2 gene corresponds to that found and normally expressed in ⁇ cells, and it is believed that this gene will ultimately provide a more physiological response than other types of glucose transporters.
• recombinant cell or even “recombinant host cell” is intended to refer to a cell into which a recombinant gene, such as a gene encoding a functional glucose transporter protein, 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, a gene or genes positioned within a recombinant adenovirus, genes produced by synthetic means, and/or 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, the invention does not exclude the possibility of employing a genomic version of a particular gene in any of the embodiments disclosed herein.
• Recombinant host 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 by budding of intracellular membranous structures known as the Golgi apparatus. Polypeptide hormones are usually synthesized as prohormones and undergo proteolytic processing to yield the shorter, mature version of the hormone.
• the initial protein product of the insulin gene in ⁇ 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. It also has a connecting peptide between the A and B chains that comprise the mature insulin molecule; this connector is known as 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 the 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 envelopes 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 and release their stores of the mature hormone.
• the important and unique features of this system are 1) the secretory granules allow a supply of a particular hormone to be built up and stored for release at the time when it is needed to perform its function and 2) the presence of processing enzymes in the granules allow efficient conversion of the precursor forms of hormones to the mature forms. Cells that lack secretory granules will thus likely not be useful for the purposes of this aspect of the invention.
• 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.
• Particularly preferred endocrine cells will be AtT-20 cells, which are derived from ACTH secreting cells of the anterior pituitary gland, GH1 or the closely related GH3 cells, which are derived from growth hormone producing cells of the anterior pituitary, or other cell lines derived from this gland. AtT-20 cells are preferred for the following reasons.
• AtT-20 ins both the parental AtT-20 cell line and the insulin expressing AtT- 20 ⁇ cell line are available from American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852
• AtT-20 jns cells are able to process the preproinsulin mRNA and protein to yield the correctly processed insulin polypeptide.
• their insulin secretory response to analogues of cAMP compares favorably with the well-differentiated hamster insulino a (HIT) cell line which is derived from hamster islet ⁇ cells.
• HIT well-differentiated hamster insulino a
• AtT-20 ins cells contain significant amounts of the islet isoform of glucokinase, making this the only tissue other than liver or islets in which glucokinase gene expression has been reported (Hughes et al . , 1991).
• GH1 and GH3 cells were originally derived from the same batch of cells isolated from a rat pituitary gland tumor.
• GH3 cells differ from GH1 cells in that they secrete more growth hormone and also secrete prolactin (both lines are available from the American Type Culture Collection) .
• These cells are believed to be preferred for the practice of the invention because it has been shown that introduction of a recombinant preprosomatostatin gene into these cells results in secretion of the mature somatostatin peptide (Stoller et al . , 1989). Processing of the endogenous preprosomatostatin gene also occurs in 5-cells of the islets of Langerhans. The finding that an islet hormone precursor can be correctly processed in growth hormone secreting cells of the anterior pituitary suggests that proinsulin processing will also occur in these cells, perhaps even more efficiently than in AtT-20 ins cells.
• a number of cell lines derived from ⁇ cells are also preferred for the practice of this invention and are readily available, particularly as concerns the therapeutic aspects of the work.
• hamster insulinoma (HIT-T15) cells are well studied and are readily available from the American Type Tissue Collection.
• a number of rat insulinoma cell lines are also available.
• the RINm5F and RINrl046-38 cell lines were derived from a radiation induced tumor of the islet ⁇ cells (Gazdar et al . , 1980; Clark et al . , 1990).
• MSL-G2 cells were derived from a liver metastasis of an islet cell tumor.
• the 0-TC insulinoma cell line has been recently derived from transgenic mice injected with a T-antigen gene driven by an insulin promoter, resulting in specific expression of T-antigen in islet ⁇ cells and consequent immortalization of these cells (Efrat et al. , 1988).
• RIN 1046-38 cells have been shown in one of the inventors' laboratory to express both GLUT-2 and glucokinase (Hughes et al . , 1991), and have been shown by Clark et al. (1990) to be responsive to glucose. Glucose stimulation of insulin release from these cells is maximal at 0.5 mM glucose, however, a level far below the stimulatory concentration of glucose required for insulin release from normal ⁇ cells. Recent studies in the inventor's laboratory have shown that this hypersensitivity to glucose in RIN 1046-38 cells may be due to high levels of hexokinase activity.
• Hexokinase performs the same function as glucokinase (glucose phosphorylation) but does so at much lower glucose concentrations (hexokinase has a Km for glucose of approximately 0.05 mM versus 8 mM for glucokinase). It is proposed that lowering of hexokinase activity by methods of recombinant DNA technology described below might make RIN cells useful for the practice of this invention.
• 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.
• the invention proposes that 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, a glucose transporter gene and a glucokinase gene. In the practice of the invention, one or more of these genes will be a recombinant gene.
• the starting cell has a functional insulin gene and a functional glucokinase gene, and these genes are expressed at levels similar to their expression in ⁇ cells, but the cell does not have a functional glucose transporter gene, introduction of a recombinant glucose transporter gene will be required.
• the starting cell expresses none of the aforementioned genes in a functional fashion, or at physiologic levels, it will be necessary to introduce all three. Since recombinant versions of all three categories of genes are available to the art, and the specific technology for introducing such genes into cells is generally known, the construction of such cells will be well within the skill of the art in light of the specific disclosure herein.
• particularly preferred endocrine cells for use in accordance with the present invention are AtT-20 ins cells, which have been stably transfected to allow the production of correctly processed human insulin.
• Engineered cells that combine both of these features have been created, and one form of cell expressing high levels of GLUT-2 mRNA, termed CTG-6 cells, are envisioned to be of particular use in aspects of the present invention.
• constitutive promoters are generally viral in origin, and include the cytomegalovirus (CMV) promoter, the Rous sarcoma long- terminal repeat (LTR) sequence, and the SV4O 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 transfection event. Once that line is chosen, 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 a specific cell type, such as the insulin promoter in insulinoma cell lines, or the prolactin, proopiomelanocortin (POMC) or growth hormone promoters in anterior pituitary cell lines.
• POMC prolactin, proopiomelanocortin
• Certain particular embodiments of the invention are directed to engineering cells with reduced hexokinase activity relative to the parent cell line.
• Hexokinases I, II, and III have very low K s (high affinities) for glucose, on the order of 0.05 mM.
• Hexokinase IV is glucokinase, which has a high Km for glucose of around 8-10 mM.
• glucokinase is the predominant glucose phosphorylating enzyme, while in most clonal cell lines grown in culture, the low Km hexokinase I isoform predominates.
• the inventor proposes that expression of hexokinases other than glucokinase at high levels in clonal cells used for engineering will tend to make the cell glucose-responsive in terms of insulin release at lower concentrations of glucose than is desirable. Thus, it is proposed that the lower the hexokinase/glucokinase ratio, the more physiologic the insulin response.
• RNA molecules may be introduced into engineered cells.
• Antisense RNA technology is now fairly well established, and involves the juxtaposition of the targeted gene in a reverse orientation behind a suitable promoter, such that an "antisense" RNA molecule is produced. More specifically, a segment of DNA encoding the protein to be inhibited is oriented in the antisense orientation relative to its controlling promoter and then expressed from that promoter. This results in the production of mRNA which is complementary to the mRNA that encodes the active protein.
• This "antisense” construct is then introduced into the engineered cell and, upon its expression, produces an mRNA molecule that will hybridize with, and prevent the processing/translation of mRNA produced by the targeted gene, in this case the hexokinase gene.
• An alternative approach to the reduction of hexokinase action is through a technique known as positive/negative selection. This technique involves selection for homologous recombination of a hexokinase gene segment that renders the endogenous hexokinase gene nonfunctional.
• the present invention is directed to a method of providing glucose-responsive insulin secreting capability to a cell.
• This method comprises obtaining an insulin producing cell and expressing a GLUT-2 and/or a glucokinase enzyme in the cell.
• a preferred cell is an islet ⁇ cell.
• the method may further comprise inhibiting the hexokinase activity in the cell.
• a preferred method of expressing the GLUT-2 and/or glucokinase activity is by transfecting the cell with an adenovirus vector expressing the protein(s) and a preferred method of inhibiting the hexokinase activity is by expressing an antisense copy of the hexokinase gene or a fragment thereof.
• an antisense fragment of less than the full length gene will inhibit expression of a gene. Therefore, as used herein a "fragment thereof" is taken to mean any contiguous stretch of bases from about 20 bases up to the full length encoding region and even including the upstream promoter region of the gene.
• Particularly preferred fragments would include as a part of the sequence, the ATG start site of the coding region of the gene.
• the present invention is directed to a method of providing a glucose-responsive insulin-secreting capability to a mammal in need of such capability.
• the method includes generally implanting engineered cells which secrete insulin in response to glucose as described in the previous paragraph into such a mammal. It is proposed that 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. In 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.
• 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 (Altman et al . , 1986).
• the cells After successful encapsulation or fiber seeding, the cells, generally approximately 1,000-10,000, may be implanted intraperitoneally, usually by injection into the peritoneal cavity through a large gauge needle (23 gauge) .
• compositions and methods related to alternative strategies by which to provide glucose- responsive insulin-secreting capability to animals or humans with non-insulin dependent diabetes (NIDDM) or other NIDDM-like syndromes are directed to restoring ⁇ cell function in such animals or patients by the application of adenovirus-mediated gene transfer, thus allowing physiological control of glucose homeostasis.
• NIDDM non-insulin dependent diabetes
• the invention generally concerns adenovirus vector constructs which comprise a recombinant insert including an expression region encoding at least one protein involved in the predisposition to diabetes, and particularly, those proteins involved in glucose sensing, such as GLUT- 2 and glucokinase, which vectors are capable of expressing these proteins in cells infected with a recombinant adenovirus.
• the construct would be administered to the mammal in a pharmacologically acceptable form as discussed below.
• a protein which is "involved in the predisposition to diabetes” is defined as a protein which is involved in insulin responsiveness, or resistance, in peripheral tissues or a protein which is involved in glucose-sensing in the ⁇ cell. Defects, mutations or alterations in such proteins will generally lead to disturbances in insulin action and glucose metabolism and homeostasis, and may lead to disease states such as various forms of diabetes and/or Maturity-Onset Diabetes of the Young (MODY) .
• Proteins which are candidates for involvement in insulin responsiveness in peripheral tissues, and in which mutations thus lead to insulin resistance include the insulin receptor itself and the GLUT-4 glucose transporter.
• other candidate proteins include hexokinase II, glycogen synthase and glycogen phosphorylase; various kinases and phosphatases which act upon the former enzymes; and various components of the insulin signal transduction pathways, such as G proteins, tyrosine kinases, the insulin-regulated substrate-1 (IRS- 1) and the like.
• the present invention is concerned with proteins which are involved in glucose-sensing in the ⁇ cell.
• particular aspects of the invention embody adenovirus vector constructs which comprise a recombinant insert including an expression region under the control of a promoter and including a coding region that encodes at least one glucose transport protein, glucose phosphorylating protein or a fragment of a glucose transport protein or a glucose phosphorylating protein.
• the expression region in the vector may comprise a genomic sequence, but for simplicity, it is contemplated that one will generally prefer to employ a cDNA sequence.
• the recombinant insert of the vector will also generally comprise a promoter region and a polyadenylation signal, such as an SV40 or protamine gene polyadenylation signal.
• the promoter may be a constitutive promoter, or it may be a jS-cell preferential promoter.
• a constitutive promoter or it may be a jS-cell preferential promoter.
• jS-cell preferential control elements such as the promoters from GLUT-2, insulin and glucokinase promoters and methods of use of these promoters for jS-cell preferential expression.
• Preferred proteins for use in such an adenoviral vector include the glucose transporter GLUT-2, the enzyme glucokinase, and the antisense versions of the hexokinase I and GLUT-1 cDNAs or fragments of the full length proteins.
• GLUT-2 glucose transporters of the invention may be of any type including e.g. the HepG2 form as described in Mueckler et al . , 1985, and the more preferred islet cell GLUT-2 glucose transporter described in Permutt et al . , 1989.
• the single glucokinase gene is also known to be alternatively regulated and processed in liver and islets (Iynedjian et al .
• ⁇ cell dysfunction in NIDDM is believed to involve a deficit in the expression of GLUT-2, glucokinase, or both such proteins, or possibly overexpression of GLUT-1 or hexokinase I.
• adenoviral vectors capable of expressing glucose transporters and/or glucose phosphorylating enzymes are particularly preferred, with the islet cell isoforms of each protein being even more preferred where applicable.
• expression units encoding normal forms of one or both of the GLUT-2 and glucokinase enzymes are used herein to refer to a form of a protein, such as an enzyme or glucose transporter, which functions essentially as that form of the protein expressed in individuals which do not have diabetes or a diabetes-associated disorder.
• adenoviral vectors capable of expressing proteins other than normally-functional GLUT-2 and glucokinase are also contemplated.
• adenoviral vectors expressing the glucose transporter GLUT-1 are contemplated to have utility as control' vectors, particularly for use as controls in in vitro or in vivo studies concerning GLUT-2 expression by adenoviral vectors.
• Adenoviral vectors capable of expressing mutant or altered proteins are also encompassed by the present invention.
• Such vectors will have utility in that they will allow the mutant form, such as the glucokinase enzyme associated with maturity onset diabetes of the young (MODY) , a form of NIDDM, to be specifically expressed in a given cell type so that the effects of the mutation may be directly assessed at the molecular level.
• mutant form such as the glucokinase enzyme associated with maturity onset diabetes of the young (MODY)
• MODY maturity onset diabetes of the young
• NIDDM a form of NIDDM
• individuals or rodent models with NIDDM and related syndromes tend to exhibit markedly elevated insulin secretion at glucose concentrations that are substimulatory for normal islets. This dysfunction may be due to overexpression of a high affinity (low Km) glucose metabolizing protein such as GLUT-1 or hexokinase I. It is proposed that this abnormality may be corrected by expression of antisense versions of hexokinase or GLUT-1.
• a promoter that will restrict expression of the gene to /3-cells.
• An example of such a promoter would be the insulin promoter, which is specifically active in / 3-cells.
• expression of the gene of interest in jS-cells and a small subset of other cell types is also preferred.
• Examples of the latter class of promoters include the GLUT-2 promoter, which directs expression in islet /3-cells, liver, and certain cells of the intestine and kidney (Takeda et al . , 1993; Bell et al . f 1990; Thorens et al .
• Specific or preferential targeting of the recombinant genes to islet jS-cells by the indicated techniques is preferred because it should prevent wholesale expression of the genes in all tissues.
• This is advantageous in that inappropriate expression of GLUT- 2 or glucokinase in other tissues such as muscle or fat, for example, tissues which normally express the insulin- regulatable transporter GLUT-4 and the glucose phosphorylating enzyme hexokinase II, might alter the glucose metabolizing properties of these tissues and affect whole-body glucose homeostasis.
• Langerhans are a highly vascularized tissue which also have some direct contact with the blood supply, suggesting that they may also be a preferred target for adenovirus.
• expression of the gene of interest could be driven by any of a host of strong constitutive promoters such as CMV, viral LTR, or SV-40, or alternatively by promoters associated with genes that are expressed at high levels in all cells such as translation elongation factor-1 or actin.
• CMV CMV
• viral LTR viral LTR
• SV-40 promoters associated with genes that are expressed at high levels in all cells such as translation elongation factor-1 or actin.
• Recent studies conducted by the inventors indeed demonstrate that infusion of recombinant adenovirus containing the reporter gene /3-galactosidase demonstrates successful gene transfer into islets in vivo without significant transfer of the gene in the surrounding exocrine tissue.
• adenoviral vectors of the present 1 invention will also have utility in embodiments other than those connected directly with gene therapy.
• Alternative uses include, for example, in vitro analyses and mutagenesis studies of various candidate genes, and the recombinant production of proteins for use, for example, in antibody generation or other embodiments.
• An important use is envisioned to be the introduction of GLUT-2 cDNA into islets isolated from ZDF rats, or other models of ⁇ cell dysfunction, allowing the issue of whether GLUT-2 underexpression is sufficient to explain ablated glucose sensing in these cells to be addressed (Johnson et al . , 1990b) .
• the adenovirus system will allow rapid introduction of genes into such cells.
• current cell lines gain glucose-stimulated insulin secretion upon transfection with GLUT-2, but respond to glucose at subphysiological concentrations of the sugar.
• hexokinase is the predominant glucose phosphorylating enzyme in the cell lines under study. Hexokinase is maximally active at subphysiological glucose concentrations, in contrast to glucokinase, which only becomes active at glucose concentrations within the physiological range.
• Adenovirus-mediated introduction of glucokinase and/or antisense hexokinase cDNAs into cells already engineered for GLUT-2 expression can allow rapid testing of this concept.
• the advantage of adenovirus- mediated gene transfer for the foregoing purpose is that it obviates the need to select clonal cell lines, a laborious and time-consuming procedure.
• the effect of genetic maneuvers on glucose-stimulated insulin secretion can be studied within 24 hours of viral infection, as opposed to cloning of stably transfected lines, which generally requires a month of work.
• a second example concerns a virus containing the cDNA for hexokinase I in antisense orientation (the virus is designated AdCMV-HKIrev) , which was used to treat GLUT-2 transfected, glucose responsive RIN 1046-38 cells.
• Adenovirus-mediated gene transfer may also have utility in the context of cells destined for implantation into animals or humans with IDDM.
• Current information suggests that adenovirus DNA integrates into chromosomal DNA with poor efficiency, meaning that most of the adenovirus genome will reside in the nucleus as extrachromosomal DNA (Van Doren and Gluzman, 1984) .
• Current experience suggests that adenovirus-directed gene expression persists at high levels for 3-4 weeks in vivo when the gene expression is directed by the CMV promoter.
• cells would be engineered for the correct levels of expression of GLUT-2, glucokinase, hexokinase and insulin by one or a combination of the methods described herein, and subsequently, gene expression could be supplemented by direct injection of relevant viruses into the implant. Since the implant is envisioned to be contained in a permselective device or capsule capable of excluding molecules of the immune system, the adenovirus will not exit the device, and gene therapy will therefore be restricted to the engineered cells within the device. In this way, relatively constant levels of expression of the genes of interest might be maintained, and therefore, relatively constant insulin secretion parameters.
• the human cyto ampleovirus (CMV) immediate early gene promoter may be used to achieve constitutive, high-level expression, as may any other viral or mammalian cellular promoters known to those of skill in the art (Sambrook et al. , 1989).
• the adenovirus vectors of the present invention have been rendered replication defective through deletion of the viral early region 1 (ElA) region such that the virus is competent to replicate only in cells, such as human 293 cells, which express adenovirus early region 1 genes from their cellular genome.
• the recombinant virus will therefore not kill normal cells which do not express adenoviral early gene products and will thus be suitable for use in human gene therapy regimens.
• Techniques for preparing replication defective adenoviruses are well known in the art (Ghosh-Choudhury and Graham, 1987; McGrory et al., 1988; Gluzman et al . , 1982; Gerard et al . , USSN 07/823,747, filed 1/22/92).
• the vectors of the present invention are replication defective, they will typically not have an adenovirus El region. Thus, it will be most convenient to introduce the region encoding the glucose-sensing protein at the position from which the El coding sequences have been removed. However, the position at which the coding region is inserted is not critical to the invention, and it may thus also be inserted in lieu of the deleted E3 region in E3 replacement vectors as described previously by Karlsson et al . (Karlsson et al ., 1986) .
• the adenovirus may be of any of the 42 different known serotypes or subgroups A-F.
• Adenovirus type 5 of subgroup C is the preferred starting material in order to obtain the conditional replication-defective adenovirus vector for use in the method of the present invention. This is because Adenovirus type 5 is a human adenovirus about which there is significant amount of biochemical and genetic information known, and which has historically been used for most constructions employing adenovirus as a vector.
• the present invention is also an adenoviral virion or adenovirus particle containing a vector construct expressing the proteins or mRNA as described above.
• recombinant, or engineered, host cells which incorporate an adenoviral vector prepared in accordance herewith.
• the recombinant adenovirus- containing host cell will be a eukaryotic or mammalian recombinant host cell, with pancreatic islet cells, such as ⁇ cells, being particularly preferred.
• pancreatic islet cells such as ⁇ cells
• engineered cells are distinguishable from naturally occurring cells in that one or more cDNAs, genes or other nucleic acid expression units, have been introduced through the hand of man.
• Recombinant host cells may be obtained from an animal or human host subsequent to the systemic in vivo administration of an adenoviral construct prepared in accordance herewith.
• the cells may be obtained from the animal, for example, by biopsy, and employed for further detailed analyses, such as to determine the degree or longevity of recombinant gene expression in vivo .
• the recombinant adenovirus may be introduced into a recombinant host cell directly in vitro , for example, in an analysis of ⁇ cell gene function regarding the introduction of normal or mutant proteins, such as transporters, enzymes or isoenzymes, or in the over-production of certain proteins.
• compositions comprising a vector construct which encodes a protein involved in glucose-sensing, such as GLUT-2, glucokinase and/or antisense hexokinase dispersed in a pharmacologically acceptable solution or buffer.
• a vector construct which encodes a protein involved in glucose-sensing, such as GLUT-2, glucokinase and/or antisense hexokinase dispersed in a pharmacologically acceptable solution or buffer.
• a vector in which the expression region of the recombinant insert is positioned under the control of a ⁇ cell-specific promoter, such as the insulin promoter, so that the resultant construct may be employed in human gene therapy, directing the expression of recombinant genes in ⁇ cells only.
• Preferred pharmacologically acceptable solutions include neutral saline solutions buffered with phosphate, lactate, Tris, and the like.
• phosphate phosphate
• lactate Tris
• a preferred means of purifying the vector involves the use of buoyant density gradients, such as cesium chloride gradient centrifugation.
• adenovirus is a virus that infects humans
• an immunological reaction is believed to be a possibility
• Such a test could be performed in a variety of accepted manners, for example, through a simple skin test or through a test of the circulating blood levels of adenovirus-neutralizing antibodies.
• the adenovirus vector employed is replication defective, it will not be capable of replicating in the cells that are ultimately infected. Moreover, it has been found that the genomic integration frequency of adenovirus is usually fairly low, typically on the order of about 1%. Thus, where continued treatment in certain individuals is required it may be necessary to reintroduce the virus every 6 months to a year. In these circumstances, it may therefore be necessary to conduct long term therapy, where the individual's plasma glucose and insulin levels are monitored at selected intervals.
• the particular cell line used to propagate the recombinant adenoviruses of the present invention is not critical to the present invention.
• the recombinant adenovirus vectors can be propagated on, e .g.
• human 293 cells or in other cell lines that are permissive for conditional replication-defective adenovirus infection, e.g., those which express adenovirus ElA gene products "in trans" so as to complement the defect in a conditional replication-defective vector.
• the cells can be propagated either on plastic dishes or in suspension culture, in order to obtain virus stocks thereof.
• the present invention is directed to methods of detecting the presence of diabetes-associated, or islet-cell directed, antibodies in a sample as a means of assessing the occurrence or risk of diabetes onset.
• numerous additional types of engineered cells will prove to be important, particularly those which exhibit an epitope of a selected antigen on their cell surface.
• Exemplary antigens include particularly GLUT-2, and also glutamic acid decarboxylase (the 64KD islet antigen and the less antigenic 67kD form) , insulin, proinsulin, islet 38KD protein, 65 kDa heat shock protein, selected immunoglobulins, insulin receptors or other types of islet cell antigens, whether cytoplasmic or surface.
• glutamic acid decarboxylase the 64KD islet antigen and the less antigenic 67kD form
• insulin proinsulin
• islet 38KD protein the 65 kDa heat shock protein
• selected immunoglobulins insulin receptors or other types of islet cell antigens, whether cytoplasmic or surface.
• it may be desirable to employ cells that do not secrete insulin in that antibody reactivity with insulin has been associated with false positive reactions.
• the cells are prepared by introducing genes expressing relevant epitopes into cultured cell lines that can be grown in unlimited quantity.
• the cells in the context of immunologically-based detection methods there is no requirement that the cells be glucose-response or have insulin-secreting capability. All that is required is that the these cells express on their surface an epitope associated either with the onset of diabetes or, more generally, an islet cell epitope.
• the cell actually express the entire protein in that all that is ultimately required is that the cell express an epitope that is recognized by the antibody that is sought to be detected. Therefore, the invention contemplates that subfragments which comprise antigenic epitopes may be employed in place of the complete antigenic protein.
• the first step of the detection methods of the invention will generally include obtaining a biological sample suspected of containing diabetes-associated or islet cell-directed antibodies.
• the biological sample will comprise serum, plasma, blood, or immunoglobulins isolated from such samples.
• the method will be applicable to any sample containing antibodies, regardless of its source or derivation.
• the sample is contacted with an engineered cell expressing a diabetes-associated or islet cell- expressed epitope, under conditions effective to allow the formation of an immunocomplex between the expressed epitope and antibodies that may be present in the sample.
• This aspect is not believed to be particularly critical to the successful practice of the invention in that any incubation technique or conditions that favor immunocomplex formation may be employed.
• Preferred conditions include incubation of the cells with serum in isotonic media such as phosphate buffered saline or Hanks balanced salt solution.
• the method is completed by testing for the formation of an immunocomplex between the diabetes- associated or islet cell epitopes expressed by the cell and antibodies present in the sample, wherein a positive immunoreaction indicates the presence of the respective antibody in the sample.
• the testing method is not believed to be crucial to the overall success of the invention.
• Many types of testing procedures for detecting immunocomplex formation are known in the art and are applicable, including RIA, EIA, ELISA, indirect immunofluorescence, and the like. In general, all that is required is a testing/detection procedure that allows one to identify an interaction of immunoglobulins present in the sample and epitopes expressed on the surface of the engineered cell.
• binding molecule is, generally speaking, any molecule that is capable of binding the immunocomplexed antibody, and that is detectable.
• exemplary binding ligands include protein A, anti- immunoglobulin antibodies, protein G, or even complement.
• the binding ligand includes an associated label that allows for the convenient detection of immunocomplexed antibodies.
• Typical labels include radioactive materials, fluorescent labels, and enzymes.
• an enzyme such as alkaline phosphatase, peroxidase, urease, ⁇ -galactosidase or others that can be detected through use of a colorimetric substrate.
• Other specific embodiments may include the use of associating ligands such as biotin, which can complex with avidin or streptavidin and thereby bring the enzyme or other label into association with the antibody or binding ligand.
• associating ligands such as biotin, which can complex with avidin or streptavidin and thereby bring the enzyme or other label into association with the antibody or binding ligand.
• the detection of immunocomplexed cells through the use of a label may be further improved, and even automated, through the application of cell sorter technology that can identify or quantify cells having associated immunocomplexed antibodies.
• Particularly preferred is the use of a fluorescent label in conjunction with sorting of cells on a fluorescence- activated cell sorter. It has been found that such a system can screen 40-50 sera per hour using a single fluorescence-activated cell sorter (Inman et al . , 1993).
• mutant or chimeric protein molecules can be constructed and expressed in recombinant AtT-20 cells, and used to investigate the binding of patients' antibodies, as described above.
• the failure of antibodies to bind to a mutant molecule after a specific deletion, or likewise, the ability of antibodies to bind to a chimeric molecule after a specific insertion, would allow the identification of the diabetes-specific epitope.
• Candidate epitopes include multiple extracellular "loop" regions of the GLUT-2 molecule. Once such an epitope is identified, synthetic peptides corresponding to the specific region of the protein sequence can be produced and used to develop simpler diagnostic procedures, for example, utilizing ELISAs or RIAs to detect the formation of an antibody/peptide complex.
• the foregoing method may be employed as a technique for selection of engineered clonal cells that express epitopes recognized by autoantibodies. That is, one may prepare a series of clones which comprise, for example, cDNA prepared to islet cell mRNA, express these DNAs in a recombinant cell and screen the resultant recombinant cells with a known antibody composition to identify diabetes associated antigens in addition to those specific antigens discussed above.
• the invention concerns a method for detecting the presence of diabetes- associated antibodies in a biological sample, such as a sample of serum, plasma, blood, or in immunoglobulins isolated therefrom.
• a biological sample such as a sample of serum, plasma, blood, or in immunoglobulins isolated therefrom.
• This method comprises contacting the sample suspected of containing diabetes-associated antibodies with intact GLUT-2-expressing cells under conditions effective to allow the interaction of any antibodies which may be present with GLUT-2, and then determining the degree of glucose uptake by the cells. Inhibition of glucose uptake indicates the presence of diabetes-associated antibodies in the sample.
• Preferred cells for use in such embodiments are GLUT-2-expressing engineered cells, and particularly,
• GLUT-2-expressing AtT20 ins cells Suitable conditions for assays of this kind include incubating the cells with an IgG sample and determining the degree of glucose uptake using 3-O-methyl- ⁇ -D-glucose.
• the engineered artificial ⁇ cells are grown in culture and then contacted with a buffer containing glucose, thus stimulating the cells to produce and secrete insulin which can be collected and purified from the surrounding media.
• CGT-6 engineered cells are contemplated to be of particular use, but any cell prepared to secrete insulin in response to glucose may be employed.
• the recombinant cells are contained within a column and subjected to perfusion with a buffer at a physiological pH, such as Krebs Ringer salt (KRS) solution, pH 7.4.
• KRS Krebs Ringer salt
• the column of cells is perfused with a glucose-containing buffer, such as KRS, 5mM glucose.
• KRS Krebs Ringer salt
• the insulin-containing eluent from the column is collected, which provides ideal starting material for the purification of increased amounts of high-quality insulin for human use.
• FIGURE 1 Northern blot demonstrating the presence of GLUT-2 mRNA in tissues and AtT-20 ins cell lines. Each lane contains 6 ⁇ g of total RNA. Samples were prepared from liver, anterior pituitary and islet tissue samples, as well as from untransfected (AtT-20 ins ) and GLUT-2 transfected (AtT-20 ins CGT-5 and CGT-6) AtT-20 ins cell lines. The blot was probed with radiolabeled antisense GLUT-2 cRNA, and as a control for gel loading, with an antisense oligonucleotide probe for 18S rRNA (Chen et al . , 1990).
• FIGURE 2 Immunoblot of GLUT-2 in tissues, untransfected cells (AtT-20 ins ) and cells transfected with the CMV/GLUT-2 construct (AtT-20 ins CGT-5, -6) .
• FIGURE 3A-C Glucose transport into AtT-20i: n ns ⁇ ! cells.
• FIGURE 3A Measurements of 3-O-CH3 glucose uptake as a function of glucose concentration for untransfected AtT-20 ins cells (parental) and GLUT-2 transfected lines CGT-5 and CGT-6. The symbol legend is shown in the upper left corner of this panel.
• FIGURE 3B Reciprocal plot of glucose uptake versus 3-O-CH3 glucose concentration for GLUT-2 transfected lines CGT-5 and CGT-6. The calculated Km and Vmax values for glucose transport and the symbol legends are given in the upper left corner of the panel.
• FIGURE 3C Reciprocal plot of glucose uptake versus 3-0-CH3 ⁇ glucose concentration for untransfected AtT-20 ins cells (parental cell line) . The calculated Km and Vmax values for glucose transport are indicated. Note the difference in the scales between figures 3B and 3C.
• FIGURE 4A-B Insulin release for AtT-20 ins cells in response to glucose, and glucose potentiation of forskolin induced secretion.
• FIGURE 4B Insulin release was measured from untransfected (AtT-20ins) and GLUT-2 transfected (CGT-6) AtT-20ins lines incubated with 0.5 ⁇ M forskolin (Fors) and 2.5mM glucose (Glc) in combinations indicated by the legend. Data are normalized to total cellular DNA in each secretion well and are expressed as the mean ⁇ SEM for 3-9 independent measurements. Statistically significant increases in secretion relative to the -Glc, -Fors control are indicated by the symbol * (p ⁇ 0.001) .
• FIGURE 5A-D ⁇ -galactosidase expression in isolated rat islets treated with AdCMV-/?GAL recombinant adenovirus.
• FIGURE 5A Light microscopic view of a representative intact islet four days after transduction with AdCMV-jSGAL and treatment of the islets with chromogenic substrate, viewed at 20 x magnification.
• FIGURE 5B An islet 21 days after viral transductions treated similarly to that in Figure 5A, viewed at 20 x magnification.
• FIGURE 5C A multicell aggregate that was prepared by trypsin-mediated dispersal of intact islets prior to incubation with the chromogenic substrate, viewed at 40 x magnification.
• FIGURE 5D A control islet treated with ⁇ - galactosidase substrate after 4 days in culture without exposure to AdCMV- ⁇ GAL, viewed at 20 x magnification.
• FIGURE 6A-B Adenovirus-mediated expression of variant glucokinases in CV-1 cells.
• FIGURE 6A Glucokinase expression was assayed by western blot hybridization analysis using 100 ⁇ g islet protein and an antibody that detects rat islet glucokinase (Antibody U343, Quaade et al . , 1991). Lanes contain the following samples: Lane 1, Islet glucokinase expressed in bacteria (Quaade et al . , 1991).
• Lane 2 Uninfected islets; Lane 3, Islets infected with recombinant adenovirus containing the / 3-galactosidase cDNA; Lane 4, Islets infected with recombinant adenovirus containing cDNA encoding a glucokinase variant containing an amber mutation (premature stop codon) in place of glutamic acid 279 that is not synthesized as a stable protein; Lane 5, Islets infected with recombinant adenovirus containing cDNA encoding a glucokinase variant with a point substitution that results in replacement of amino acid glycine 261 with arginine; Lane 6, Islets infected with recombinant adenovirus containing cDNA encoding a glucokinase variant with a point substitution that results in replacement of amino acid glutamic acid 279 with glutamine.
• FIGURE 6B The glucokinase enzymatic activity measured in crude homogenates of the CV-1 cells described in the western blot.
• FIGURE 7 Adenovirus-mediated expression of variant glucokinases in primary rat islets. Lanes contain the following samples: Lane 1, Uninfected islets; Lane 2, Islets infected with recombinant adenovirus containing the jS-galactosidase cDNA; Lane 3, Islets infected with recombinant adenovirus containing cDNA encoding a glucokinase variant containing an amber mutation (premature stop codon) in place of glutamic acid 279 that is not synthesized as a stable protein; Lane 4, Islets infected with recombinant adenovirus containing cDNA encoding a glucokinase variant with a point substitution that results in replacement of amino acid glycine 261 with arginine; Lane 5, Islets infected with recombinant adenovirus containing cDNA encoding a glucokinase variant with a point substitution that
• FIGURE 8A-F Delivery of recombinant adenovirus to the islets of Langerhans in vivo .
• FIGURE 8A Five days after termination of the infusion, fresh-frozen 15 ⁇ m pancreatic sections were prepared, incubated with the j ⁇ -galactosidase substrate solution.
• FIGURE 8B Five days after termination of the infusion, fresh-frozen 15 ⁇ m pancreatic sections were prepared, incubated with the jS-galactosidase substrate solution, and then treated with an anti-insulin antibody.
• FIGURE 8C Five days after termination of the infusion, fresh-frozen 15 ⁇ m pancreatic sections were prepared, incubated with the jS-galactosidase substrate solution.
• FIGURE 8D Five days after termination of the infusion, fresh-frozen 15 ⁇ m pancreatic sections were prepared, incubated with the 3-galactosidase substrate solution, and then treated with an anti-insulin antibody.
• FIGURE 8E A representative islet isolated from a virally infused animal that was treated with ⁇ - galactosidase substrate and then embedded and sectioned into 5 ⁇ m sections. Multiple blue nuclei are evident.
• FIGURE 8F In contrast to Figure 8E, islets isolated from a control animal are completely devoid of blue color.
• Insulin dependent diabetes mellitus is caused by autoimmune destruction of insulin producing jS-cells. Islet transplantation has been extensively investigated as a strategy for curing IDDM, but suffers from the difficulties associated with procuring enough tissue.
• the present invention is based in part on the recognition that the problem of islet supply could potentially be circumvented if a non-islet cell type could be engineered to secrete insulin in response to metabolic signals, since such cells could be grown in unlimited quantity in vitro . Such cells could ultimately replace daily insulin injections as therapy for Type I diabetes.
• pancreatic islets of Langerhans in fuel homeostasis are mediated in large part by their ability to respond to changes in circulating levels of key metabolic fuels by secreting peptide hormones. Accordingly, insulin secretion from islet ⁇ - cells is stimulated by amino acids, three-carbon sugars such as glyceraldehyde, and most prominently, by glucose. While these diverse secretagogues may ultimately work through a final common pathway involving alterations in K+ and Ca++ channel activity and increases in intracellular Ca++ (Prentki et al . , 1987; Turk et al . , 1987) , the biochemical events leading from changes in the levels of a particular fuel to insulin secretion are initially diverse.
• IDDM has traditionally been treated by insulin replacement, either classically, by external administration, or experimentally, by transplantation of islets or pancreas fragments.
• the latter strategy is not likely to be broadly applicable because of the difficulty and expense associated with the isolation of large numbers of islets.
• the present invention is directed to an alternative approach, that of using molecular techniques to engineer an "artificial /3-cell", i.e., a non-islet cell capable of performing glucose-stimulated insulin secretion, which can be grown in unlimited quantity in vitro.
• the anterior pituitary cell line AtT-20 i ⁇ s is preferred because of important similarities to 3-cells.
• these cells have been modified for insulin gene expression by stable transfection with a viral promoter/proinsulin cDNA construct (Moore et al . , 1983).
• AtT-20 ins cells are able to process the preproinsulin mRNA and preprotein to yield the correctly processed insulin polypeptide.
• their secretory response to analogues of cAMP compares favorably with the well differentiated hamster insulinoma (HIT) cell line (Moore et al . , 1983).
• HIT well differentiated hamster insulinoma
• AtT-20 ins cells contain significant amounts of the islet isoform of glucokinase (Hughes et al . , 1991), making this the only tissue other than liver or islets in which glucokinase gene expression has been reported.
• AtT-20 ins cells differ from islet j ⁇ -cells in two important ways. First, they do not secrete insulin in response to glucose, and second, they express the low Km GLUT-1 glucose transporter mRNA and not GLUT-2 (Hughes et al. , 1991). The inventors hypothesized that the lack of glucose responsiveness in At ⁇ -20 ins cells could be explained either by deficient capacity or altered affinity of glucose uptake relative to normal islets. To test this hypothesis, AtT-20 ins cells were stably transfected with GLUT-2 cDNA.
• pCB-7 was constructed by Drs. Michael Roth and Colleen Brewer of the Biochemistry Department, University of Texas Southwestern Medical Center. It differs from pCMV-4 in that it contains a hygromycin resistance gene; thus, cells transfected with the pCB7/GLUT-2 construct can be selected for stable integration of the vector DNA into the cell's genome by treatment with hygromycin. AtT-20 ins cells were transfected with this construct using electroporation, and stable transfectants were selected with hygromycin.
• AtT-20 ins cell line with glucose-stimulated insulin secretion Although the engineering of an AtT-20 ins cell line with glucose-stimulated insulin secretion has been accomplished, maximal insulin secretion from these cells occurs at a much lower glucose concentration than observed for normal islets, which do not respond at levels less than the fasting glucose concentration of approximately 4-5 mM, and which have not reached maximum secretion at the upper range of physiological glucose (10 mM) .
• the potentiating effect of glucose on forskolin, dibutyl cAMP, or IBMX induced insulin secretion from AtT- 20 ins ce H s ⁇ s a l so maximal at low glucose.
• the rat insulinoma cell line RIN 1046-38 is responsive to glucose when studied after short periods of time in cell culture (between passages 6-17) , albeit with a maximal response at sub-physiological glucose levels, as in transfected AtT-20 ins cells. With longer time in culture (passage number greater than 50) , all glucose-stimulated insulin secretion is lost (Clark et al . , 1990).
• Low passage RIN 1046-38 cells contain both glucokinase and GLUT-2, but lose expression of these genes when studied at higher passages.
• normal islets have both sufficient glucokinase activity and inhibited hexokinase (the levels of glucose-6-phosphate, an inhibitor of hexokinase, increase during glucose stimulation) to allow the control of glucose metabolism to be tied directly to glucokinase activity (Km of - 10 mM in islets) (Meglasson and Matchinsky, 1986) .
• GLUT-2 transfected RIN 1046-38 cells were preincubated with 50 mM 2-deoxyglucose for 30 minutes.
• 2-deoxglucose is readily transported into mammalian cells and phosphorylated by glucokinase and hexokinase, but once phosphorylated, this glucose analog is not metabolized further.
• administration of 2-deoxyglucose to cells will result in accumulation of 2-deoxyglucose-6- phosphate, a potent inhibitor of hexokinase, but not glucokinase.
• glucokinase/hexokinase ratio In addition to the chemical approach to reduction of hexokinase activity with 2-deoxyglucose, the inventors have also demonstrated the importance of control of the glucokinase/hexokinase ratio by molecular approaches.
• a recombinant adenovirus containing hexokinase I in sense orientation (AdCMV-HKI) was used to overexpress the enzyme in normal islets.
• AdCMV-HKI a recombinant adenovirus containing hexokinase I in sense orientation
• the inventors have also constructed a virus containing the cDNA for hexokinase I ⁇ in antisense orientation (the virus is designated AdCMV-HKIrev) and used it to treat GLUT-2 transfected, glucose responsive RIN 1046-38 cells.
• AdCMV-HKIrev a virus containing the cDNA for hexokinase I ⁇ in antisense orientation
• AdCMV-HKIrev a virus containing the cDNA for hexokinase I ⁇ in antisense orientation
• AtT-20 ins cells have glucokinase activity, but it represents only 9% of total glucose phosphorylation in these cells, and only 32% of the activity measured in normal islets (Table 1 in Example I below) ; the proportions of glucose phosphorylating enzymes in RIN1046-38 cells are similar to those found in AtT-20 jns cells.
• Hexokinase I the isoform that is expressed in most clonal cell lines (Arora et al . , 1990) is found bound to mitochondria and in a free cytosolic form (Lynch et al . , 1991). In the former state, the enzyme is less sensitive to glucose-6-phosphate inhibition (Wilson,
• AtT-20 ⁇ and RIN cells have reduced glucokinase activity, they may also have altered regulation of hexokinase such that it becomes the predominant glucose phosphorylating enzyme at any concentration of glucose studied.
• hexokinase/glucokinase ratio may at times result in maximal insulin secretory response at subphysiological glucose concentrations. It is proposed that a more physiologic glucose response may be achieved by "knocking out" hexokinase activity in engineered cells of the present invention.
• One approach is to co-transfect these cells with antisense hexokinase constructs.
• plasmid will contain an alternate resistance gene, such as puromycin or histidinol, since the AtT-20 ins cell line is resistant to both neo ycin (due to stable integration of the SV40-insulin-neo chimeric construct) and hygromycin (due to stable integration of the CMV- GLUT-2-hygromycin chimeric construct) .
• alternate resistance gene such as puromycin or histidinol
• recombinant adenoviruses can be used for introduction of multiple genes at one time.
• hexokinase isozyme expressed in mouse hepatoma cells has been cloned and characterized (Arora et al . , 1990) and shown to be approximately 92% identical to the hexokinase I sequences derived from rat brain (Schwab et al . , 1989) and human kidney (Nishi et al . , 1988).
• the hexokinase variant present in the cell was converted to cDNA by reverse transcribing the mRNA and amplification of the DNA product, a procedure recently employed in one of the inventors' laboratory for amplification of glucokinase mRNA from islets, RIN cells, AtT-20 ins cells, and primary anterior pituitary cells (Hughes et al., 1991).
• the oligonucleotides used for amplification were based on the published sequence of the mouse hepatoma hexokinase I (Arora et al . , 1990).
• the oligonucleotides included restriction enzyme recognition sequences at their 5' ends to facilitate directional cloning of the amplified cDNA into the selected vector in an antisense orientation.
• engineered lines may be transfected with antisense constructs by electroporation. After appropriate selection to obtain colonies that have stably integrated the antisense hexokinase construct into their genome, expression of the antisense mRNA can be evaluated by hybridization to labeled sense RNA, e.g., prepared with the pGEM vector system (Promega) .
• antisense constructs can be introduced via adenovirus. Blot hybridization analysis may be carried out not only with the probe corresponding to the antisense construct, but to regions outside as well, since cellular factors known to unwind RNA:RNA duplexes result in modification of that RNA, thus interfering with its detection on Northern blots (Walder, 1988) .
• modified antisense oligonucleotides may be employed.
• an antisense oligonucleotide may be prepared to sequences surrounding and/or containing the ATG initiation codon, for example, and introduced into cells by simply incubating the cells in media containing the oligonucleotide at high concentration.
• This approach bypasses uncertainties about the stability of longer antisense hexokinase transcripts synthesized from the construct and should provide suppression of hexokinase activity for a period of time sufficient to assess the functional consequences.
• the oligonucleotide antisense procedure can only cause a transient reduction in endogenous expression, and is thus not applicable to the engineering of a stable "artificial" ⁇ cell.
• ribozyme modification of the antisense approach.
• the ribozyme catalytic domain which is a piece of RNA with the capacity to cleave other RNA molecules (Forster and Symons, 1987; Hasellof and Gerlach, 1988) is inserted between fragments of antisense RNA complementary to the gene to be targeted (hexokinase I, for example).
• the RNA degrading activity is thereby targeted to the gene of interest, theoretically providing more efficient reduction in the level of expression of the targeted RNA.
• a second alternative that bypasses the issue of effectiveness of antisense strategies altogether would be to knock out the endogenous hexokinase gene of interest in the cells using a positive/negative selection protocol (Mansour et al . , 1988; Capecchi, 1989; Zheng et al . , 1990) to select for homologous recombination of a hexokinase gene segment that renders the endogenous hexokinase gene nonfunctional.
• This approach involves cloning of at least a segment of the hexokinase gene(s) expressed in the engineered cells either by library screening or PCR amplification, and construction of a vector that contains a genomic fragment, preferably containing exons that encode the putative ATP or glucose binding sites (Arora et al . , 1990; Schwab et al . , 1989; Nishi et al . , 1988; Andreone et al . 1989).
• antibiotic resistance gene e.g., puromycin
• HSV herpes simplex virus
• the plasmid is then introduced into cells by electroporation and homologous recombination events are selected for by incubation of the cells in puromycin and FIAU, a recently described thymidine kinase substrate (Capecchi, 1989; available from Dr. Richard White, Bristol Myers/Squibb, Walingford, CT) .
• FIAU a recently described thymidine kinase substrate
• the action of FIAU is exerted as follows. If recombination occurs at a nonhomologous site, the viral thymidine kinase gene is retained in the genome and expressed, rendering cells extremely sensitive to FIAU.
• the disrupted gene is inserted at its homologous site (the endogenous hexokinase gene) , in contrast, the viral thymidine kinase gene is lost, and the cells are tolerant of the drug. While homologous recombination in mammalian cells is a relatively rare event, the selection strategy is sound, and has recently been applied to mammalian tissue culture cells (Zheng et al . , 1990).
• AtT-20ins cells the activity of 0.7 U/g protein is only about 25% of the activity in normal islet cells, which contain approximately 3.1 U/g protein.
• a GLUT-2 + , insulin + , glucokinase- overexpressing, hexokinase " cell line will require cotransfection of some of the relevant constructs, since limited numbers of resistance-gene containing plasmids are available. Efficient cotransfection can be expected when using either electroporation or CaP0 4 precipitation transfection strategies (Sambrook et al. , 1989). Again, recombinant adenovirus vectors can also be used for cases in which the introduction of multiple gene products is desired. As described above, the complication of increasing glucokinase expression may not be relevant to GLUT-2 transfected RIN cells, which undergo a 2-3 fold increase in glucokinase activity upon transfection with GLUT-2.
• NIDDM NON-INSULIN DEPENDENT DIABETES MELLITUS
• the engineered cells of the present invention will be particularly advantageous in the treatment of insulin dependent diabetes following their introduction into such diabetic animals or human patients.
• these cells are also contemplated to be of use in the treatment of non-insulin dependent diabetes mellitus (NIDDM; type 2 diabetes) , particularly for the reasons set forth below.
• NIDDM non-insulin dependent diabetes mellitus
• the inventors have demonstrated in rat and human models of type 2 diabetes that the ratio of amylin to insulin is extremely high.
• Amylin has been shown to cause insulin resistance in muscle and to increase the production of glucose by the liver, effects that lead to hyperglycemia.
• Amylin is normally co-secreted by the ⁇ cells with insulin (Ogawa et al . , 1990). In nondiabetics the ratio of amylin to insulin is always below 2%. In animals or humans with type 2 diabetes the ratio of amylin to insulin exceeds 2%.
• the ⁇ cells By implanting a source of insulin secretion into type 2 diabetic individuals, i.e., the engineered insulin-secreting cells of the present invention, the ⁇ cells will become hypoactive and the insulin levels will be derived not from ⁇ cells that co-secrete amylin but rather from the engineered cells that secrete only insulin. This should greatly enhance the effectiveness of the insulin by eliminating amylin- mediated insulin resistance.
• amylin levels are high in obese individuals and low in thin persons.
• the present inventors propose that amylin directs the anabolic actions of insulin towards an increase in the formation of fat from dietary glucose. Based on this hypothesis, they predict that suppression of the relative concentration of amylin to insulin will reduce the flow of glucose into the formation of fat.
• IDDM TREATMENT OF INSULIN DEPENDENT DIABETES MELLITUS
• the engineered cells of the present invention may be particularly advantageously employed in the treatment of animals or human patients with insulin dependent diabetes in that such cells can sense glucose and respond by secreting insulin.
• cells are engineered to achieve glucose dose responsiveness more closely resembling that of islets, it is believed that implantation of the CGT-5 or CGT-6 GLUT-2 expressing cells will also achieve advantages in accordance with the invention.
• Madsen and coworkers have shown that implantation of poorly differentiated rat insulinoma cells into animals results in a return to a more differentiated state, marked by enhanced insulin secretion in response to metabolic fuels (Madsen et al . , 1988) . These studies suggest that exposure of engineered cell lines to the in vivo milieu enhances some of their response(s) to secretagogues.
• Engineered cells may be implanted using the alginate-polylysine encapsulation technique of O'Shea and Sun (O'Shea and Sun,1986), with modifications as recently described by Fritschy (Fritschy et al . , 1991).
• the engineered cells are suspended in 1.3% sodium alginate and encapsulated by extrusion of drops of the cell/alginate suspension through a syringe into CaC ⁇ . After several washing steps, the droplets are suspended in polylysine and rewashed.
• the alginate within the capsules is then reliquified by suspension in 1 mM EGTA and then rewashed with Krebs balanced salt buffer.
• Each capsule should contain several hundred cells and have a diameter of approximately l mm.
• Implantation of encapsulated islets into animal models of diabetes by the above method has been shown to significantly increase the period of normal glycemic control, by prolonging xenograft survival compared to unencapsulated islets (O'Shea and Sun, 1986; Fritschy et al . , 1991). Also, encapsulation will prevent uncontrolled proliferation of clonal cells.
• Capsules containing cells are implanted (approximately 1,000- 10,000/animal) intraperitoneally and blood samples taken daily for monitoring of blood glucose and insulin.
• An alternate approach to encapsulation is to simply inject glucose sensing cells into the scapular region or peritoneal cavity of diabetic mice or rats, where these cells are reported to form tumors (Sato et al . , 1962). Implantation by this approach may circumvent problems with viability or function, at least for the short term, that may be encountered with the encapsulation strategy. This approach will allow testing of the function of the cells in animals but will require further safety testing before it is used as a strategy for treating human diabetes.
• the cells of the bone marrow are derived from a common progenitor, known as pluripotent stem cells, which give rise to a variety of blood borne cells including erythrocytes, platelets, lymphocytes, macrophages, and granulocytes.
• these cells are capable of secreting peptides such as tumor necrosis factor and interleukin 1 in response to specific stimuli.
• these cells contain granules similar in structure to the secretory granules of ⁇ cells, although there is no clear evidence that such granules are collected and stored inside macrophages as they are in ⁇ cells (Stossel, 1987). Nevertheless, it may ultimately be possible to use the recombinant DNA for glucose transporters and glucose phosphorylating enzymes in combination with the recombinant insulin gene in a manner described for clonal cells to engineer primary cells that perform glucose- stimulated insulin secretion.
• Adenovirus gene transfer systems are based upon recombinant, engineered adenovirus which is rendered replication-incompetent by deletion of a portion of its genome, and yet still retains its competency for infection (Sen et al . , 1988).
• Adenovirus-mediated gene transfer has recently been investigated as a means of mediating gene transfer into eukaryotic cells and into whole animals. For example, in treating mice with the rare recessive genetic disorder ornithine transcarbamylase (OTC) deficiency, it was found that adenoviral constructs could direct the expression of normal levels of the OTC enzyme in 4 out of 17 instances (Stratford-Perricaudet et al . , 1990).
• OTC rare recessive genetic disorder ornithine transcarbamylase
• adenovirus vectors can transfer a cDNA encoding rabbit muscle glycogen phosphorylase into primary hepatocytes with very high efficiency (Gomez-Foiz et al . , 1992) such that 86% of the primary hepatocyte expressed the muscle phosphorylase gene as determined by in situ hybridization.
• the remarkable efficiency of phosphorylase gene transfer into primary, non-dividing cells suggested to the inventors that this system had great potential for use in gene therapy protocols for the treatment of diabetes.
• a recombinant adenovirus containing the bacterial ⁇ - galactosidase gene has been used to demonstrate a 75-80% efficiency of gene transfer into isolated islets of Langerhans (Becker et al . , 1993), see figures 5-8.
• infusion of this virus into animals results in / 3-galactosidase expression in islets but not in surrounding exocrine tissue.
• the inventors further determined that luciferase, a reporter gene, is actively expressed in multiple tissues when a recombinant adenovirus is injected into the systemic circulation. It is proposed that the various properties of adenovirus may be adapted for use in therapeutic strategies directed to the treatment of diabetes, particularly in the treatment of NIDDM, but also in IDDM.
• NIDDM in contrast to IDDM, is a disease with two coexisting but distinct derangements: (l) Insulin resistance, or the inability of insulin to exert its normal metabolic effect on fat, muscle, and liver, its primary target tissues, and (2) ⁇ cell failure, in which the insulin secretory response to glucose is lost, resulting in the inability to correct hyperglycemia.
• the challenges for therapy in NIDDM are thus complex, in that the genetic and physiological bases for insulin resistance and ⁇ cell failure are still incompletely understood.
• the reduced levels of muscle glucose-6-phosphate in patients with NIDDM may be due to a reduction in glucose transporter activity, or to altered hexokinase II activity.
• glycogen synthase, glycogen phosphorylase and their attendant regulatory kinases and phosphatases have been considered as candidate genes, due in large part to demonstrated impairment in non-oxidative glucose metabolism (i.e., glycogen synthesis) in muscle tissues of NIDDM patients.
• candidate genes are likely to emerge from further studies on the mechanism of insulin-receptor mediated signal transduction. Possible players could include G proteins, which have been implicated in insulin action, or proteins with tyrosine kinase activity and their substrates that may participate in an insulin-activated "phosphorylation cascade", leading to altered target enzyme functions.
• LTR long-terminal repeat
• adenovirus is known to infect mouse skeletal muscle cells in vivo (Stratford- Perricaudet et al . , 1992; Ragot et al . , 1993; Herz and Gerard, 1993) following either intravenous or intramuscular injection. Given these limitations, the use of adenovirus-mediated gene transfer in supplying copies of normal genes involved in insulin responsiveness is therefore very attractive.
• / 3-cell function in the early stages of NIDDM is that insulin secretion is elevated in the presence of basal, normally non-stimulatory concentrations of glucose.
• /3-cell failure progresses through a phase of insulin hypersecretion at fasting glucose levels and continues to an exacerbated state in which stimulatory concentrations of glucose no longer cause increased insulin release.
• the first hypersensitive phase of /3-cell dysfunction may be a consequence of overexpression of a low Km glucose metabolizing protein such as GLUT-1 or hexokinase. If so, this condition could be treated by gene therapy with adenovirus-mediated introduction of antisense hexokinase or GLUT-1 cDNAs as discussed above.
• adenovirus containing ⁇ cell-specific promoter/enhancer elements, such as the insulin promoter, followed immediately by a suitable gene (or genes) involved in glucose sensing, particularly either the GLUT-2 and/or glucokinase gene, and then a suitable polyadenylation signal, such as the mouse protamine polyadenylation signal.
• a suitable gene or genes involved in glucose sensing, particularly either the GLUT-2 and/or glucokinase gene
• a suitable polyadenylation signal such as the mouse protamine polyadenylation signal.
• Administration of such a recombinant virus via injection into animals, and after proper testing, into humans, will result in infection of multiple tissues.
• the virus may be delivered directly into the pancreatic circulation via a catheter, providing for preferential expression in the highly vascularized islet cells.
• the recombinant glucose-sensing gene(s) such as GLUT-2 or a suitable glucokinase gene, would be preferably expressed only in islet ⁇ cells, since the expression of the transgene(s) is driven by islet-selective or islet preferred promoter/enhancer elements (e .g. , the insulin, GLUT-2 or glucokinase promoter elements) .
• restoration of GLUT-2 and/or glucokinase in ⁇ cells of animals or humans with NIDDM-like syndromes may restore normal ⁇ cell function, thus correcting the diabetic state. Since the virus is replication incompetent, there will be no deleterious effect of the virus itself on subject health.
• Viruses containing GLUT-l or hexokinase in sense orientation can serve as important controls and have also served to validate the concept that overexpression of one or both of these low Km glucose metabolizing proteins predisposes to hypersensitivity to glucose when introduced into normal islets.
• IDDM is caused by autoimmune destruction of the islet ⁇ cells, leading to complete insulin deficiency and uncontrolled glucagon secretion.
• the transplantation of appropriately encapsulated insulin- secreting islet tissue into patients has been proposed.
• the major problem with this approach is that it is very difficult to obtain large numbers of islets, in part because isolated islets do not proliferate in vitro.
• the implantation of recombinant cells engineered to have regulated insulin secretion, such as those disclosed herein, is one particularly advantageous manner in which to replace islet ⁇ cells in IDDM patients.
• the present inventors contemplate other ways in which this may be achieved, for example, by utilizing adenovirus- linked protocols, such as those described below, to expand islet cells in culture after their isolation and to thus increase the number of cells available for transplantation.
• This aspect of the present invention was developed from a consideration of work on neurotrophic factors and their receptors.
• a number of neurotrophic factor receptors including molecules named gpl30, LIFR ⁇ , CNTFR, TrkA, TrkB, and TrkC have been cloned. These receptors, and their corresponding peptide ligands such as brain- derived neurotrophic factor (BDNF) , nerve growth factor (NGF) , neurotrophin-3 (NT-3) , and neurotrophin-4 (NT-4) appear to be involved in survival, growth and differentiation of cells derived from the nervous system.
• BDNF brain- derived neurotrophic factor
• NGF nerve growth factor
• NT-3 neurotrophin-3
• NT-4 neurotrophin-4
• TrkB receptor neurotrophin receptor molecules
• the essence of this novel idea is to isolate islets by standard techniques from either human or large animal sources, and infect these cells in vitro with a recombinant adenovirus containing the gene or cDNA for one of the neurotrophin receptors listed above. Genes or cDNAs encoding other growth factor or cytokine receptors or cloned islet cell-specific receptors could also be employed. This efficient mode of gene transfer would result in a large percentage of islet cells expressing the desired receptor molecule. It is proposed that administration of the corresponding ligand would then lead to a large expansion of the number of islet cells in the culture, thus increasing the number of cells available for transplantation and overcoming the obstacle imposed by repeated islet isolation procedures.
• a further aspect of this new concept is that islet cells infected with one of these receptors will exhibit enhanced survival in the presence of the cognate peptide ligand. If so, a supply of the peptide may be maintained in close proximity to the transplanted cells expressing the receptor when the cells are transplanted into diabetic patients. It is proposed that this could be achieved by transfecting endocrine cells, such as cells derived from islet cells or anterior pituitary tissue with the cDNA encoding the appropriate peptide ligand.
• the recombinant secretory cells of the present invention are ideally suited for delivery of neurotrophic peptides.
• the cells engineered for expression of the peptide could be co-transplanted with primary islet cells expressing the neurotrophin receptor, thus maintaining a supply of the necessary ligand in close proximity to the target tissue.
• proinsulin Kuglin et al . , 1988
• islet 64 Kd and 38 Kd protein Boekkeskov et al . , 1982
• immunoglobulins DiMario et al . , 1988
• mammalian 65 Kd heat shock protein Elias et al . , 1991
• insulin receptors Lidwig et al . , 1987
• the inventors propose that cells engineered for specific expression of one of the foregoing epitopes, or for any epitope that may subsequently be identified in autoimmune diabetes, may be employed in diagnostic tests for diabetes.
• the principle of such a test involves reaction of the antibodies in a patients' serum with cells expressing the antigen(s) of choice, or epitope(s) of such an antigen, and subsequent detection of the antigen/antibody complex by reaction with a second antibody that recognizes human immunoglobulins (antibodies) .
• a test would be scored as positive if the serum being tested reacts with the cells engineered for expression of the antigen of interest, but not with the parental (non-engineered) cell line.
• the reaction of the patient's serum with the expressed antigen is measured indirectly by virtue of the fact that the anti- immunoglobulin antibody used is "labeled” or “tagged” with a molecule that readily allows its detection by direct inspection or mechanical measurement.
• the most common "tags" that are linked to commercially available preparations of anti-human immunoglobulin are fluorescent molecules such as fluorescein or tetramethyl rhodamine or enzymes such as horseradish peroxidase or alkaline phosphatase.
• engineered cells expressing the GLUT-2 antigen in diagnostic assays is greatly advantageous in that it allows rapid, efficient and reproducible analyses of patients' sera.
• Engineered GLUT-2-expressing cells such as GLUT-2- expressing AtT20 ins cells may be used in diagnostic assays based either on immunocomplex formation, or on the inhibition of glucose uptake, for example, using 3-0- methyl- ⁇ -D-glucose.
• engineered cells expressing the GLUT-1 antigen will also have utility. In particular, they may be used as control' cells in diagnostic tests since no reaction of IDDM sera is detected with GLUT-1-expressing cells in these assays.
• the first is simple direct inspection of cells by fluorescence microscopy. In this procedure, cells are adhered to poly-L-lysine coated microscope slides or cover slips. The cells are then fixed lightly by treatment with 0.5% paraformaldehyde or left untreated. Treatment of the cells with paraformaldehyde will cause changes in membrane structure of cells, resulting in changes in the conformation of antigen molecules. For some, but not all antibodies, alteration of antigen conformation in this way will allow a tighter association of the antibody and antigen.
• Engineered and control cells are then exposed to either crude serum or purified immunoglobulins (IgGs) from patients to be tested for antibodies against the expressed antigen. After washing, the cells are exposed to an appropriately "tagged” or labeled antibody recognizing human IgGs and the antigen/antibody/anti-antibody complexes are visualized in a microscope by excitation of the fluorescent tag by exposure to light of an appropriate wavelength.
• IgGs immunoglobulins
• FACS fluorescence activated cell sorter
• FACS fluorescence intensities
• GLUT-2 transfected AtT-20 ins cells treated with sera from patients with IDDM compared with normal sera with both the microscopic and FACS techniques.
• an antibody raised against an exposed (extracellular) region of the GLUT-2 molecule has been found by the inventors to cause a shift (increase) in fluorescence that is similar to the shift caused by the diabetic sera.
• GLUT-2 appeared to be a particularly useful epitope for the identification of new-onset IDDM patients and even prediction of diabetes onset.
• this flow cytometry-based immunofluorescence assay was found to be particularly useful in distinguishing the sera of patients with new-onset IDDM from non-diabetic subjects. It was found that 29 of 31 (94%) of the nondiabetic population were negative for IgG binding to GLUT-2 while 23 of 30 (77%) of sera from IDDM patients were positive. Thus, 81% of negative results were from nondiabetic patients and 92% of positive results were from patients with IDDM (Table 2) . USE OF ENGINEERED CELLS IN THE IDENTIFICATION OF SPECIFIC EPITOPES
• GLUT-1 transfected AtT-20 ins cells do not discriminate diabetic from normal sera in FACS-based diagnostic tests, providing strong evidence that diabetic sera contain an antibody specific for the islet GLUT-2 glucose transporter (Inman et al . , 1993). It is therefore envisioned that the artificial / 3-cells of the present invention will be of use in the identification of the specific epitope or segment of protein within GLUT-2 that is responsible for interacting with the antibody. Comparison of the GLUT-1 and GLUT-2 sequences reveals that the 2 putative membrane spanning regions in the two molecules are highly hydrophobic and of very similar sequence.
• GLUT-2 contains a very large extracellular loop between membrane spanning regions 1 and 2, while GLUT-1 contains a much smaller loop with little sequence homology to the GLUT-2 loop.
• the inventors propose that construction of chimeric GLUT molecules in which individual or multiple "loop" regions are substituted could lead to identification of the specific epitope of GLUT-2 that reacts with diabetic sera.
• the DNA encoding the large extracellular loop of GLUT-2 can be inserted in place of the small extracellular loop of GLUT-1 in the GLUT-1 cDNA sequence, and this chimeric molecule expressed in AtT- 20 ⁇ cells. If the chimera reacts with diabetic serum (as the native GLUT-1 molecule does not) , the added GLUT- 2 extracellular loop would be the specific epitope.
• synthetic peptides corresponding to this region of the protein sequence can be produced and used to develop simpler diagnostic procedures. Examples would include a simple test in which the peptide epitope is reacted with test serum and the formation of an antibody/peptide complex is monitored by well established techniques such as ELISA or RIA.
• An alternative means of identifying the reactive epitope of GLUT-2 is to synthesize the individual peptide sequences that constitute the entire GLUT-2 sequence as individual segments of 15-50 amino acids in length. This analysis would initially be focused on the regions that are most dissimilar in comparing GLUT-2 and GLUT-1, such as the extracellular "loop" region described above.
• the peptide or peptides corresponding to this epitope can be synthesized and tested for utility in screening of diabetic (and pre- diabetic) sera. This can be approached by rapid solid- phase assays such as ELISA, in which the peptide is aliquoted into a multi-well plastic plate and tested for reactivity with diabetic and non-diabetic sera by using a "tagged" second antibody and well-established colorimetric procedures. The intensity of the colorimetric reaction (and thus, the reactivity of the serum) would then be evaluated with an automated plate scanner that reads the optical density of the colorimetric solution at desired wavelengths.
• rapid solid- phase assays such as ELISA, in which the peptide is aliquoted into a multi-well plastic plate and tested for reactivity with diabetic and non-diabetic sera by using a "tagged" second antibody and well-established colorimetric procedures. The intensity of the colorimetric reaction (and thus, the reactivity of the serum) would then be evaluated with an
• GLUT-2 transfection is herein shown to cause an increase in intracellular insulin of approximately 5-fold in the AtT-20 ins cell line, CGT-6.
• CGT-6 cells contain approximately 1 mUnit/10° cells of human insulin when grown on gelatin beads in solution.
• the average IDDM patient requires approximately 30 Units of insulin per day for control of blood glucose levels.
• Cell densities of 5 x 10 ⁇ cells/liter cell culture media are readily achieved in the current liquid culture configuration, meaning that 5 Units of insulin/liter can be produced. Much higher densities can be achieved using currently commercially available bioreactor technology (e.g., that available from New Brunswick Scientific) .
• bioreactor technology e.g., that available from New Brunswick Scientific
• the intracellular insulin content of the cells can be further increased by one of the following methods: 1) Retransfection of AtT-20 cells with the Rous sarcoma virus/human proinsulin cDNA plasmid that was originally used to generate the AtT-20 ins cell line. The level of expression of a transfected gene appears to be dependent on the site of insertion of the plasmid in the chromosome. Thus, it is highly likely that higher levels of insulin expression will be achieved by simply reintroducing the plasmid and isolating new clones, that are rendered resistant to neomycin by the neomycin resistance gene in the plasmid.
• plasmids in which human proinsulin cDNA expression is directed by alternate promoters examples include the CMV promoter, which was used to achieve very high levels of expression of GLUT-2 in the creation of the CGT-6 cell line in the inventor's laboratory, or 3) Amplification of the viral promoter/human proinsulin cDNA (Sambrook et al . , 1989) by cloning next to a resistance gene such as dihydrofolate reductase (DHFR) , adenosine deaminase, or glutamine synthetase (Cockett et al . , 1990).
• DHFR dihydrofolate reductase
• adenosine deaminase adenosine deaminase
• glutamine synthetase Cockett et al . , 1990.
• DHFR is the most commonly used system, but is generally of limited usefulness in cell lines that have endogenous expression of DHFR (this is true of the AtT- 20 ins cell line) .
• the glutamine synthetase system allows amplification of the gene of interest even in the presence of endogenous expression of glutamine synthetase.
• Cells are stably transfected with a plasmid containing the transcription unit (i.e., viral promoter fused to the human proinsulin gene) adjacent to the hamster glutamine synthetase coding sequences. Selection of clones and amplification of the integrated transcription unit/GS gene is then carried out by addition of methionine sulfoxide to the tissue culture media (Cockett et al . , 1990). Resulting clones contain greatly increased copy numbers of the transcription unit, by virtue of its association with the amplified glutamine synthetase gene. As a result, much greater quantities of insulin are produced by the recombinant cell, making it an even more viable source for human insulin production. 4) Use of a strong and ubiquitously active cellular promoter, such as that for elongation factor el ⁇ .
• the transcription unit i.e., viral promoter fused to the human proinsulin gene
• the invention contemplates that correctly-folded human insulin could be produced relatively simply and rapidly using clonal cells that secrete insulin in response to glucose.
• the most appropriate method to accomplish this has been found by the inventors to be the perfusion of a column containing engineered AtT-20 ins cells adhered to gelatin beads. Passing a glucose-containing buffer, such as Hanks Balanced Salt solution, with 5mM glucose, pH 7.4, over such a column of insulin producing ⁇ cells has been found to stimulate the increased secretion of insulin into the surrounding media, which can then be collected and used as a starting material for the purification of recombinant insulin.
• a glucose-containing buffer such as Hanks Balanced Salt solution
• purification of insulin from the perfusion media can be rapidly achieved by one or a combination of the following approaches: 1) Affinity chromatography, for example, passage of the insulin containing media over a column containing anti-insulin antibodies. After removal of non-insulin proteins and other impurities by washing of the column, insulin can be specifically eluted by using a buffer with an increased salt concentration or decreased pH. 2) Preparative high performance liquid chromatography. 3) Size selection by conventional size-exclusion column chromatography.
• AtT-20 ins cells used were provided by Dr. Regis Kelly, University of California San Francisco, and were similar to the line that was originally described (Moore et al . , 1983) except that the Rous sarcoma virus long terminal repeat was substituted for the SV40 early gene promoter for directing insulin cDNA expression.
• the cells were grown in Dulbecco's modified Eagles' medium (DMEM) , supplemented with 10% fetal calf serum, 100 ⁇ g/ml streptomycin, and 250 ⁇ g/ml neomycin.
• DMEM Dulbecco's modified Eagles' medium
• Anterior pituitary and liver samples were excised from normal ad-lib fed Wistar rats, and islets were isolated from groups of 10- 20 animals as previously described (Johnson et al . , 1990a, 1990c) and pooled for RNA extraction or homogenization for glucose phosphorylation assays.
• the rat islet GLUT-2 cDNA (Johnson et al . , 1990a) was cloned into the vector pCB-7, a derivative of vector pCMV4 (Andersson et al . , 1989), immediately downstream of its cytomegalovirus (CMV) promoter.
• the cDNA was cleaved at its 3' end with Hind III, resulting in the removal of 635 base pairs of 3' untranslated region.
• AtT-20 ins cells were transfected with this construct using electroporation.
• Cells were harvested from pre-confluent plates by light trypsinization, washed twice in phosphate buffered saline, and resuspended at 3 x 10 6 cells/ml in a solution containing 20 mM Hepes (pH 7.05), 137 mM NaCl, 5 mM KCl, 0.7 mM Na 2 HP0 4 , 6 mM glucose, and 0.5 mg/ml salmon testis DNA. After equilibration of the cells to room temperature in electroporation cuvettes (Bio-Rad Labs; electrode gap width 0.4 cm), a single pulse was delivered using a capacitance setting of 960 ⁇ F and voltage settings between 0.2 and 0.3 kV.
• the cells remained in the buffer for five minutes and were then plated onto tissue culture dishes. Stable transfectants were selected with hygromycin, since the plasmid also contains a resistance gene for this drug. Four colonies were obtained and passaged several times in the presence of hygromycin to obtain pure cultures.
• RNA was prepared by guanidinium isothiocyanate extraction, resolved on a formaldehyde/agarose gel and transferred to a nylon membrane (Micron Separations Inc.) as previously described (Newgard et al . , 1986). Blots were hybridized sequentially with *P labeled antisense GLUT-2 or 18S rRNA probes, prepared as described (Chen et al . , 1990), with stripping of the blot between hybridizations by boiling in 0.1% SDS for 30 minutes.
• Liver plasma membranes were prepared by the method of Axelrod and Pilch (1983) and only the light plasma membrane fraction was used. Islet and AtT-20 ins cell membranes were prepared as previously described (Johnson, 1990b) , except that the sucrose gradient was deleted and the homogenization buffer consisted of 50 mM Tris, pH
• AtT-20 jns cells or transfected lines CGT-5 and CGT-6 were grown to a density of 5 x 10° cells per 100 mm dish and harvested by incubation at 37°C with a solution of 0.02% EDTA in PBS. After three washes in DMEM containing 20 mM HEPES, approximately 1.5 x 10 5 cells were transferred onto 12 mm poly-L-lysine coated glass coverslips, to which they adhered during a 30 minute incubation at 37°C. The cells were then fixed with 3% paraformaldehyde in PBS for 30 minutes at room temperature, and incubated with 0.1 M NH 4 C1 in phosphate buffered saline (PBS), pH 7.9 for 30 minutes.
• PBS phosphate buffered saline
• Glucose phosphorylation and glucokinase activities were measured by conversion of U- 14 C glucose to U-* 4 C glucose-6-phosphate, as previously described (Method "B” in Kuwajima et al . , 1986).
• Cultured cells or tissues were homogenized in 5 volumes of buffer containing 10 mM Tris, 1 mM EDTA, 1 mM MgCl 2 , 150 mM KCl, and 1 mM DTT, pH 7.2. The homogenate was cleared by centrifugation at 12,000 x g and the supernatant used for assays of glucose phosphorylation.
• Reactions were carried out at 37°C in a total volume of 150 ⁇ l, and initiated by addition of 10- 30 ⁇ l of extract to a reaction mix containing 100 mM Tris, 5 mM ATP, 10 mM MgCl 2 , 100 mM KCl, 1 mM DTT, pH 7.2, 15 or 50 mM glucose, and 6.2 ⁇ Ci of U- 14 C glucose (300 mCi/mmol; New England Nuclear) .
• assays were performed in the presence and absence of 10 mM glucose-6-phosphate, which potently inhibits hexokinase but not glucokinase activity.
• Reactions were carried out for 90 minutes and terminated by addition of 50 ⁇ l of reaction mix to 100 ⁇ l of 3% methanol in 95% ethanol. An aliquot of this mixture was transferred to nitrocellulose filter circles (Grade NA 45, Schleicher & Schuell) , which bind phosphosugars, and after air drying, washed extensively in water to remove labeled glucose. Radioactivity on the paper was then detected by liquid scintillation counting, and glucose phosphorylating activities are expressed in terms of the total protein content of the extracts.
• GLUT-2 transfected lines CGT-5 and CGT-6 were removed from growth plates by light trypsinization and reseeded in 6 well dishes (Costar) at a density of 5 x 10 5 cells per well. The cells were then grown for three days in culture media containing lmM glucose (see above) . On the third day, cells were washed twice for 10 minutes each in HEPES balanced salt solution containing 1% BSA (HBSS) , but lacking glucose. Secretion studies were initiated by addition of HBSS plus a range of glucose concentrations (0 - 2OmM) or in the presence of one of three non-glucose secretagogues, forskolin
• Cells were collected in 1ml of 5M acetic acid, lysed by three cycles of freeze-thawing, and lyophilized. The dried lysate was then reconstituted in 5ml of insulin assay buffer (50mM NaH 2 P0 4 , 0.1% BSA, 0.25% EDTA, 1% aprotinin, pH 7.1) and aliquots were assayed for insulin by radioimmunoassay.
• insulin assay buffer 50mM NaH 2 P0 4 , 0.1% BSA, 0.25% EDTA, 1% aprotinin, pH 7.1
• GLUT-2 mRNA Expression of GLUT-2 mRNA was evaluated by blot hybridization analysis of AtT-20 ins cells, either transfected or untransfected with a cytomegalovirus (CMV) promoter/GLUT-2 hybrid gene, and in extracts of rat liver, islets of Langerhans, and anterior pituitary tissues.
• CMV cytomegalovirus
• RNA from four GLUT- 2 transfected AtT-20 ins cell lines (CGT-1, CGT-2, CGT-5, CGT-6) , untransfected AtT-20 ins cells, and the three primary tissues ( Figure 1) .
• Steady state levels of GLUT- 2 mRNA were highest in CGT-5 and CGT-6; the former contained approximately half as much and the latter an equal amount of GLUT-2 mRNA as rat islets, and they contained 10 and 16 times as much, respectively, as rat liver, measured by densitometric scanning and normalization to the signal obtained with an 18S rRNA probe.
• the transfected lines contained a smaller GLUT-2 transcript than liver or islets (2.2 versus 2.8 kb) because 635 base pairs of the 3' untranslated region were removed in the course of cloning the GLUT-2 cDNA into the pCB-7 vector.
• Lines CGT-1 and CGT-2 exhibited less active expression of GLUT-2.
• Untransfected AtT-20j_ c cells and primary anterior pituitary cells did not contain detectable amounts of GLUT-2 mRNA, consistent with previous studies (Hughes et al . , 1991). 2. Expression of GLUT-2 protein in tissues and cell lines
• GLUT-2 protein in transfected AtT-20 ins cells was studied by immunofluorescent staining techniques, using an antibody raised against the C- terminal hexadecapeptide of GLUT-2 (Johnson et al . , 1990b) .
• CCT-5 and CGT-6 abundant GLUT-2 immunofluorescence was detected at the cell membrane as well as some intracellular signal that was mostly localized to regions of cell-cell contact. The fluorescent signal was blocked by preincubation of the antibody with the antigenic peptide and was not seen in untransfected cells or in cells transfected with the vector lacking the GLUT-2 insert.
• AtT- 20 ⁇ cells not only have the capacity to produce GLUT-2 mRNA and protein but also sort the protein to the cell membrane, as occurs in both islets and liver (Thorens et al . , 1988; Orci et al . , 1989; Tal et al . , 1990; Orci et al . , 1990).
• Preferential expression at regions of cell- cell contact is in keeping with a recent report (Orci et al .
• the GLUT-2 cDNA has been cloned from both liver (Thorens et al . , 1988) and islets (Permutt et al . , 1989; Johnson et al . , 1990a), two tissues with high Km glucose transport activity. Although the cDNA has been expressed in bacteria (Thorens et al . , 1988) and oocytes (Permutt et al . , 1989), these systems have not been used for kinetic studies. Thus, direct evidence that the GLUT-2 cDNA encodes a protein that confers the high Km glucose transport activity has not been presented to date.
• FIG. 3A shows a plot of the concentration dependence of glucose uptake in the AtT-20 ins cell lines, and demonstrates the dramatically increased rates of glucose transport in lines CGT-5 and CGT-6 relative to the untransfected (parental) AtT-20 ins cells.
• Lineweaver- Burke analysis of the data showed that the CGT-5 and CGT- 6 lines had apparent Kms for glucose of 16 and 17 mM and Vmax values of 25 and 17 mmoles/min/liter cell space, respectively ( Figure 3B) .
• the untransfected parental AtT-20 ⁇ line had an apparent Km for glucose of 2 mM and a Vmax of 0.5 mmoles/min/liter cell space ( Figure 3C) , consistent with its expression of the GLUT-1 mRNA (Hughes et al . , 1991), which encodes the low Km glucose transporter found in most clonal cell lines (Flier et al . , 1987; Birnbaum et al . , 1987).
• the transfected AtT-20 ⁇ cells have glucose transport kinetics that are remarkably similar to isolated, dispersed islets of Langerhans, which have a Km of 18 mM for glucose and a Vmax of 24 mmoles/min/liter cell space (Johnson et al . , 1990a).
• the GLUT-2 cDNA clearly encodes the protein responsible for the high Km glucose transport activity in islets and liver, and is capable of transferring this activity into the AtT-20 ins cell line.
• FIG. 4A compares glucose-stimulated insulin release from AtT-20ins cells and CGT-6 cells, expressed as mU insulin released/mg total cellular protein. Consistent with previous results (Hughes et al . , 1991), glucose had no significant effect on insulin release from parental AtT-20ins cells. AtT-20ins cells transfected with the pCB7 vector lacking a GLUT-2 insert were also found to be unresponsive to glucose. GLUT-2 transfected cells, in contrast, are clearly glucose responsive (data are shown for line CGT-6 only; results for line CGT-5 were qualitatively identical) .
• glucose potentiates the insulin secretory response to various /3-cell secretagogues, including agents that increase intracellular cAMP levels (Ullrich and Wollheim, 1984; Malaisse et al . , 1984).
• the potentiating effect of glucose on insulin secretion in the presence of forskolin, dibutyryl cAMP, and IBMX was therefore studied.
• Glucose had a modest stimulatory effect on forskolin stimulated insulin release from parental AtT-20ins cells, expressing the data either as insulin release/mg cellular protein (Figure 4A) or as insulin release/mg cellular DNA ( Figure 4B) .
• the response was unchanged by glucose concentration over the range of l-5mM, and similar potentiating effects of glucose on dibutyl cAMP and IBMX induced secretion were also observed.
• Insulin secretion studies involved static incubation of cells with the secretagogue for three hours, and thus provided little information about the dynamics of insulin release.
• the inventors succeeded in growing the parental and transfected AtT-20ins cell lines on gelatin beads in liquid culture, thus allowing their secretory properties to be studies by perfusion with glucose containing media. Cells grown in this configuration released insulin within minutes of glucose stimulation. Furthermore, the insulin secretory response exhibits a first intense and a second less intense but sustained phase, as is characteristic of normal ⁇ cells.
• AtT-20ins cells with the GLUT-2 cDNA results in a substantial increase in intracellular insulin content, despite the fact that insulin gene expression is driven by the glucose insensitive Rous sarcoma virus long-terminal repeat enhancer/promoter in these cells.
• Native AtT-20ins cells and the GLUT-2 transfected CGT-6 cells were grown for 3 days in media supplemented with low (lmM) or high (25mM) glucose. The CGT-6 cells were found to contain 3.6-fold and 5.4-fold more insulin than the AtT-20ins cells when studied at low and high glucose, respectively (p ⁇ 0.001 for both comparisons) .
• the enhanced sensitivity to glucose is not explained by the kinetics of glucose transport, since both the CGT-5 and CGT-6 lines transport glucose with a velocity and concentration dependence that is virtually identical to islets.
• stimulation of insulin secretion at low glucose concentrations might be explained by differential regulation of glucose phosphorylation in AtT-20 ins cells relative to ⁇ cells.
• the ratio of hexokinase:glucokinase activity in these cells was therefore compared with activities found in normal islets of Langerhans and liver. Studies from this and other laboratories (Iynedjian et al .
• glucokinase gene is alternatively regulated and processed in liver and islets, resulting in distinct transcripts that predict proteins with unique N-termini; the Km for glucose of both isoforms is in the range of 8-10 mM.
• AtT-20 ⁇ ns cells express the islet isoform of glucokinase (Hughes et al . , 1991) .
• Both lines have a total glucose phosphorylating capacity that is similar to that in liver and islets.
• glucokinase activity in AtT-20 ins cells is only 32% of the glucokinase activity in islets and 10% of that in liver.
• glucokinase represents only 9% of the total glucose phosphorylating activity of AtT-20 ⁇ ns cells (the remaining 91% is presumably due to hexokinase activity) , as compared to 24% in normal islets and 86% in normal liver.
• the altered hexokinase:glucokinase ratio in AtT- 20 ⁇ cells may result in low Km glucose metabolism that accounts for the insulin secretory response at low glucose concentrations.
• Glucokinase activity was determined with the same assay as used for total glucose phosphorylation at 50 ( a ) or 15 ( b ) mM glucose, except in the presence of 10 mM glucose-6-phosphate to inhibit hexokinase. Values represent the means + SEM for 3 independent determinations for liver and islets and 4 independent determinations for untransfected (parental) and GLUT-2 transfected (line CGT-6) AtT-20 ins cells.
• AtT-20 ins cells are grown to a density of 5 x 10" cells per 100 mm dish and harvested by incubation at 37°C with a solution of 0.02% EDTA in phosphate buffered saline (PBS) . After washing the cells in DMEM media containing 20 mM Hepes, approximately 1.5 x 10 ⁇ cells are transferred onto 12 mm poly-L-lysine coated glass coverslips, to which they adhere during a 30 minute incubation at 37°C. The cells are then fixed for 30 minutes with varying amounts (0.5-3.0%) of paraformaldehyde, depending on the extent of fixation that is desired.
• PBS phosphate buffered saline
• a light fixation (0.5% paraformaldehyde) for studies with anti-GLUT-2 antibodies or serum, the inventors have found a light fixation (0.5% paraformaldehyde) to be most appropriate.
• a serum sample usually diluted 1:1 in BSA
• an antibody designated X617 raised against the unique extracellular loop peptide of the rat GLUT-2 transporter is used, diluted 1:100 in PBS (the antibody is raised against a peptide with sequence DAWEEETEGSAHIV (SEQ ID NO:l), as found at amino acids 64-77 of the rat GLUT-2 primary structure) .
• FACS fluorescence activated cell sorter
• Cells are prepared for FACS analysis essentially as described for the microscope slide approach except that incubations are done with cells in suspension rather than attached to microscope slides. Briefly, near-confluent tissue culture plates containing parental AtT-20 ins cells or GLUT-2 expressing CGT-6 cells are washed with PBS, and then exposed to 0.02% EDTA for 15 minutes at 37°C to dislodge cells from the plate. The dispersed cells are washed with culture media followed by PBS and used as intact, live cells or fixed gently in 0.5% paraformaldehyde/PBS for 15 minutes at room temperature. The live or fixed cells are then incubated in 100 ⁇ l of patient serum: PBS in a ratio of 1:1, with 0.002% EDTA added to keep the cells dispersed.
• the cells After a one hour incubation at 4°C, the cells are washed 3 times with PBS and incubated with anti-human IgG or anti-human globulin fraction labeled with phycoerythrin for 1 hour at 4°C. Subsequently, the cells are washed with PBS and run through a flow cytometer in the red channel. Phycoerythrin is chosen as the fluorescent marker because it was found that the AtT-20 ins cells have a natural fluorescence in the green channel that is used for FITC- labeled antibodies. B. Results
• the FACS method was found to be appropriate for detecting the presence of a specific immune complex.
• GLUT-2 expressing AtT-20 ins cells were treated with the anti-GLUT-2 antibody X617 and with anti-rabbit IgG second antibody labeled with phycoerythrin (set one) .
• Cells were also incubated with antibody X617 after it had been preincubated with GLUT-2 expressing AtT-20ins cells (set two) .
• the cells are loaded into the FACS, which passes the cells one-by-one past a light source set at a wavelength that will excite the fluorescent marker of the second antibody.
• the cells then pass a detector which measures the fluorescence emission from the cells. Data are plotted as a histogram of fluorescence intensity.
• the following example is directed to an analysis of serum samples from diabetic patients and non-diabetic subjects.
• the interactions of purified IgG samples with rat islet cells and engineered AtT20 ins cells was investigated using both binding assays and assays based on the inhibition of glucose uptake.
• the following results demonstrate the usefulness of such analyses in diagnostic and prognostic tests.
• AtT20 ins cells and GLUT-2-expressing AtT20 jns cells were harvested by removal of cells from plates with a rubber policeman in Dulbecco's phosphate-buffered saline, pH 7.6. Following two washes in Dulbecco's phosphate- buffered saline by sedimentation at 500 x g for 30 seconds at room temperature, the cells were divided into
• R-PEAb R-phycoerythrin-labeled goat antihuman, heavy chain-specific IgG
• Flow cytometry was performed on a FACScan (Becton Dickinson) flow cytometer.
• Forward scatter threshold was set at 100 using the E-01 forward scatter detector.
• Linear amplifier gains were 6.18 for forward scatter and 1.22 for 90° angle light scatter with a photomultiplier setting of 274 volts.
• Forward and 90° angle light scatter were read on linear scale and fluorescence measurements were made on logarithmic scale. Setting adjustments were made by using a sample of unstained cells and increasing the photomultiplier voltage so that events were on-scale during observation of 530 ⁇ 15 nm (FL1) histogram.
• a sample of cells stained only with R- phycoerythrin-labeled goat antihuman IgG (R-PEAb) was then used to adjust the photomultiplier voltage so that events were on-scale during measurement of a 575 ⁇ 13 n (FL2) histogram.
• a control specimen was then used to adjust the FL2 photomultiplier tube voltage such that FL2 histogram events remain minimally on scale.
• the FL2-FL1 compensation was adjusted to minimize fluorescence overlap and for these cells a setting of 45.9% was used. Acquisition of 10 4 events per specimen were required and data were stored on floppy discs for analysis.
• the following assays were performed to investigate the effects of human IgG on glucose uptake by intact cells.
• the assays were performed as described by Johnson and Unger, PCT Patent Application WO 91/13361, incorporated herein by reference.
• Rat islet cells exhibit two kineticall'y distinct facilitated diffusion glucose transporter functions, a high Kr ⁇ function ascribed to GLUT-2 and a low I ⁇ transport function attributed to unidentified transporter. Results from a detailed kinetic analysis of the inhibition of glucose transport into islet cells induced by diabetic IgG indicated that the inhibition was directed against the high J ⁇ or GLUT-2 mediated function. As a test of the specificity of inhibition of GLUT-2, additional measurements in GLUT-1 expressing AtT20 jns cells were made.
• GLUT-1-transfected AtT20 ins cells express GLUT-1 constitutively, the GLUT-1-transfected cell line overexpresses this protein and exhibits a greater than 10-fold increase in the velocity of glucose uptake which increases the accuracy of the transport measurement.
• Glucose uptake in GLUT-1-transfected AtT20 ins cells treated with IgG from new-onset IDDM patients was indistinguishable from transport in the presence of IgG from nondiabetic individuals. These data indicate that IgG from new-onset IDDM patients does not inhibit glucose transport in AtT20 ins cells that express a facilitative glucose transporter other than GLUT-2.
• Nondiabetic 2/31 (6%) 29/31 (94%) Sensitivity 23/30 (77%)
• Insulin secretion from CGT-6 (GLUT-2 expressing AtT-20 jns ) cells was evaluated using a column perfusion technique (Knudsen et al . , 1983). Cells were grown in liquid culture on microcarrier beads (InvitroGen) .
• Approximately 50 x 10 6 cells were harvested by gentle centrifugation (500 rpm in a Sorvall RT6000B desk top centrifuge) , resuspended in 4ml Krebs-Ringer salt (KRS) solution, pH 7.4, and loaded onto a Pharmacia PlO/10 column. A cell count was obtained immediately before loading the column in the following manner. An aliquot of cells was taken, the beads digested with 1.2 U/ml Dispase (Boehringer Mannheim) , the cell clumps were dispersed by extrusion through a 25 gauge needle and the cells were counted directly.
• KRS Krebs-Ringer salt
• the top plunger of the column was gently inserted and the whole apparatus was submerged in a 37°C water bath.
• the cells were then perfused as described below.
• Native AtT-20 ⁇ cells, as well as GLUT-2 and GLUT-1 transfected lines were grown in liquid culture on microcarrier beads (InvitroGen) , harvested into a Pharmacia PlO/10 column, and washed with HBSS lacking glucose for 15 minutes.
• the capacity of lines CGT-6 (GLUT-2 transfected) , CGT1-15 (GLUT-1 transfected) and the parental AtT-20 ins cells to secrete insulin in response to glucose was compared. Perfusion with HBSS lacking glucose was continued after the 15 minute wash-out for an additional 25 minutes. During this period, there was a gradual decline in insulin release from all three cell lines. Phase II was initiated by switching to HBSS buffer containing 5mM glucose.
• Insulin secretion from the CGT-6 cells persisted at the glucose-stimulated level for approximately 10 minutes after the switch to buffer lacking glucose, but then declined rapidly. The low level of insulin release from parental AtT-20 ⁇ cells and CGT1-16 cells was further reduced during perfusion with glucose free media. In phase IV, cells were switched back to buffer containing 5 mM glucose. The CGT-6 cells again showed a much stronger secretory response to glucose, but the response was less rapid (requiring 15 minutes to reach maximum) , and was without an obvious first phase and second phase.
• Recombinant adenovirus (Gluzman et al . , 1982) containing distinct cDNAs (AdCMV-cDNA) were prepared according to the following method, which relates particularly to the preparation of recombinant adenovirus containing /3-galactosidase (AdCMV- ⁇ GAL) (Herz and Gerard, 1993) .
• a cDNA encoding the E. coli / 3-galactosidase carrying the SV40 T antigen nuclear targeting signal (Bonnerot et al . , 1987) was inserted into pACCMV to create a novel construct.
• the resulting expression cassette comprises the cytomegalovirus (CMV) promoter, the / 3-galactosidase cDNA and the mouse protamine polyadenylation signal, and is flanked by adenovirus type 5 sequences.
• CMV cytomegalovirus
• the El region of adenovirus is replaced by the foreign gene.
• the resulting plasmid was cotransfected into 293 cells (Graham et al . , 1977) together with a plasmid carrying the complete adenovirus type 5 genome (pJM17) (McGrory et al . , 1988). Plasmid sequences conferring ampicillin and tetracycline resistance are inserted into the virus genome at map position 3.7.
• the molecular strategy employed to produce recombinant adenovirus is based upon the fact that, due to the packaging limit of adenovirus, pJM17 cannot efficiently form plaques on its own.
• homologous recombination between the pAC-cDNA plasmid and pJM17 within a transfected cell results in a viable virus that can be packaged and form plaques only on 293 cells which express adenovirus ElA proteins.
• Co-transfection was performed as follows: 293 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, from GIBCO-BRL) containing 10% fetal bovine serum (FBS, from Hyclone) in a humidified 5% C0 2 atmosphere. Confluent 10 cm dishes were split to three 6 cm dishes the day before calcium phosphate cotransfection of an appropriate amount of DNA, such as 4 ⁇ g pJM17, 4 ⁇ g pACCMV- ⁇ GAL, and 12 ⁇ g HeLa DNA as carrier.
• DMEM Dulbecco's modified Eagle's medium
• FBS fetal bovine serum
• a 15% glycerol shock was used to boost transfection efficiency and the cells were overlaid with 0.65% Noble agar in DMEM containing 2% FBS, 50 ⁇ g/ml penicillin G, 10 ⁇ g/ml streptomycin sulfate, and 0.25 ⁇ g/ml fungizone (GIBCO) .
• Monolayers were incubated for approximately 10 days until the appearance of viral plaques.
• plaques were picked, suspended in DMEM containing 2% FBS, and used to infect a new monolayer of 293 cells. When greater than 90% of the cells showed infection, viral lysates were subjected to a freeze/thaw cycle and were designated as primary stocks. Recombinant virus with the correct structure was verified by preparation of viral DNA from productively-infected 293 cells, restriction analysis, and Southern blotting. Secondary stocks were subsequently generated by infecting 293 cells with primary virus stock at a multiplicity of infection of 0.01 and incubation until lysis.
• the large scale production of recombinant adenovirus can be performed in 293 cells grown either in 15 cm culture dishes or in suspension using Joklik's calcium-free MEM (GIBCO) supplemented with 10% FBS, as follows. Infected cells may be lysed 48 hours post ⁇ infection with Dulbecco's PBS (GIBCO) containing 1 mM MgCl 2 and 0.1% NP-40. Virus-containing extracts should then be centrifuged, such as at 12,OOOxg for 10 minutes, to remove debris before precipitation of the virus particles by addition of 0.5 vol 20% polyethylene glycol (PEG) 8000, 2.5 M NaCl and incubated on ice' for 1 hour.
• PEG polyethylene glycol
• Virus can be collected by centrifugation at 12,OOOxg for 10 minutes, resuspended in isotonic saline (135 mM NaCl, 5 mM KCl, 1 mM MgCl 2 , 10 mM Tris-HCl, pH 7.4), and dialyzed against the same buffer overnight before sterilization through a 0.22 ⁇ m filter.
• Virus can then be collected from the lower interface and dialyzed overnight at 4°C versus isotonic saline.
• AdCMV- ⁇ GAL recombinant virus
• AdCMV/3gal titer of approximately 5 x 10 7 pfu/ml
• virus was applied to cells at a multiplicity of infection of approximately 10:1.
• ⁇ -galactosidase expression was scored following dispersal of the. islets and incubation with substrate and colorimetric reagents.
• recombinant adenoviruses containing genes involved in glucose sensing, including GLUT-2, GLUT-1, glucokinase and mutant glucokinases associated with Maturity-Onset Diabetes of the Young (MODY) were prepared according to the methodology set forth above. The said genes are under the control of the CMV promoter and adenovirus containing the insulin promoter have also been prepared. The structures of the recombinant viruses generated were verified by restriction enzyme digestion and Southern blotting.
• /3-galactosidase expression in isolated rat islets treated with AdCMV-/3GAL recombinant adenovirus is demonstrated in Figure 5.
• /3-galactosidase expressing cells are indicted by their blue color.
• Figure 5C shows a multicell aggregate that was prepared by trypsin-mediated dispersal of intact islets prior to incubation with the chromogenic substrate to allow quantitation of the efficiency of gene transfer, viewed at 40 x magnification.
• Figure 5D a control islet treated with / 3-galactosidase substrate after 4 days in culture without exposure to AdCMV-/3GAL, is viewed at 20 x magnification.
• Adenovirus-mediated delivery of /3-galactosidase to islets in vivo was also investigated.
• Recombinant adenovirus containing the /3-galactosidase gene directed by the CMV promoter was infused via the jugular vein into normal rats over 2 four hour periods at a concentration of approximately 1 x 10' pfu/ml.
• Pancreas tissue was removed, frozen and sliced into thin sections. Sections were fixed with 0.5% glutaraldehyde and then incubated with the /3-galactosidase chromogenic substrate X-gal. Animals were infused with the AdCMV-/3gal virus for five days.
• Islets from Zucker diabetic fatty (ZDF) rats which completely lack GLUT-2 expression and which fail to respond to levels of glucose that stimulate normal islets to secrete insulin, were isolated for use in gene transfer studies. Recombinant adenovirus containing the GLUT-2 cDNA was administered to cultured islets from such animals allowing the effects of this maneuver on the insulin secretion response to glucose to be assayed.
• FIG. 6 Adenovirus-mediated expression of variant glucokinases in CV-1 cells was studied ( Figure 6) .
• CV-1 cells were infected with recombinant adenoviruses at a multiplicity of infection of 10.
• Glucokinase expression was assayed by western blot hybridization analysis using 100 ⁇ g islet protein and an antibody that detects rat islet glucokinase (Antibody U343, Quaade et al . , 1991). Lanes contain the following samples: Lane 1, Islet glucokinase expressed in bacteria (Quaade et al .
• Lane 2 Uninfected islets; Lane 3, Islets infected with recombinant adenovirus containing the 3-galactosidase cDNA; Lane 4, Islets infected with recombinant adenovirus containing cDNA encoding a glucokinase variant containing an amber mutation (premature stop codon) in place of glutamic acid 279 that is not synthesized as a stable protein; Lane 5, Islets infected with recombinant adenovirus containing cDNA encoding a glucokinase variant with a point substitution that results in replacement of amino acid glycine 261 with arginine; Lane 6, Islets infected with recombinant adenovirus containing cDNA encoding a glucokinase variant with a point substitution that results in replacement of amino acid glutamic acid 279 with glutamine ( Figure 6A) .
• glucokinase enzymatic activity measured in crude homogenates of the CV-1 cells is described in the western blot ( Figure 6B) .
• Expression of the Gly261Arg and Glu270Gln variants increase glucokinase activity in CV-1 cells in accord with their reported enzymatic properties as assayed in bacteria (Gidh-Jain et al . , 1993). These data show that recombinant adenovirus allows highly efficient glucokinase gene transfer into cell lines such as CV-1.
• Lanes contain the following samples: Lane 1, Uninfected islets; Lane 2, Islets infected with recombinant adenovirus containing the /3-galactosidase cDNA; Lane 3, Islets infected with recombinant adenovirus containing cDNA encoding a glucokinase variant containing an amber -Ill- mutation (premature stop codon) in place of glutamic acid 279 that is not synthesized as a stable protein; Lane 4, Islets infected with recombinant adenovirus containing cDNA encoding a glucokinase variant with a point substitution that results in replacement of amino acid glycine 261 with arginine; Lane 5, Islets infected with recombinant adenovirus containing cDNA encoding a glucokinase variant with a point substitution that results in replacement of amino acid glutamic acid 279 with glutamine; Lane 6, empty; Lane
• Diabetic patients for whom the medical indication for adenovirus-mediated gene transfer treatment has been established would be tested for the presence of antibodies directed against adenovirus. If antibodies are present and the patient has a history of allergy to either pharmacological or naturally occurring substances, application of a test dose of on the order of 10 ⁇ to 10 ⁇ recombinant adenovirus under close clinical observation would be indicated.
• recombinant adenovirus expressing a suitable candidate gene or genes involved in glucose sensing particularly either the GLUT-2 and/or glucokinase genes, under the control of ⁇ cell specific promoter/enhancer elements, such as the insulin promoter, would be prepared and purified according to a method that would be acceptable to the Food and Drug Administration (FDA) for administration to human subjects.
• FDA Food and Drug Administration
• Such methods include, but are not limited to, cesium chloride density gradient centrifugation, followed by testing for efficacy and purity.
• Virus may be administered to patients by means of intravenous administration in any pharmacologically acceptable solution, either as a bolus or as an infusion over a period of time. Generally speaking, it is believed that the effective number of functional virus particles to be administered would range from 1 x 10 10 to 5 x 10 1 . If warranted or desi.red, recombi.nant adenovi.rus could be delivered closer to the site of the target cells using a catheter.
• adenovirus employed will be replication incompetent, no deleterious effect of the virus itself on subject health is anticipated. However, patients would remain hospitalized during the treatment for at least 48 hours to monitor acute and delayed adverse reactions. Glucose-tolerance tests would be monitored twice daily to follow the efficacy of the gene transfer.
• pancreatic biopsy in which the pattern of expression of the transferred gene could be directly assessed. This would also supply information about the number of islet cells that have taken up the transferred gene and about the relative promoter strength in the human pancreas. Based on the data obtained adjustments to the treatment may be desirable. These adjustments might include adenovirus constructs that use different promoters or a change in the number of pfu injected to ensure a infection of more, or all, islet cells without unphysiological overexpression of the recombinant genes.
• exogenous genes transferred in vivo by adenovirus can persist for extended periods of time.
• Therapeutically effective long-term expression of virally transferred exogenous genes will have to be addressed on a case by case basis. Marker genes are limited in their usefulness to assess therapeutically relevant persistence of gene expression as the expression levels required for the amelioration of any given genetic disorder might differ considerably from the level required to completely cure another disease.
• Marker genes are limited in their usefulness to assess therapeutically relevant persistence of gene expression as the expression levels required for the amelioration of any given genetic disorder might differ considerably from the level required to completely cure another disease.
• the relatively high expression necessary to treat ⁇ j -antitrypsin deficiency which results from consumption of molecule whilst executing its desired function, has not yet been obtained (Jaffe et al . , 1992).
• correction of normal islet cell function in NIDDM patients will require considerably lower expression of the transferred gene(s), as such intracellular molecules fulfill their physiological roles without any consequent inactivation.
• adenovirus type 5 This infiltration phenomenon has been previously observed in the mouse lung following intranasal inoculation of 10 pfu of adenovirus type 5 (Ginsberg et al . , 1991). Plasma levels of the liver marker enzymes alanine aminotransferase and aspartate aminotransferase show a 20 fold increase following administration of 2x10 pfu intravenously. Therefore, the dose of adenovirus to be administered must be appropriately controlled so as to minimize untoward side effects of the gene therapy regimen, and a more extensive careful evaluation will be necessary to ensure that adenovirus is safe for human in vivo gene therapy.
• compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the composition, methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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