EP1497305A2 - Proteine 1 de liaison a l'adn sensible a l'insuline et methodes destinees a reguler des genes sensibles a l'insuline - Google Patents

Proteine 1 de liaison a l'adn sensible a l'insuline et methodes destinees a reguler des genes sensibles a l'insuline

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Publication number
EP1497305A2
EP1497305A2 EP02794127A EP02794127A EP1497305A2 EP 1497305 A2 EP1497305 A2 EP 1497305A2 EP 02794127 A EP02794127 A EP 02794127A EP 02794127 A EP02794127 A EP 02794127A EP 1497305 A2 EP1497305 A2 EP 1497305A2
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EP
European Patent Office
Prior art keywords
nucleic acid
erdbp
cell
irdbp
seq
Prior art date
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EP02794127A
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German (de)
English (en)
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EP1497305A4 (fr
Inventor
Betty C. Villafuerte
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Emory University
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Emory University
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Publication of EP1497305A4 publication Critical patent/EP1497305A4/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present mvention relates generally to an Insulin-Responsive Sequence DNA Binding Protein (IRSBP) gene, specifically IRDBP-1, of mammals, and to corresponding IRDBP-1 proteins and coding sequences thereof. More specifically, the invention relates to a nucleotide sequence encoding an IRDBP-1 protein, to methods and compositions that employ this coding sequence and protem, to the use of therapeutic agents that mimic or facilitate the action of IRDBP-1, and to nucleotide sequences useful in diagnostic methods and treatment of diabetes, obesity, insulin resistance syndrome and other pathological ailments.
  • IRSBP Insulin-Responsive Sequence DNA Binding Protein
  • Type II diabetes mellitus Although ⁇ -cell dysfunction is detectable in all diabetic patients whose pancreas exhibits an inability to produce sufficient insulin to maintain glucose levels in the normal range , the rapid increase in the prevalence of diabetes over the past several decades is apparently more likely to be due to insulin resistance
  • diabetes did not affect insulin action on target tissues.
  • the current epidemic of Type II diabetes in the United States is usually attributed to the aging of the population, the increased prevalence of obesity and sedentary activity, and the enrichment of the population with ethnic groups that may have a genetically predisposed inability ofthe pancreas to meet the challenge of increased insulin resistance or pancreatic dysfunction.
  • the high incidence of diabetes represents a significant economic burden, such that approximately $92 billion in health care expenditures in 1992 were diverted to the treatment of diabetes.
  • Insulin resistance is a key factor in the pathogenesis of Type U diabetes, and can precede by decades abnormal insulin secretion and the onset of clinical diabetes. Resistance to insulin action involves all major target tissues, i.e., skeletal muscle, liver and fat. Although insulin resistance appears to involve defects in insulin signaling at the post-receptor level, the mechanism of insulin resistance remains poorly understood.
  • IRS-1 insulin receptor substrate-1
  • SH2 Src homology 2
  • ATLANTA 333137vl 9 effects.
  • Each of these cytoplasmic substrates and the activating regulatory loop involved represents a potential linkage to the development of insulin resistance.
  • the substantial number of signaling circuits involved including interacting, bypassing and overlapping pathways, the involvement of numerous serine/threonine kinases and phosphatases, and still uncharacterized links, characterize the complexity ofthe signaling from the insulin signal at the cell surface receptor to targets within the cell.
  • One approach to the study of insulin interactions with cells is to select a physiological action of insulin and then trace back toward the receptor, an approach known as the target backward approach.
  • This target backward approach has yielded information concerning the mechanism of insulin regulation by focusing on the genetic regulation of the insulin-regulated gene insulin-like growth factor binding protein-3 (IGFBP-3).
  • IGFBP-3 insulin-regulated gene insulin-like growth factor binding protein-3
  • NTDDM non-insulin dependent Type II diabetes mellitus
  • a series of adapter proteins or substrates link the receptor tyrosine kinases to gene transcription, and determine the response to insulin in a given cell or tissue.
  • IRBPs insulin- responsive binding proteins
  • IGF-I and -II Insulin-like growth factors I and II
  • IGFs are proteins that have insulin-like metabolic and trophic effects and mediate some of the peripheral actions of growth hormone. IGFs also have a role in wound healing by stimulating fibroblasts to produce collagen and induce hematopoiesis through an erythropoietin-like activity. Studies have also shown that certain cancer cells, such as from breast and kidney, produce IGFs. IGF production in cancer cells auto-regulates cell proliferation and the production of a vascular system required for growth of the tumor mass. IGFs have also been implicated in diabetic retinopathy by stimulating endothelial and fibroblast proliferation.
  • IGFBPs IGF-binding proteins
  • IGFBP-3 is inhibitory to IGF action. Furthermore, IGFBP-3 can: (a) mediate the growth inhibitory actions of transforming growth factor- ⁇ (TGF- ⁇ ), retinoic acid, anti-estrogens and fibroblast growth factor, (b) mediate the induction of apoptosis by the tumor suppressor gene p53, and (c) travel to TGF- ⁇ , transforming growth factor- ⁇ , retinoic acid, anti-estrogens and fibroblast growth factor, (b) mediate the induction of apoptosis by the tumor suppressor gene p53, and (c) travel to TGF- ⁇
  • TGF- ⁇ transforming growth factor- ⁇
  • retinoic acid retinoic acid
  • anti-estrogens fibroblast growth factor
  • ATLANTA 333137vl A the cell nucleus, potentially directly regulating the transcription of critical growth inhibitory genes independent of IGF-I.
  • IGFBP-3 The levels of IGFBP-3 in serum and liver mRNA are highest during puberty and adult life. Unlike other IGFBPs, IGFBP-3 levels increase in the presence of anabolic hormones such as insulin and growth hormone. Dependence on growth hormone (GH) has been inferred from the deceased levels of IGFBP-3 in hypopituitary subjects and GH-deficient children and increased levels in acromegalic patients. Additionally, IGFBP-3 production is inhibited at the level of gene expression by glucocorticoids. The mechanisms by which IGFBP-3 is regulated are complex. IGFBP-3 may undergo post-translational processing to yield various proteolytically cleaved, phosphorylated, and glycosylated products.
  • GH growth hormone
  • IGFBP-3 can also associate with the cell surface and extracellular matrix; dissociation of cell-associated IGFBP-3 is one mechanism by which IGF-1 promotes release of IGFBP-3 into conditioned medium by fibroblasts and breast cancer cells.
  • Insulin increases IGFBP-3 expression by stimulating the rate of gene transcription rather than by stabilization of mRNA transcripts. This enhancement is mediated through a cis-regulatory insulin-responsive element (IRE) localized to the - 1150 to -1124 bp region ofthe gene encoding IGFBP-3.
  • the IGFBP-3 IRE comprises the nucleotide dyad ACC(A/G)A which has a strong resemblance to the recognition sequence of ETS-related transcription factors, namely AGGAA, which is within the IRE of both the prolactin and somatostatin genes.
  • the 10-bp core sequence of the IGFBP-3 IRE that is most critical for insulin responses had no significant consensus sequence similarity to previously identified transcription factor binding sites. What was not known, however, was any protein or other factor that would mediate a cellular response to insulin and which directly binds to such insulin-response elements like the IRE of IGFBP-3.
  • the present invention relates to a novel protein called Insulin-Responsive DNA Binding Protein- 1 (IRDBP-1) and nucleotide sequences that encode it.
  • IRDBP-1 is capable of binding to nucleic acid regions associated with genes that respond when cells are exposed to insulin or insulin-like factors. IRDBP-1 regulates genes important in mediating the insulin response in humans and animals and in regulating pathological conditions such as diabetes, obesity, insulin-resistant syndrome and cell proliferative disorders.
  • One aspect of the present invention relates to isolated or non-naturally occurring nucleic acid molecules that encode at least a portion of a human or animal
  • ERDBP-1 protein or a variant thereof The present invention provides isolated molecules that can hybridize to nucleic acid sequences of the genome of a human or animal and which encode an IRDBP-1 protein or variants thereof.
  • the present invention further provides nucleic acids that are fragments or derivatives of cDNA molecules comprising at least in part a region of the ERDBP-1 coding region and/or an untranslated region of the cDNA, wherein the fragments may be used as probes specific for hybridizing to, and detecting, nucleic acid molecules that encode at least in part a region ofthe IRDBP-1 protein.
  • recombinant cells, tissues and animals containing non-naturally occurring recombinant nucleic acid molecules encoding IRDBP-1 including expression vectors for the expression of IRDBP-1, antibodies to the IRDBP-1 proteins, assays utilizing the IRDBP-1 polypeptide, and methods relating to all of the foregoing. Also within the scope of the present invention is the development of therapeutic and diagnostic agents that mimic, facilitate or inhibit the action of IRDBP-1, and/or are based on relationships to the structure and action of IRDBP-1.
  • the invention further provides non-naturally occurring recombinant nucleic acid molecules encoding IRDBP-1 that can be delivered to a cell or an organism.
  • the recombinant nucleic acid may comprise IRDBP-1 -related sequences, functional derivatives thereof, and a vector or a promoter effective to initiate transcription in a
  • the recombinant nucleic acid molecule can alternatively contain transcription regulatory sequences functional in a particular cell, a sequence complementary to a mRNA sequence encoding an IRDBP-1 polypeptide and transcriptional control sequences functional in that cell.
  • the present invention still further provides oligopeptides having amino acid sequences derived from the amino acid sequence of a human or animal IRDBP-1 protem that may be used to induce the formation of polyclonal or monoclonal antibodies that specifically bind to at least one region of the IRDBP-1 protein from human or animal.
  • the antibodies may be used for, but are not limited to, the detection and assay of IRDBP-1 in biological samples, or the purification of the IRDBP-1 protem. Diagnostic kits for the detection of ERDBP-1 in biological samples are also within the scope ofthe present invention.
  • the invention also provides a recombinant cell or tissue containing non- naturally occurring recombinant nucleic acid molecules coding for an ERDBP-1 polypeptide or a portion thereof.
  • the ERDBP-1 coding sequence may be expressed under the control of its genomic regulatory elements, or may be under the control of exogenous regulatory elements including an exogenous promoter.
  • the present invention further provides for the production of animals that have modified nucleic acids encoding at least a portion ofthe IRDBP-1 protein, or have the IRDBP-1 gene inactivated.
  • the present invention further provides for methods of gene therapy and pharmaceutical compositions including antisense and sense nucleic acids that will modulate, in the human or animal, the activity of the IRDBP-1 gene or the ERDBP-1 protein encoded therein.
  • the invention features methods for identifying mammalian cells containing an IRDBP-1 polypeptide, or a related sequence.
  • Such methods comprise identifying the ERDBP-1 polypeptide in mammalian cells using techniques that are routine and standard, in the art, for example, PCR amplification, and Northern, Western, Southern and Southwestern blotting using oligonucleotides and derivatives thereof, or antibodies specific to the JRDBP-1 protein.
  • the present invention also relates to methods of detecting and treating proliferating cells, and the cells of humans or animals having diabetic disorders.
  • the present invention further relates to methods of activating or inhibiting the expression of the gene in humans or animals that encode an IRDBP-1 protein, wherein the proliferation of cells may be modified.
  • the present invention further contemplates that modulation of the activity of the ERDBP-1 protein or the expression thereof may be used to relieve the symptomatic effects of diabetes, particularly type II diabetes.
  • Fig. 1 shows the nucleotide sequence ofthe rat clone 52 cDNA SEQ ID NO: 2 with the translated protein sequence SEQ ED NO: 3 therefrom depicted in Fig. 2.
  • Fig. 2 shows the protein sequence of the rat clone 52 SEQ ED NO: 3 translated from the cDNA nucleic acid sequence SEQ ID NO: 2 depicted in Fig. 1.
  • Fig. 3 shows the nucleotide sequence of a nucleic acid probe SEQ ID NO: 4 derived from the rat clone 52 cDNA SEQ ID NO: 2 depicted in Fig. 1 that was subcloned into a transcription plasmid vector, transcribed and used as a riboprobe.
  • Figs. 4A-4B show the nucleotide sequence of the rat cDNA SEQ ED NO: 5 encoding a rat IRDBP-1 protein having the translated protein sequence SEQ ID NO: 11 as depicted in Fig. 9, wherein Fig. 4A shows the rat ERDBP-1 coding region (SEQ ED NO: 8) with the corresponding 3 -letter amino acid designation listed below its respective nucleotide triplet, and Fig. 4B shows the untranslated region that is 3' ofthe coding region in Fig. 4A.
  • Figs. 5A-5B show the nucleotide sequence of a truncated rat cDNA SEQ ID NO: 6 wherein Fig. 5 A is a partial region of a rat ERDBP-l coding region with the corresponding 3 -letter amino acid designation listed below its respective nucleotide triplet, and Fig. 5B is the untranslated region that is 3' ofthe coding region in Fig. 5 A.
  • Figs. 6A-6C show the nucleotide sequence ofthe human cDNA SEQ ED NO: 7 encoding a human ERDBP-1 protein having the translated protein sequence SEQ ID NO: 12 as shown in Fig. 10, wherein Fig. 6A shows a region 5 1 to the coding region, Fig. 6B is the coding region SEQ ID NO: 9, with the corresponding 3 -letter amino acid designation listed below each respective nucleotide triplet, and Fig. 6C is the untranslated region that is 3' ofthe coding region shown in Fig. 6B.
  • Fig. 7 shows the exons SEQ ED NOS: 16-41 that comprise SEQ ED NO: 7.
  • Figs. 8A-8B show the nucleotide sequence SEQ ED NO: 10 of a truncated variant human cDNA, wherein Fig. 8 A is a partial region of a human variant ERDBP-1 coding region with the corresponding 3 -letter amino acid designation listed below its respective nucleotide triplet, and Fig. 8B is the untranslated region that is 3' of the coding region in Fig. 8A.
  • Fig. 9 shows the amino acid sequence SEQ ED NO: 11 of the rat ERDBP-1 protein translated from the coding region of the rat IRDBP-1 cDNA, the sequence (SEQ ID NO: 8) of which is depicted in Fig. 4 A.
  • Fig. 10 shows the amino acid sequence SEQ ID NO: 12 ofthe human IRDBP-
  • Fig. 11 shows the amino acid sequence SEQ ED NO: 13 of the variant human ERDBP-1 protein translated from the coding region ofthe human ERDBP-1 cDNA, the sequence SEQ ED NO: 10 of which is depicted in Fig. 8 A.
  • Figs. 12A-12B show the nucleotide sequence of the truncated rat cDNA SEQ ID NO: 14, wherein Fig. 12A is a partial region ofthe rat ERDBP-1 coding region with the corresponding 3 -letter amino acid designation listed below its respective nucleotide triplet, and Fig. 12B is the untranslated region that is 3' of the coding region in Fig. 12 A.
  • Fig. 13 shows the nucleotide sequence of the rat ERDBP-1 -encoding DNA SEQ ID NO: 44.
  • Fig. 14 shows the nucleotide sequence of the human IRDBP-1 -encoding DNA SEQ ID NO: 45.
  • Fig. 15 shows the amino acid sequence ofthe rat ERDBP-1 SEQ ED NO: 47.
  • Fig. 16 shows the amino acid sequence of the human ERDBP-1 SEQ ED NO: 48.
  • Fig. 17A illustrates gel mobility shift of the -1150/-1117 bp IRE fragment of IGFBP-3 (SEQ ID NO: 1) by polypeptides derived from cDNA clones isolated using the yeast one-hybrid system.
  • Fig. 17B illustrates gel mobility shift analysis ofthe -1150/-1117 ERE fragment of IGFBP-3 (SEQ ED NO: 1) by cDNAs expressed as thioredoxin fusion proteins.
  • Fig. 18 illustrates a competition-binding assay of the polypeptide encoded by clone 52 binding to the -1150/- 1117 bp ERE fragment of IGFBP-3 (SEQ ID NO: 1).
  • ATLANTA 333137vl ⁇ Q Fig. 19 illustrates EREs derived from other genes competing with the IRE of IGFBP-3 (SEQ ED NO: 1) for binding to the protein product of clone 52.
  • Fig. 20 illustrates the expression of ERDBP-1 in the liver and other tissues.
  • RNAs from hepatic nonparenchymal cells 50 ⁇ g/lane treated with or without insulin for 24 hours (left panel) and from normal and streptozotocin- diabetic rat livers (right panel) were probed with the 3.4 kb ERDBP-1 probe.
  • Fig. 21 A shows ribonuclease protection assays of rat tissues using the 170 bp
  • nucleic acid SEQ ID NO: 5 as the probe.
  • Fig. 2 IB shows the relative expression of ERDBP-1 mRNA in different rat tissues.
  • Fig. 21C illustrates the tissue distribution of IRDBP-1 -specific mRNA in adipose tissue.
  • Fig. 22 illustrates the tissue distribution of IRDBP-1 -specific mRNA in human digestive tissue.
  • Fig. 23 illustrates IGFBP- 1 ERE reporter activity in CHO cells transfected with a vector containing HBP 1 -encoding sequence or a rat ERDBP-1 -encoding sequence (SEQ ED NO: 5 cotransfected with vectors), or the two vectors (each vector having the ERDBP-1 or HBP 1 -encoding sequence) were cotransfected.
  • Fig. 24 illustrates the effect of insulin on IGFBP-3 IRE-regulated reporter gene activity in response to the addition of insulin to cultured cells containing either expression vector or expression vector with SEQ ED NO: 5 therein.
  • ATLANTA 333137vl ⁇ ⁇ Fig. 25 A shows the detection of IRDBP-1 with an attached Trx-His tag using anti -His antibody or anti-IRDBP-1 cAb antibody.
  • Fig. 25B shows a Western blot of hepatic nuclear extracts from normal and streptozotocin-dibetic rats.
  • Fg. 26 shows a gel-shift assay with Trx-IRDBP-1 fusion protein and IGFBP-3 ERE.
  • Fig. 27 shows binding of rat hepatic nuclear extracts to IGFBP-3 ERE and secondarily probed with anti-IRDBP-1 cAb or nAb antibody.
  • Fig. 28 illustrates the effect of an ERDBP-1 expressing clone (SEQ TD NO: 5) on insulin regulation of glucose uptake in myoblasts.
  • Fig. 29 illustrates cofransfection of COS7 cells with 3.4, 4.8 and 5.043 kb IRDBP-1 -encoding cDNA and the IGFBP-3 ERE luciferase reporter.
  • Fig. 30 illustrates the effect of IRDBP-1 (SEQ ED NO: 14) on the glycogen content of cultured cells.
  • Fig. 31 illustrates the detection of ERDBP-1 mRNA within the hypothalamic portion ofthe brain by in-situ hybridization.
  • Fig. 32 illustrates the detection of ERDBP-1 mRNA in the lateral hypothalamus by in-situ hybridization.
  • Fig. 33 shows a comparison of ERDBP-1 mRNA in the lateral hypothalamic area of obese and lean rats by in-situ hybridization.
  • Fig. 34 illustrates ERDBP-1 expression in the solitary tract nuclei of lean and obese rats.
  • Fig. 35 illustrates an in-situ hybridization analysis of a rat brain section showing that ERDBP-l -specific mRNA is expressed in the pyramidal tract and decussations ofthe pyramidal tract in obese Zucker rats.
  • Fig. 36 illustrates an in-situ hybridization analysis showing the expression level of IRDBP-1 in the pyramidal area ofthe brain of a lean Zucker rat.
  • Fig. 37 illustrates ERDBP-1 expression in the olfactory bulb.
  • Fig. 38 illustrates ERDBP-l expression in the amygdala.
  • Fig. 39 illustrates immunohistochemical staining with an anti-ERDBP-1 antibody, or with pre-immune serum, of a section of rat pancreas.
  • Fig. 40 illustrates immunoflourescent detection of ERDBP-1 in HIT ⁇ cells.
  • Fig. 41 illustrates the detection of Glut4 and Glutl expression and the translocation of Glut4 in L6 cells with ERDBP-1 expression.
  • Fig. 42A illustrates immunohistochemical staining of rat kidney mesangial cells, using anti-IRDBP-1 antibody (right) or pre-immune serum (left).
  • Fig. 42B illustrates immunohistochemical staining ofthe endothelial lining of blood vessels ofthe kidney, using anti-IRDBP-1 antibody (right) or pre-immune serum (left).
  • ATLANTA 333137vl 13 Fig. 42C illustrates immunohistochemical staining ofthe rat brain, using anti- IRDBP-1 antibody.
  • Fig. 43 illustrates a Western blot analysis using anti-rat ERDBP-1 peptide polyclonal antibody and cell extracts from human vascular endothelial cells treated with and without insulin for 6 hours.
  • Fig. 44A illustrates a Western blot analysis showing IRDBP-1 in COS 7 cell extracts and co-immunoprecipitation of IRDBP-1 with both anti-Akt 1 (lanes 1, 2) and anti-Erk2 (lanes 3 , 4) that is increased with the addition of insulin.
  • Fig. 44B illustrates differential phosphorylation of IRDBP-1 in normal and streptozotocin-induced diabetic rats, and in Zucker lean and obese rats.
  • Upper panel western blot; lower panel: Erk2-phosphorylation of IRDBP-1.
  • Fig. 45 A illustrates probing with anti-ERDBP-1 and anti-phosphoErk antibodies using Western immunoblot of cell extracts from differentated 3T3-L1 adipocytes.
  • Fig. 45B shows in vitro phosphorylation of ERDBP-1 by Akt and Erk.
  • Fig. 45C shows the effects of Aktl on insulin-mediated IRDBP-1 transcription from the IFGBP-3 ERE
  • Fig. 45D shows the effects of Erk2 and Aktl on IGFBP-3 ERE transcription.
  • Figs. 46A and 46B illustrate the proteolysis and cell distribution of ERDBP-1 in adipocytes and COS7 (46A) and HepG2 cells (46B).
  • ATLANTA 333137vl 14 Fig. 47 shows the confocal microscopy of HepG2 cells grown with or without insulin and probed for the n-region or the c-region specific antibodies of ERDBP-1.
  • Fig. 48 shows the results of using anti-phospohotyrosine and antiphosphoserine/threonine antibodies to precipitate phosphorylated ERDBP-1 with cell extracts from COS7 cells with and without exposure to insulin.
  • Fig. 49 illustrates the DNA of various constructs.
  • the bottom schema shows the proposed proteolysis ofthe IRDBP-1 protein.
  • Fig. 50 shows a Western blot wherein recombinant adenovirus encoding IRDBP-1 (Ad-ERDBP-1) and Ad-GFP were transfected into 3T3-L1 cells and cell lysates were probed with anti-ERDBP-1 antibody.
  • Fig. 51A illustrates ERDBP-1 increases glucose uptake in L6 myoblasts.
  • Fig. 5 IB illustrates IRDBP-1 increases glucose uptake in human adipocytes transfected with Ad-IRDBP-1.
  • Fig. 52 shows that ERDBP-1 is a target for thiazolidinedione in 3T3-L1 adipocytes.
  • Fig. 53A shows a Northern analysis of hepatic tissues from rats infected with an Ad-IRDBP-1 and Ad-GFP constructs.
  • Fig. 53B shows the blood glucose levels of Ad-ERDBP-1 and Ad-GFP injected ZDF rats before and after treatment with the recombinant virus.
  • Fig. 53C shows the change in the body weight of Ad-IRDBP-1 injected ZDF rats ad libitum fed.
  • Fig. 53D shows the rate of food uptake of Ad-GFP and Ad-ERDBP-1 treated ZDF rats.
  • Fig. 53E illusfrates the serum glucose levels after gene therapy treatment on 12 week rats injected with Ad-ERDBP-1
  • Fig. 53F illustrates the serum glucose levels after gene therapy treatment on 18 week rats injected with Ad-ERDBP-1.
  • Fig. 54 illustrates the effects of ERDBP-l on glycogen content in two cell lines with stable expression of ERDBP-1 in transfected L6 cells.
  • Fig. 55A illustrates the effect of administered ERDBP-1 antisense (AS) or sense (S) oligonucleotides on the body weight of rats.
  • Fig. 55B illustrates the loss in body weight of females rats receiving sense IRDBP-1 DNA.
  • Fig. 56 illusfrates phase contrast micrographs of L6 cells stably fransfected with control vector alone (left) or with vector containing the ERDBP-1 nucleic acid sequence (right) and grown in soft agar for 4 weeks.
  • Fig. 57A illustrates the expression of human IRDBP-1 in tumor and normal tissues.
  • Fig. 57B illusfrates the expression of human IRDBP-1 in kidney and breast tumor tissues.
  • Fig. 57C illustrates the expression of human ERDBP-1 in colon tumor tissues.
  • ATLANTA 333137vl ⁇ Fig. 58 illustrates ERDBP-1 induces apoptosis in HepG2 cells.
  • Fig. 59 illustrates the decreased DNA synthesis in HCT-116 and SW620 cells with IRDBP-1 overexpression.
  • Fig. 60 illustrates analysis by flow cytometry of he cell cycle compartments of L6 cells with (right panel) or without (left panel) over-expression of the ERDBP-1 nucleic acid sequence.
  • Fig. 61 illustrates the increase in Go cells after expression of ERDBP-1 therein.
  • ATLANTA 333137vl ⁇ normal type then the symbol and name of the mutant gene appear in italicized lower case letters.
  • animal is used herein to include all vertebrate animals, including humans. It also includes an individual animal in all stages of development, including embryonic and fetal stages.
  • a "transgenic animal” is any animal containing one or more cells bearing genetic information altered or received, directly or indirectly, by deliberate genetic manipulation at a subcellular level, such as by targeted recombination or microinjection or by infection with recombinant virus.
  • transgenic animal is not intended to encompass classical cross-breeding or in vitro fertilization, but rather is meant to encompass animals in which one or more cells are altered by, or receive, a recombinant nucleic acid molecule.
  • This recombinant nucleic acid molecule may be specifically targeted to a defined genetic locus, may be randomly integrated within a chromosome, or it may be exfrachromosomally replicating nucleic acid.
  • the term "germ cell line transgenic animal” refers to a transgenic animal in which the genetic alteration or genetic information was introduced into a germ line cell, thereby conferring the ability to transfer the genetic information to offspring. If such offspring in fact possess some or all of that alteration or genetic information, they are transgenic animals as well.
  • mammalian refers to any species, subspecies or race of organism of the taxonomic class mammalia, such as, but not limited to, such organisms as mice, rats, rabbits, sheep, cattle, and primates, including humans.
  • insulin-responsive protein- 1 refers to an Insulin-Responsive DNA Binding Protein- 1 capable of binding to at least one insulin responsive element associated with a gene or genes, and by so doing may regulate the expression of an insulin-responsive gene.
  • the term “ERDBP-1” is also intended to apply to proteins, peptides or polypeptides capable of binding to at least one insulin-responsive element of eukaryotic organisms, including fungi or animals.
  • nucleic acid refers to any natural and synthetic linear and sequential arrays of nucleotides and nucleosides, for example cDNA, genomic DNA, mRNA, RNA, oligonucleotides, oligonucleosides and derivatives thereof.
  • nucleic acids may be collectively referred to herein as "constructs,” "plasmids,” or “vectors.”
  • Representative examples of the nucleic acids of the present invention include bacterial plasmid vectors including expression, cloning, cosmid and transformation vectors such as, but not limited to, pBR322, animal viral vectors such as, but not limited to, modified adenovirus, influenza virus, polio virus, pox virus, retrovirus, and the like, vectors derived from bacteriophage nucleic acid, and synthetic oligonucleotides like chemically synthesized DNA or RNA.
  • nucleic acid further includes modified or derivatised nucleotides and nucleosides such as, but not limited to, halogenated nucleotides such as, but not only, 5-bromouracil, and derivatised nucleotides such as biotin-labeled nucleotides.
  • isolated nucleic acid refers to a nucleic acid with a structure (a) not identical to that of any naturally occurring nucleic acid or (b) not identical to that of any fragment of a naturally occurring genomic nucleic acid spanning more than three separate genes, and includes DNA, RNA, or derivatives or variants thereof.
  • the term covers, for example, (a) a DNA which has the sequence of part of a naturally occurring genomic molecule but is not flanked by at least one ofthe coding sequences that flank that part of the molecule in the genome of the species in which it naturally occurs; (b) a nucleic acid inco ⁇ orated into a vector or into the genomic nucleic acid of a prokaryote or eukaryote in a manner such that the resulting molecule is not identical to any vector or naturally occurring genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), ligase chain reaction (LCR) or chemical synthesis, or a restriction fragment; (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein, and (e) a recombinant nucleotide sequence that is part of a hybrid sequence that is
  • ATLANTA 333137vl 19 allelic variants as well as nucleic acid molecules modified by nucleotide deletions, insertions, inversions, or substitutions such that the resulting nucleic acid molecule still essentially encodes an ERDBP-1 protein or a variant thereof of the present invention.
  • enriched in reference to nucleic acid it is meant that the specific DNA or RNA sequence constitutes a significantly higher fraction of the total DNA or RNA present in the cells or solution of interest than in normal or diseased cells or in the cells from which the sequence was taken. Enriched does not imply that there are no other DNA or RNA sequences present, just that the relative amount of the sequence of interest has been significantly increased.
  • the other DNA may, for example, be derived from a yeast or bacterial genome, or a cloning vector, such as a plasmid or a viral vector.
  • a cloning vector such as a plasmid or a viral vector.
  • nucleotide sequence is in purified form.
  • purified in reference to nucleic acid represents that the sequence has increased purity relative to the natural environment.
  • polypeptide and protein refer to a polymer of amino acids of three or more amino acids in a serial array, linked through peptide bonds.
  • polypeptide includes proteins, protein fragments, protein analogues, oligopeptides and the like.
  • polypeptide contemplates polypeptides as defined above that are encoded by nucleic acids, produced through recombinant technology, isolated from an appropriate source such as a mammal, or are synthesized.
  • polypeptide further contemplates polypeptides as defined above that include chemically modified amino acids or amino acids covalently or noncovalently linked to labeling ligands.
  • fragment refers to a nucleic acid that is an isolated portion of the subject nucleic acid and may be constructed artificially (e.g., by chemical synthesis) or by cleaving a natural product into multiple pieces, using restriction endonucleases or mechanical shearing, or a portion of a nucleic acid synthesized by PCR, DNA polymerase or any other polymerizing technique well
  • fragment as used herein may also refer to an isolated portion of a polypeptide, wherein the portion ofthe polypeptide is cleaved from a naturally occurring polypeptide by proteolytic cleavage by at least one protease, or is a portion of the naturally occurring polypeptide synthesized by chemical methods well known to one of skill in the art.
  • modulates refers to the ability of a compound to alter the function of an ERE binding protein.
  • a modulator preferably increases the binding or activating potential of an ERDBP-1.
  • a modulator can alternatively decrease the binding or activating potential of IRDBP-1 polypeptide or fragments thereof.
  • the terms “regulating” and “modulating' as used herein also refer to increasing or decraesing any parameter such as, but not limited to, the infracular level of gene expression, the intracellular level of mRNA or polypeptide, the proliferationof a cell or the metabolic rate or uptake of glucose and the like.
  • gene or “genes” as used herein refers to nucleic acid sequences
  • RNA or DNA genes that are not naturally part of a particular organism's genome
  • foreign genes Genes that are not naturally part of a particular organism's genome
  • heterologous genes or “exogenous genes”
  • genes that are naturally a part of a particular organism's genome are referred to as “endogenous genes”.
  • endogenous genes The term “gene product” refers to RNAs or proteins that are encoded by the gene.
  • Foreign gene products are RNA or proteins encoded by “foreign genes” and “endogenous gene products” are RNA or proteins encoded by endogenous genes.
  • Heterologous gene products are RNAs or proteins encoded by "foreign, heterologous or exogenous genes” and which, therefore, are not naturally expressed in the cell.
  • expressed refers to the transcription from a gene to give an RNA nucleic acid molecule at least complementary in part to a region of one of the two nucleic acid strands of the gene.
  • expression refers to the transcription from a gene to give an RNA nucleic acid molecule at least complementary in part to a region of one of the two nucleic acid strands of the gene.
  • ATLANTA 333137vl 21 "expression" as used herein may also refer to the translation from said RNA nucleic acid molecule to give a protein or polypeptide or a portion thereof.
  • locus refers to the site of a gene on a chromosome. Pairs of genes control hereditary traits, each in the same position on a pair of chromosomes. These gene pairs, or alleles, may both be dominant or both be recessive in expression of that trait. In either case, the individual is said to be homozygous for the trait controlled by that gene pair. If the gene pair (alleles) consists of one dominant and one recessive trait, the individual is heterozygous for the trait controlled by the gene pair.
  • allelic variants Natural variation in genes or nucleic acid molecules caused by, for example, recombination events or resulting from mutation, gives rise to allelic variants with similar, but not identical, nucleotide sequences. Such allelic variants typically encode proteins with similar activity to that of the protein encoded by the gene to which they are compared, because natural selection typically selects against variations that alter function. Allelic variants can also comprise alterations in the untranslated regions of the gene as, for example, in the 3' or 5' untranslated regions or can involve alternate splicing of a nascent transcript, resulting in alternative exons being positioned adjacently.
  • genomic sequence refers to the total DNA as found in the genome of an organism, and may include non-coding regions like introns, enhancers, promoters and the like.
  • transcription regulatory sequences refers to nucleotide sequences that are associated with a gene nucleic acid sequence and which regulate the transcriptional expression of the gene.
  • the "transcription regulatory sequences” may be isolated and incorporated into a vector nucleic acid to enable regulated transcription in appropriate cells of portions of the vector DNA.
  • the "transcription regulatory sequence” may precede, but is not limited to, the region of a nucleic acid sequence that is in the region 5' of the end of a protein coding sequence that may be transcribed into mRNA.
  • Transcriptional regulatory sequences may also be located within a protein coding region, in regions of a gene that are identified as
  • ATLANTA 333137vl 22 "intron" regions or may be in regions of nucleic acid sequence that are in the region of nucleic acid.
  • coding region refers to a continuous linear arrangement of nucleotides which may be translated into a protein.
  • a full length coding region is translated into a full length protein; that is, a complete protein as would be translated in its natural state absent any post-translational modifications.
  • a full length coding region may also include any leader protein sequence or any other region ofthe protein that may be excised naturally from the translated protein.
  • complementary refers to two nucleic acid molecules that can form specific interactions with one another.
  • an adenine base within one strand of a nucleic acid can form two hydrogen bonds with thymine within a second nucleic acid strand when the two nucleic acid strands are in opposing polarities.
  • a guanine base within one strand of a nucleic acid can form three hydrogen bonds with cytosine within a second nucleic acid strand when the two nucleic acid strands are in opposing polarities.
  • Complementary nucleic acids as referred to herein may further comprise modified bases wherein a modified adenine may form hydrogen bonds with a thymine or modified thymine, and a modified cytosine may form hydrogen bonds with a guanine or a modified guanine.
  • the probe may be modified with labels such as, but not only, radioactive groups, biotin, or any other label that is well known in the art.
  • stringent hybridization conditions typically permit the hybridization of nucleic acid molecules having at least 70% nucleic acid sequence
  • ATLANTA 333137vl 23 identity with the nucleic acid molecule being used as a probe in the hybridization reaction.
  • substantially similar in the context of the present invention refers to sequences that are detectable under at least medium stringency conditions and typically remain hybridized under high-stringency conditions, there being at least 75% similarity between the hybridizing sequences
  • the first nucleic acid may be a test sample or probe
  • the second nucleic acid may be the sense or antisense strand of a ERDBP-1 gene or a fragment thereof.
  • the first nucleic acid may be a target nucleic acid derived from the genome, or RNA transcript therefrom, of a first species and the second nucleic acid may be isolated from a second animal species.
  • Hybridization of the first and second nucleic acids may be conducted under stringent conditions, e.g., high temperature and/or low salt content that tend to disfavor hybridization of dissimilar nucleotide sequences.
  • hybridization of the first and second nucleic acid may be conducted under reduced stringency conditions, e.g.
  • Low stringency hybridization conditions may be followed by high stringency conditions to increase the selectivity of the binding of the first and second nucleic acids.
  • the hybridization conditions may further include reagents such as, but not limited to, dimethyl sulfoxide (DMSO) or formamide to disfavor still further the hybridization of dissimilar nucleotide sequences.
  • DMSO dimethyl sulfoxide
  • a suitable hybridization protocol may, for example, involve hybridization in 6X SSC (wherein IX SSC comprises 0.015 M sodium turite and 0.15 M sodium chloride), at 65° Celsius in aqueous solution, followed by washing with IX SSC at 65° Celsius.
  • stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) from about pH 7.0 to about pH 8.3 and the temperature is at least about 30° C for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C for long probes (e.g., greater than 50 nucleotides).
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCI, 1% SDS (sodium dodecyl sulphate) at 37° Celsius, and a wash in lx to 2x SSC at 50 to 55° Celsius.
  • Exemplary medium stringency conditions include hybridization in 40 to 45% formamide, 1 M NaCI, 1% SDS at 37° Celsius, and a wash in 0.5x to lx SSC at 55 to 60° Celsius.
  • Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCI, 1% SDS at 37° Celsius, and a wash in 0. lx SSC at 60 to 65° Celsius.
  • nucleic acid region refers to a sequence present in a nucleic acid that is not present in any other nucleic acid sequence.
  • conserved nucleic acid region is a nucleotide sequence present in two or more nucleic acid sequences, to which a particular nucleic acid sequence can hybridize under low, medium or high stringency conditions. The greater the degree of conservation between the conserved regions of two or more nucleic acid sequences, the higher the hybridization stringency that will allow hybridization between the conserved region and a particular nucleic acid sequence.
  • ATLANTA 333137vl 25 100, wordlength 12, to obtain nucleotide sequences homologous to a nucleic acid molecule ofthe invention.
  • Gapped BLAST is utilized as described in Attschul et al. (1997) Nuc. Acids Res. 25: 3389-3402.
  • the default parameters of the respective programs e.g. XBLAST and NBLAST are used. See http://www.ncbi.nlm.nih.gov.
  • sense strand refers to a single stranded DNA molecule from a genomic DNA that may be transcribed into RNA and translated into the natural polypeptide product ofthe gene.
  • antisense strand is used herein to mean the single strand DNA molecule of a genomic DNA that is complementary with the sense strand ofthe gene.
  • antisense DNA refers to a gene sequence DNA that has a nucleotide sequence complementary to the "sense strand" of a gene when read in reverse orientation, i.e., DNA read into RNA in a 3' to 5' direction rather than in the 5' to 3' direction.
  • antisense RNA is used to mean an RNA nucleotide sequence (for example that encoded by an antisense DNA or synthesized complementary with the antisense DNA).
  • Antisense RNA is capable of hybridizing under stringent conditions with an antisense DNA.
  • the antisense RNA of the invention is useful for regulating expression of a "target gene” either at the transcriptional or translational level.
  • transcription ofthe subject nucleic acids may produce antisense transcripts that are capable of inhibiting transcription by inhibiting initiation of transcription or by competing for limiting transcription factors; or, the antisense transcripts may inhibit transport of the "target RNA", or, the antisense transcripts may inhibit translation of "target RNA”.
  • antisense therapy refers to the administration or in situ generation of oligonucleotide probes or their derivatives that specifically hybridize (e.g. bind) under cellular conditions, with the cellular mRNA and/or genomic DNA encoding a ERDBP-1 protein so as to inhibit expression of that protein, e.g. by inhibiting transcription and/or translation.
  • the binding may be by conventional base pair complementation, or, for example, in the case of binding to DNA duplexes, through specific interactions in the major groove of the double helix.
  • antisense refers to the range of techniques generally employed in the art, and includes any therapy that relies on specific binding to oligonucleotide sequences.
  • vector and "nucleic acid vector” as used herein refers to a natural or synthetic single or double stranded plasmid or viral nucleic acid molecule that can be fransfected or transformed into cells and replicate independently of, or within, the host cell genome.
  • a circular double stranded plasmid can be linearized by treatment with an appropriate restriction enzyme based on the nucleotide sequence of the plasmid vector.
  • a nucleic acid can be inserted into a vector by cutting the vector with restriction enzymes and ligating the pieces together.
  • the nucleic acid molecule can be RNA or DNA.
  • expression vector refers to a nucleic acid vector that may further include at least one regulatory sequence operably linked to the nucleotide sequence coding for the ERDBP-1 protein. Regulatory sequences are well recognized in the art and may be selected to ensure good expression of the linked nucleotide sequence without undue experimentation by those skilled in the art.
  • regulatory sequences includes promoters, enhancers, and other elements that may control expression. Standard molecular biology textbooks such as Sambrook et al. eds "Molecular Cloning: A Laboratory Manual” 2nd ed. Cold Spring Harbor Press (1989) may be consulted to design suitable expression vectors, promoters, and other expression confrol elements. It should be recognized, however, that the choice of a suitable expression vector depends upon multiple factors including the choice ofthe host cell to be transformed and/or the type of protein to be expressed.
  • transformation and “transfection” as used herein refer to the process of inserting a nucleic acid into a host.
  • Many techniques are well known to those skilled in the art to facilitate transformation or transfection of a nucleic acid into a prokaryotic or eukaryotic organism. These methods involve a variety of techniques, such as treating the cells with high concentrations of salt such as, but not only a calcium or magnesium salt, an electric field, detergent, or liposome mediated transfection, to render the host cell competent for the uptake of the nucleic acid molecules.
  • salt such as, but not only a calcium or magnesium salt, an electric field, detergent, or liposome mediated transfection
  • nucleic acid refers to combinations of at least two nucleic acid sequences that are not naturally found in a eukaryotic or prokaryotic cell.
  • the nucleic acid sequences may include, but are not limited to nucleic acid vectors, gene expression regulatory elements, origins of replication, sequences that when expressed confer antibiotic resistance, and protein-encoding sequences.
  • recombinant polypeptide it is meant to include a polypeptide produced by recombinant DNA techniques such that it is distinct from a naturally occurring polypeptide either in its location, purity or structure. Generally, such a recombinant polypeptide will be present in a cell in an amount different from that normally observed in nature.
  • recombinant cell refers to a cell that has a new combination of nucleic acid segments that are not covalently linked to each other in nature.
  • a new combination of nucleic acid segments can be introduced into an organism using a wide array of nucleic acid manipulation techniques available to those skilled in the art.
  • a recombinant cell can be a single eukaryotic cell, or a single prokaryotic cell, or a mammalian cell.
  • the recombinant cell can harbor a vector that is exphanomic. An exminomic nucleic acid vector does not insert into the cell's genome.
  • a recombinant cell can further harbor a vector or a portion thereof that is inphanomic.
  • intomic defines a nucleic acid construct inco ⁇ orated within the recombinant cell's genome.
  • antibody refers to polyclonal and monoclonal antibodies and fragments thereof, and immunologic binding equivalents thereof that
  • ATLANTA 333137vl 28 are capable of selectively binding to the ERDBP-1 polypeptides and fragments thereof, including epitopes thereof, or to polynucleotide sequences from the IRDBP-1 region, particularly from the ERDBP-1 locus or a portion thereof.
  • the term "antibody” also refers to a homogeneous molecular entity, or a mixture such as a serum product made up of a plurality of different molecular entities, and may further comprise any modified or derivatised variant thereof that retains the ability to specifically bind to IRDBP-1 -related polypeptides.
  • Such antibodies may include, but are not limited to polyclonal antibodies, monoclonal antibodies (mAbs), humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab') 2 fragments, fragments produced by a FAb expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any ofthe above.
  • mAbs monoclonal antibodies
  • Such antibodies may be used, for example, in the detection of a finge ⁇ rint, target, or pathway gene in a biological sample, or, alternatively, as a method for the inhibition of normal or abnormal target gene activity.
  • such antibodies may be utilized as part of body weight disorder treatment methods, and/or may be used as part of diagnostic techniques whereby patients may be tested for abnormal levels of finge ⁇ rint, target, or pathway gene proteins, or for the presence of abnormal forms of the such proteins.
  • various host animals may be immunized by injection with a differentially expressed or pathway gene protein, or a portion thereof.
  • host animals may include but are not limited to rabbits, mice, and rats, to name but a few.
  • adjuvants may be used to increase the immunologic response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum.
  • BCG Bacille Calmette-Guerin
  • Corynebacterium parvum bacille Calmette-Guerin
  • polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen, such as a target gene product, or an antigenic functional derivative thereof.
  • an antigen such as a target gene product, or an antigenic functional derivative thereof.
  • host animals such as those described above, may be immunized by injection with differentially expressed or pathway gene product supplemented with adjuvants as also described above.
  • Monoclonal antibodies which are homogeneous populations of antibodies to a particular antigen, may be obtained by any technique that provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to the hybridoma technique of Kohler & Milstein (1975) Nature 256: 495-497; and U.S. Patent No. 4,376,110), the human B-cell hybridoma technique (Kosbor et al. (1983) Immunology Today 4: 72; Cole et al. (1983) Proc. Natl. Acad. Sci. 80: 2026- 2030), and the EBV-hybridoma technique (Cole et al. (1985) Monoclonal Antibodies And Cancer Therapy Alan R. Liss, Inc. pp.
  • spleen cells are harvested from an immunized mouse and fused with immortalizing cells (i.e., myeloma cells) to yield antibody-producing hybridomas.
  • immortalizing cells i.e., myeloma cells
  • Hybridomas can be screened immunochemically for production of monoclonal antibodies specifically reactive with the ERDBP-1 protein.
  • Protocols for producing, isolating and purifying conventional and monoclonal antibodies may be analogous to those described in Cassone et al. (1988) J. Med. Microbiol. 27: 233-238; Hancock & Evan Production and Characterization of Antibodies against Synthetic Peptides pp23-33 in Immunochemical Protocols ed. M.M. Manson, (1992) (Humana Press, Totowa, NJ); Goding, J. W., Monoclonal Antibodies: Principles and Practice, 2d ed., (1986) (Academic Press Ltd., London) and Lam & Mutharia, "Antigen- Antibody Reactions," pp 104- 132 in Methods for General and Molecular Bacteriology, ed.
  • Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof.
  • ATLANTA 333137vl 3Q invention may be cultivated in vitro or in vivo. Production of high titers of mAbs in vivo makes this the presently preferred method of production.
  • chimeric antibodies In addition, techniques developed for the production of "chimeric antibodies" (Morrison et al. (1984) Proc. Natl. Acad. Sci. 81: 6851-6855; Neuberger et al. (1984) Nature 312: 604-608; Takeda et al. (1985) Nature 314: 452-454) by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used.
  • a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region.
  • single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide.
  • Antibody fragments which recognize specific epitopes may be generated by known techniques.
  • such fragments include but are not limited to: the F(ab') 2 fragments which can be produced by pepsin digestion ofthe antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges ofthe F(ab') 2 fragments.
  • Fab expression libraries may be constructed (Huse et al. (1989) Science 246: 1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.
  • pharmaceutical compositions refers to compositions comprising agents that will modulate the physiological activity of the IRDBP-1 gene product or the regulation of the expression of the IRDBP-1 gene. Such compositions can be administered in dosages and by techniques well known to those skilled in the medical or veterinary arts, taking into consideration such factors as the age, sex, weight, species and condition ofthe particular patient, and the route of administration.
  • the route of administration can be percutaneous, via mucosal administration (e.g., oral, nasal, anal, vaginal) or via a parenteral route (intradermal, intramuscular, subcutaneous, intravenous, or infraperitoneal).
  • Pharmaceutical compositions can be administered alone, or can be co-administered or sequentially administered with other treatments or therapies.
  • Forms of administration may include suspensions, syrups or elixirs, and preparations for parenteral, subcutaneous, intradermal, intramuscular or intravenous administration (e.g., injectable administration) such as sterile suspensions or emulsions.
  • compositions may be administered in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, or the like.
  • a suitable carrier diluent, or excipient
  • the compositions can contain auxiliary substances such as wetting or emulsifying agents, pH buffering agents, adjuvants, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired.
  • Standard pharmaceutical texts such as "Remmington's Pharmaceutical Science," 17th edition, 1985 may be consulted to prepare suitable preparations, without undue experimentation.
  • the effective dosage and route of adminisfration are determined by the therapeutic range and nature of the compound, and by known factors, such as the age, weight, and condition of the host, as well as LD 50 and other screening procedures that are known and do not require undue experimentation. Dosages can generally range from a few hundred milligrams to a few grams.
  • a "transgenic animal” is any animal, preferably a non-human mammal, in which one or more ofthe cells ofthe animal contain heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art.
  • the nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus.
  • the term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the infroduction of a recombinant DNA molecule. This molecule may be integrated within a chromosome, or it may be exfrachromosomally replicating DNA.
  • the fransgene causes
  • non-human animals include vertebrates such as rodents, non-human primates, sheep, dog, cow, birds, amphibians, reptiles, etc. Preferred non-human animals are selected from the rodent family including rat and mouse, most preferably mouse.
  • chimeric animal is used herein to refer to animals in which the recombinant gene is found, or in which the recombinant is expressed in some but not all cells of the animal.
  • tissue- specific chimeric animal indicates that the recombinant IRDBP-1 gene is present and/or expressed in some tissues but not others.
  • transgene means a nucleic acid sequence (encoding, e.g., a ERDBP-1 polypeptide) that is partly or entirely heterologous, i.e., foreign, to the transgenic animal or cell into which it is infroduced, or, is homologous to an endogenous gene of the transgenic animal or cell into which it is infroduced, but which is designed to be inserted, or is inserted, into the animal's genome in such a way as to alter the genome of the cell into which it is inserted (e.g., it is inserted at a location which differs from that of the natural gene or its insertion results in a knockout).
  • a transgene can include one or more transcriptional regulatory sequences and any other nucleic acid, such as introns, that may be necessary for optimal expression of a selected nucleic acid.
  • an "ERDBP-l nucleic acid molecule” includes nucleic acid sequences related to a natural ERDBP-1 gene and may include all or some regions such as regulatory regions that confrol production of an RNA nucleic acid encoding the IRDBP-1 protein or production ofthe ERDBP-1 protein encoded by the gene (such as, but not limited to, transcription, translation or post-translation regulatory sequences) as well as the coding region itself, and any introns or non-translated coding regions or fragments thereof.
  • an ERDBP-1 variant is an ERDBP-1 polypeptide that differs from an exemplified sequence in that one or more amino acids have been changed, added or deleted.
  • An ERDBP-1 variant retains its useful function, i.e., for example, ability to bind EREs, activate or suppress insulin-regulating genes.
  • epipe refers to a part of the protein that can specifically bind to an antibody by fitting into the antigen-binding site ofthe antibody.
  • thiazolidinedione refers to "insulin sensitizers" that can bind to peroxisome proliferator-activated receptors such as, but not limited to, PPAR ⁇ ) resulting in increased glucose production.
  • exemplary thiazolidinediones include, but are not limited to, troglitazone, rosiglitazone and pioglitazone.
  • cancer and “tumor” as used herein refer to a pathological condition of uncontrolled replication of a transformed cell.
  • a cancer or tumor may be of any tissue such as, but not limited to, heart, breast, kidney, colon, intestinal and the like.
  • the techniques used to isolate and characterize the nucleic acids and proteins of the present invention are well known to those of skill in the art and standard molecular biology and biochemical manuals may be consulted to select suitable protocols without undue experimentation. See, for example, Sambrook et al (1989) Molecular Cloning: A Laboratory Manual 2nd ed. Cold Spring Harbor Press, the content of which is herein inco ⁇ orated by reference in its entirety.
  • Abbreviations used in the present specification include the following: aa, amino acid(s); bp, base pair(s); cDNA, DNA complementary to RNA; ERDBP-1, insulin responsive DNA-binding protein- 1; IGF, insulin-like growth factor; IGFBP, IGF- binding protein; ERE, insulin response element; PEPCK, phosphoenol pryuvate carboxykinase; GAPDH, glyceraldehyde-3 -phosphate dehydrogenase enzyme; nt, nucleotide(s); SSC, sodium chloride-sodium frante ; DMSO, dimethyl sulfoxide.
  • One aspect ofthe present invention provides isolated nucleic acids, derivatives and variants thereof that encode human or rat ERDBP-1 proteins, derivatives or variants thereof.
  • ERDBP-1 protein or functionally active derivatives or fragments thereof are particularly useful as direct or indirect modulators of gene expression wherein the genes so modulated comprise an ERE and are capable of responding to fluctuations in insulin levels.
  • the present invention further provides an isolated nucleic acid encoding a fragment of a rat IRDBP-1 protein isolated based on the ability of the expressed protein product thereof to bind to the nucleic acid Insulin Responsive Element (IRE) associated with the rat IGFBP-3 and which has the nucleotide sequence 5'-AATTCAAGGGTATCCAGGAAAGTCTCC-3' (SEQ ED NO: 1).
  • EREs are regulatory nucleic acid sequences of insulin-regulated genes that are necessary to enable an insulin-dependent response.
  • the nucleotide sequence of SEQ ID NO: 1 is localized between the -1150 and the -1124 bp positions of the promoter region of the IGFBP-3 encoding gene of the rat.
  • rat liver cDNA library using the yeast one-hybrid system was screened using concatemerized EREs of rat IGFBP-3, using methods described by Wang & Reed (1993) Nature 364: 121-126, inco ⁇ orated herein by reference in its entirety, and discussed in Example 1 below.
  • the cDNA library screening provided a novel 952-bp cDNA (clone 52) encoding a portion ofthe Insulin-Responsive DNA Binding Protein- 1 (ERDBP-1) that was identified and sequenced (SEQ ED NO: 2) (GenBank Accession No. AF439714) , as illustrated in Fig. 1.
  • the nucleic acid sequence of clone 52 (SEQ ID NO: 2) encodes a polypeptide having the amino acid sequence of SEQ ID NO: 3, as shown in Fig. 2, capable of binding to the ERE region of the rat IGFBP-3 (SEQ ID NO: 1), as described in Example 1 and 4.
  • a clone 52-thioredoxin (Trx) fusion protein also binds to the rat IGFBP-3 ERE SEQ ED NO: 1.
  • the amino acid sequence SEQ ED NO: 3 deduced from the nucleotide sequence (SEQ ED NO: 2) of clone 52 comprises a homeodomain motif typical of transcription factors. Binding by the polypeptide SEQ ED NO: 3 to the ERE of IGFBP- 3 (SEQ LD NO: 1) could be competed away by IGFBP-3 ERE nucleic acids but not by
  • the ERDBP-1 polypeptide fragment (SEQ ED NO: 3) encoded by clone 52 also interacts with EREs associated with other insulin-responsive genes besides IGFBP-3, as shown in Examples 4 and 7 below.
  • the polypeptide interacts with the EREs from insulin-responsive genes encoding IGF-1, IGFBP- 1, phosphoenol pyruvate carboxykinase (PEPCK), amylase, and glyceraldehyde-3 -phosphate dehydrogenase (GAPDH).
  • IGFBP- 1 is a hepatic acute phase reactant protein that coordinates the level of
  • IGF-1 in response to changes in insulin levels (Lee et al. (1993)).
  • Amylase is important for intestinal hydrolysis of carbohydrates.
  • GAPDH catalyzes the conversion of glyceraldehyde-3 -phosphate to 1,3-diphosphoglycerate, a rate-limiting step in adipose tissue glycolysis.
  • the naturally occurring IRDBP-1 protein is likely a franscription factor that coordinates the responses of several genes to insulin.
  • the ERDBP-1 polypeptide (SEQ ID NO: 3) of clone 52 can regulate critical genes in target tissues implicated in insulin resistance and insulin secretion.
  • ERDBP-1 polypeptide modulates the pleiofropic actions of insulin in the normal metabolism and storage of ingested carbohydrate and other fuels, in the modulation of intermediary metabolism, and in normal cellular growth and differentiation.
  • Ribonuclease protection assays (discussed in Example 3) using an antisense RNA probe obtained by transcribing a Kpnl-Xhol fragment of clone 52 and having the nucleic acid sequence SEQ ED NO: 4, as shown in Fig. 3, showed that at least one gene, encoding at least one nucleic acid with sequence similarity to a region of the clone 52 cDNA sequence SEQ ED NO: 2 is expressed in at least liver, kidney, brain, small intestine, muscle, and fat pads.
  • ATLANTA 333137vl 36 the addition of physiological concentrations of insulin (10 ⁇ 9 M) in cell culture. It was also decreased in the livers of diabetic rats, as described in Example 8.
  • Another aspect of the present invention provides for the use of the isolated cDNA clone 52 (SEQ ED NO: 2) as a probe to screen rat and human cDNA libraries to obtain isolated nucleic acids capable of hybridizing with clone 52, as discussed in Example 5. Nucleic acid regions extending the cDNA sequences in the 5' direction from the isolated human and rat partial cDNA clones were obtained by primer extension reactions such as 5' RACE, and then sequenced.
  • the present invention further provides rat cDNA clones that hybridize to the clone 52 probe, and were identified and sequenced as SEQ ED NOS: 5, 6, 14 and 44
  • a first rat ERDBP-1 cDNA clone (SEQ ED NO 5; shown in Fig 4A and 4B) comprises about 4998 bp, and includes at least one open reading frame (ORF) (SEQ ID NO: 8) as in Fig. 4A and which encodes a rat ISRBP-1 protein (SEQ ID NO: 11 ; Fig. A).
  • a second rat cDNA clone (SEQ ID NO: 6, shown in Fig. 5A and 5B) is a partial cDNA comprising a partial open-reading frame (ORF) (Fig. 5A) having sequence similarity to a region of SEQ ED NO: 5 (Fig. 4A), and a 3 'untranslated region (Fig. 5B) longer than that of SEQ ED NO: 5 (shown in Fig. 5B).
  • Nucleic acid SEQ ED NO: 44 and the protein sequence encoded therein (SEQ ED NO: 47) are shown in Figs. 6 and 15 respectively.
  • the present invention also provides for the human cDNA clones having the nucleic acid sequences SEQ ED NO: 7 as shown in Fig. 6A-6C, SEQ ED NO: 10 as shown in Fig. 8A and 8B) that were also identified by hybridization with a probe comprising the clone 52 nucleic acid sequence (SEQ ED NO: 2) during the screening of a human cDNA library, and SEQ ED NO: 45 (shown in Fig. 45) (GenBank Accession No. AF ) generated by 5' RACE extension of the isolated clone SEQ ID NO: 7.
  • any nucleic acid of the present invention can comprise one or more regulatory regions, full-length or partial coding regions such as, but not limited to, the fragments SEQ ED NOS: 16-41 (Fig. 7) derived from the ERDBP-1 gene, or any combinations thereof. It is contemplated to be within the scope of the present invention for a probe to be derived from any of SEQ ED NOS: 2, 5-10, 14, 16- 41 and 44-45 or a variant or truncated variant thereof.
  • the minimal size of a nucleic acid molecule of the present invention is a size sufficient to allow the formation of a stable hybridization product with the complementary sequence of another nucleic acid molecule under selected stringency conditions.
  • Embodiments of the present invention may, therefore, include, but are not limited to, nucleic acid molecules such as: a) an IRDBP-1 cDNA molecule derived from the rat and comprising the protein coding region (SEQ ID NO: 8, shown in Fig.
  • SEQ ID NO: 5 or the coding region of SEQ ID NO: 44, and a 3' non-coding, or untranslated, region of SEQ ED NOS: 5 or 44; b) an ERDBP-1 cDNA molecule derived from the rat nucleic acid SEQ ED NOS: 5 or 44 and comprising the isolated coding region (SEQ ED NO: 8), or a substantial region thereof; or nucleic acid molecules representing degenerate variants, derivatives, modified sequences and truncated variants such as, but not limited to, SEQ ED NO: 6 shown in Figs.
  • ERDBP-1 -encoding cDNA molecule derived from the human comprising the protein coding region and a 5' and/or 3' non-coding regions of the sequence SEQ ID NO: 7 (GenBank Accession No. AF439717) as shown in Figs. 7A-C, or SEQ ED NO: 45 shown in Fig. 14; d) a nucleic acid molecule derived from the human ERDBP-1 cDNA sequence SEQ ID NO: 7 and comprising the human ERDBP-1 coding region alone (SEQ ED NO: 9), as depicted in Fig.
  • nucleic acid molecules representing degenerate variants, derivatives, alternatively spliced variants and modified variants thereof.
  • a variant may be, but is not limited to, the sequence SEQ ID NO: 10 (GenBank Accession No. AF439718) as shown in Fig. 8 A and 8B.
  • nucleic acid molecules can include nucleotides in addition to those included in SEQ ED NOS: 2, 5-10, 14, and 44-45 such as, but not limited to, a full-length gene, a full-length coding region, or a nucleic acid molecule
  • ATLANTA 333137vl 38 encoding a fusion protein.
  • BLASTN algorithm searching of the Genbank database using the human ERDBP-l nucleic acid sequence SEQ ED NO: 7 or 45 as the search target found that there was almost 100%) identity with regions of the human genomic DNA sequence GenBank Accession No. AC005237 from the human chromosome lp31.31.3-32.2 and at least one human gene encoding the ERDBP-1 transcribed nucleic acid and protein derived therefrom is comprised of at least 26 exons as shown in Table 1, Example 5.
  • the present invention is intended also to provide isolated nucleic acids comprising at least one exon, or a fragment, variant or derivative thereof, capable of hybridizing with at least one region of the sequences SEQ ID NOS: 2, 5-10, 14, and 44-45 under low, medium or high stringency conditions, wherein the hybridization is specific for an ERDBP-1 -encoding nucleic acid, or a fragment, variant or derivative thereof.
  • One aspect ofthe invention therefore also provides nucleic acids that hybridize under selected high, medium or low stringency conditions to a nucleic acid that encodes a peptide having all of, a derivative of, or a portion of an amino acid sequence derived from the nucleic acid sequences SEQ ED NOS: 2, 5-10, 14, and 44- 45.
  • Appropriate stringency conditions which promote DNA hybridization for example, 6 x SSC at about 45°C, followed by a wash of 2 x SSC at 50°C, are well known to those skilled in the art or can be found, for example, in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
  • the salt concentration in the wash step can be selected from a low stringency of about 2 x SSC at 50°C. to a high stringency of about 0.2 x SSC at 50°C.
  • the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22°C, to high stringency conditions at about 65°C.
  • Isolated nucleic acids that differ in sequence from the nucleotide sequences represented in SEQ ED NOS: 2, 5-10, 13, and 44-45 due to degeneracy in the genetic code are also within the scope of the invention.
  • Such nucleic acids can encode functionally equivalent peptides (i.e., a polypeptide having a biological activity of a ERDBP-1 protein).
  • Isolated nucleic acid sequence variants may also encode non- functional polypeptides, the sequences of which are substantially similar, but not
  • ATLANTA 333137vl 39 identical, to those of functional variants of ERDBP-1. These isolated nucleic acids may be used to generate variant animals with inactive or functionally modified ERDBP-1 polypeptides or fragments, variants or derivatives thereof.
  • a number of amino acids are designated by more than one triplet. Codons that specify the same amino acid, or synonyms (for example, CAU and CAC are synonyms for histidine) may result in "silent" mutations which do not affect the amino acid sequence of the subject protein.
  • CAU and CAC are synonyms for histidine
  • nucleotides up to about 3-4% of the nucleotides
  • nucleic acids encoding peptides having an activity of, for example, an ERDBP-1 protein may exist among individuals due to natural allelic variation. Any and all such nucleotide variations and resulting amino acid polymo ⁇ hisms are within the scope of this invention.
  • Nucleic acid variants having sequence differences of about 3-4% may be readily detectable under high or medium stringency hybridization conditions using, for example, any of SEQ ED NOS: 2, 5-10, 13 or 44-45 or fragments thereof, such as SEQ ED NO: 4, as the probe.
  • Fragments of a nucleic acid encoding an active portion of one of the subject ERDBP-1 proteins are also within the scope of the invention.
  • a fragment ofthe nucleic acid encoding an active portion of a ERDBP-1 protein refers to a nucleotide sequence having fewer nucleotides than the nucleotide sequence encoding the entire amino acid sequence of the protein but which encodes a peptide that possesses agonistic or antagonistic activity relative to a naturally occurring form of the protein.
  • Nucleic acid fragments within the scope of the invention also include those capable of hybridizing under high or low stringency conditions with nucleic acids from other species for use in screening protocols to detect ERDBP-1 homologs. Comparison of the nucleic acid sequences of rat and human ERDBP-1 show that oligonucleotide primers can be generated that are suitable for detecting and isolating
  • ATLANTA 333137vl 4Q ERDBP-1 clones in other eukaryotes could be used to detect ERDBP-1 homologs in other vertebrate species, such as, but not only, human, mice, rats, chickens.
  • SEQ ED NO:2 was used to identify a hybridizing human ERDBP-1 -encoding cDNA SEQ ED NO: 7 under medium stringency hybridization conditions.
  • nucleic acid comprising a nucleic acid sequence substantially similar to the clone 52 cDNA sequence (SEQ ED NO: 2) encoding at least a region of a rat IRDBP-1 protein (SEQ ED NO: 3) as shown in Figs. 1 and 2 respectively, or any variants thereof.
  • the nucleic acid molecules of the present invention can include an isolated deletion mutation conesponding to the IRDBP-1 phenotype, a natural IRDBP-1 gene, an ERDBP-1 cDNA molecule, a degenerate variant, a truncated form thereof, a homolog thereof or any other modified versions.
  • a nucleic acid comprising a nucleic acid sequence substantially similar to the cDNA sequence for a rat IRDBP-1 (SEQ ED NO: 5) as shown in Figs. 4A and 4B, or any variant thereof.
  • the nucleic acid molecules of the present invention can include an isolated deletion mutation conesponding to the ERDBP-1 phenotype, a natural IRDBP-1 gene, an ERDBP-1 cDNA molecule, a degenerate variant thereof, a truncated variant thereof or a homolog thereof or any other variant thereof, including a human IRDBP-1 -encoding nucleic acid having at least 75% sequence similarity to SEQ ED NOS: 2 or 5.
  • a nucleic acid comprising a nucleic acid sequence substantially similar to the cDNA sequence for a rat IRDBP-1 (SEQ ID NO: 6) shown in Figs. 5A and 5B comprising a variant of SEQ ID NO: 5.
  • an isolated nucleic acid comprises the nucleic acid sequence conesponding to a human ERDBP- 1 sequence SEQ ED NO: 7 as shown in Figs. 6A-6C.
  • an isolated nucleic acid comprises the nucleic acid sequence conesponding to a variant human ERDBP-1 (SEQ ED NO: 10) as shown in Figs. 8A and 8B.
  • a mammalian IRDBP-1 gene or nucleic acid molecule can be allelic variants of SEQ ED NOS: 2, 5-10 and 44-
  • allelic variant is a gene that occurs essentially at the same locus or loci in the mammalian genome as the genes comprising SEQ ED NOS: 5-10, 14 and 44-45, but which has similar, but not identical, sequences to that of SEQ ID NO: 5-10 and 44-45.
  • an isolated nucleic acid molecule ofthe present invention includes a nucleic acid that is at least about 75%, preferably at least about 80%, more preferably at least about 85%, even more preferably at least about 90%), and even more preferably at least about 95% identical to a rat-derived IRDBP-1 -encoding nucleic acid molecule as depicted in SEQ ED NO: 5 or 44, and/or a variant thereof, such as, but not limited to, SEQ ED NOS: 6 and 14 or the human ERDBP-1 nucleic acids SEQ ED NOS: 7, 10 and 45.
  • an isolated nucleic acid molecule of the present invention includes a nucleic acid that is at least about 75%, preferably at least about 80%, more preferably at least about 85%, even more preferably at least about 90%>, and even more preferably at least about 95% identical to a human-derived nucleic acid molecule as depicted in SEQ ED NOS: 7 and 45, and/or a variant thereof, such as, but not limited to, SEQ ED NO: 10.
  • ERDBP-1 nucleic acid molecules SEQ ID NOS: 2, 5-10, 14 and 44-45
  • the nucleic acid sequences of a ERDBP-1 nucleic acid molecules (SEQ ID NOS: 2, 5-10, 14 and 44-45) of the present invention allow one skilled in the art to, for example, (a) make copies of those nucleic acid molecules by procedures such as, but not limited to, insertion into a cell for replication by the cell, by chemical synthesis or by procedures such as PCR or LCR, (b) obtain nucleic acid molecules which include at least a portion of such nucleic acid molecules, including full-length genes, full-length coding regions, regulatory confrol sequences, truncated coding regions and the like, (c) obtain ERDBP-1 nucleic acid homologs in other mammalian species such as the dog, cat, cow, pig or primates other than human and, (d) to obtain
  • ATLANTA 333137vl 42 isolated nucleic acids capable of hybridizing to a mammalian JJRDBP-1 nucleic acid and be used to detect the presence of ERDBP-1 nucleic acid sequences by complementation between the probe and the target nucleic acid.
  • nucleic acid homologs can be obtained in a variety of ways including by screening appropriate expression libraries with antibodies of the present invention; using traditional cloning techniques employing oligonucleotide probes made according to the present invention to screen appropriate libraries; amplifying appropriate libraries or DNA using oligonucleotide primers ofthe present invention in a polymerase chain reaction or other amplification method; and screening public and/or private databases containing genetic sequences using nucleic acid molecules of the present invention to identify targets.
  • prefened libraries to screen, or from which to amplify nucleic acid molecules include but are not limited to mammalian BAC libraries, genomic DNA libraries, and cDNA libraries.
  • preferred sequence databases useful for screening to identify sequences in other species homologous to IRDBP-1 include, but are not limited to, GenBank and the mammalian Gene Index database of The Institute of Genomics Research (TIGR).
  • Another aspect of the present invention is to provide protein sequences that comprise a mammalian ERDBP-1 protein, and derivatives and fragments thereof.
  • One embodiment of the present invention therefore, comprises a protein sequence (SEQ ID NO: 1
  • a rat ERDBP-1 protein having an amino acid sequence (SEQ ID NO: 11, illustrated in Fig. 9) derived from the coding region SEQ ED NO: 8, as in Fig. 4A, of the rat cDNA clone ERDBP-1 SEQ ID NO: 5.
  • a rat ERDBP-1 protein sequence SEQ ID NO: 47 illustrated in Fig. 15 is provided that is encoded by the coding region of human nucleic acid sequence SEQ ID NO: 44 (Fig. 13).
  • a human ERDBP-1 protein sequence (SEQ ED NO: 12, illustrated in Fig 10) is provided that is encoded by a coding region SEQ ED NO: 9 of the human nucleic acid sequence SEQ ED NO: 7, as shown in Fig. 6B.
  • a human ERDBP-1 protein sequence SEQ ID NO: 48, illusfrated in Fig. 16 is provided that is encoded by the coding region of human nucleic acid sequence SEQ ED NO: 45 (Fig. 14).
  • peptide fragments of a human or animal IRDBP-1 protein are provided, wherein the fragments may be immunogenic peptides, capable of inducing an immune response when administered to an animal, and which will be recognized and bound by an antibody or not immunogenic when administered to an animal.
  • the peptide fragment is an epitope essentially within the carboxy-region ofthe rat ERDBP-1 protein SEQ ED NO: 3 (as in Fig. 2) and has the amino acid sequence: AcetylatedCys-Thr-Ser-Gln-Asn-Thr-Lys-
  • Ser-Arg-Ty-Iso-Pro-Asn-Gly-Lys-Leu (SEQ ID NO: 15) at amino acid positions 62-76 ofthe rat ERDBP-1 amino acid sequence SEQ ED NO: 3 shown in Fig. 2.
  • the epitope is substantially within the N-region of the ERDBP-1 protein between amino acid positions 233-247 of SEQ ED NO: 44 and having the sequence AcetylatedCys-Arg-Asn-Gly-Gly-Thr-Tyr-Lys-Glu-Thr-Gly-Asp- Glu-Tyr-Arg (SEQ ED NO: 46).
  • Isolated peptides and polypeptides of the present invention may also include any protein fragments thereof, a protein analogue, or any immunologic fragments thereof.
  • an IRDBP-1 nucleic acid molecule of the present invention encodes a protein having an amino acid sequence that is at least about 75%, preferably at least about 80%, more preferably at least about
  • ATLANTA 333137vl 44 85%, even more preferably at least about 90%, and more preferably still at least about 95% identical to a rat ERDBP-l protein whose amino acid sequence is disclosed in SEQ ED NO: 11 or 47, as well as allelic variants of an IRDBP-1 nucleic acid molecule encoding a protein having these sequences, including nucleic acid molecules that have been modified to accommodate codon usage properties of the cells in which such nucleic acid molecules are to be expressed.
  • an IRDBP-1 nucleic acid molecule of the present invention encodes a protein having an amino acid sequence that is at least about 75%, preferably at least about 80%, more preferably at least about 85%, even more preferably at least about 90%>, and more preferably still at least about 95% identical to a human ERDBP-1 protein whose amino acid sequence is disclosed in SEQ ID NO: 12 and 48, as well as allelic variants of an IRDBP-1 nucleic acid molecule encoding a protein having these sequences, including nucleic acid molecules that have been modified to accommodate codon usage properties ofthe cells in which such nucleic acid molecules are to be expressed.
  • Isolated peptidyl portions ofthe subject ERDBP-1 proteins within the scope of the present invention can be obtained by screening peptides recombinantly produced from the conesponding fragment of the nucleic acid encoding such peptides.
  • fragments can be chemically synthesized using techniques known in the art such as conventional Merrifield solid phase f-Moc or t-Boc chemistry.
  • one of the subject ERDBP-1 proteins may be arbitrarily divided into fragments of desired length with no overlap of the fragments, or preferably divided into overlapping fragments of a desired length.
  • the fragments can be produced recombinantly or by chemical synthesis and tested to identify those peptidyl fragments which can function as either agonists or antagonists of, for example, IRDBP-1 binding to nucleic acids.
  • Other fragments such as, for example, SEQ ED NOS: 15 and 46 are especially useful for the generation of antibodies specific for the ERDBP-1 protein or selected regions thereof.
  • peptidyl portions of ERDBP-1 can tested for nucleic acid-binding activity, as well as preventing inhibitory ability, by expression as, for example, thioredoxin fusion proteins each of which contains a
  • ATLANTA 333137vl 45 discrete fragment of the IRDBP-1 protein see, for example, U.S. Patent Nos. 5,270,181 and 5,292,646; and PCT publication WO94/02502) inco ⁇ orated herein by reference in their entireties.
  • an ERDBP-1 polypeptide for such pu ⁇ oses as enhancing therapeutic or prophylactic efficacy, or stability (e.g., shelf life ex vivo and resistance to proteolytic degradation in vivo).
  • modified peptides are considered functional equivalents of peptides having an activity of, or which antagonize, a IRDBP-1 protein as defined herein.
  • a modified polypeptide can be produced in which the amino acid sequence has been altered, such as by amino acid substitution, deletion, or addition.
  • Whether a change in the amino acid sequence of a peptide results in a functional ERDBP-1 homolog can be readily determined by assessing the ability of the variant peptide to, for instance, mediate ubiquitination in a fashion similar to the wild-type
  • a host cell is transformed with a nucleic acid comprising the sequences SEQ ED NOS: 5-10, 14 or 44-45, or variants thereof.
  • the transformed cell may, but not necessarily, express the transformed nucleic acid to yield rat (SEQ ID NOS: 3, 11, and 47) or human (SEQ ID NOS: 12-13 and 48) ERDBP-1 polypeptides respectively, or any fragment or derivative thereof.
  • a recombinant expression vector suitable for transformation of a host cell means that the recombinant expression vector contains a nucleic acid molecule, or an oligonucleotide fragment thereof, of the present invention coupled to a regulatory sequence selected on the basis of the host cell used for expression.
  • the nucleic acid sequence coding for the ERDBP-1 protein of the present invention may be operatively linked to a regulatory sequence selected to direct expression ofthe desired protein in an appropriate host cell.
  • the protein ofthe present invention may be produced in purified form by any known conventional techniques. For example, rat or human cells may be homogenized and centrifuged. The supernatant is then subjected to sequential ammonium sulfate precipitation and heat treatment. The fraction containing the protein ofthe present invention is subjected to gel filtration in an appropriately sized dextran or polyacrylamide column to separate the proteins. If necessary, the protein fraction may be further purified by HPLC.
  • the present invention provides novel compositions comprising nucleotide sequences encoding ERDBP-1 fragments. Also provided are recombinant proteins produced using the novel coding sequences, and methods of using the recombinant proteins.
  • DNA nucleic acid molecules of the present invention can be inco ⁇ orated into cells using conventional recombinant DNA technology. Such techniques are
  • ATLANTA 333137vl 47 especially useful, for example, for producing ERDBP-1 polypeptides in cells, or to regulate the expression ofthe naturally occurring IRDBP-1 gene in the recipient cells.
  • the DNA molecule may be inserted into an expression system to which the DNA molecule is heterologous (i.e. not normally present). Alternatively, as described more fully below, the DNA molecule may be introduced into cells which normally contain the DNA molecule, as, for example, to conect a deficiency or defect in ERDBP-1 expression, or where over-expression ofthe ERDBP-1 protein is desired.
  • the heterologous DNA molecule can be inserted into the expression system or vector in proper sense orientation and conect reading frame.
  • the vector contains the necessary elements for the transcription and translation ofthe inserted protein-coding sequences.
  • Recombinant genes may also be introduced into viruses, such as vaccinia virus or adenovirus.
  • Recombinant viruses can be generated by transfection of plasmids into cells infected with virus.
  • Suitable vectors include, but are not limited to, the following viral vectors such as lambda vector system gtl 1, gt WES.tB, Charon 4, and plasmid vectors such as pBR322, pBR325, ⁇ ACYC177, pACYC184, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pKC37, pKClOl, SV 40, pBluescript H SK +/- or KS +/- (see "Stratagene Cloning Systems” Catalog (1993) from Stratagene, La Jolla, Calif, which is hereby inco ⁇ orated by reference), pQE, pEH821, PGEX, pET series (see Studier
  • Recombinant molecules can be infroduced into cells via transformation, particularly transduction, conjugation, mobilization, or elecfroporation.
  • the DNA sequences are cloned into the vector using standard
  • host-vector systems may be utilized to express the protein- encoding sequence(s).
  • the vector system must be compatible with the host cell used.
  • Host-vector systems include but are not limited to the following: bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA; microorganisms such as yeast containing yeast vectors; mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus).
  • the expression elements of these vectors vary in their strength and specificities. Depending upon the host-vector system utilized, any one of a number of suitable transcription and translation elements can be used.
  • RNA polymerase messenger RNA
  • Transcription of DNA is dependent upon the presence of a promoter which is a DNA sequence that directs the binding of RNA polymerase and thereby promotes mRNA synthesis.
  • the DNA sequences of eukaryotic promoters differ from those of prokaryotic promoters.
  • eukaryotic promoters and accompanying genetic signals may not be recognized in or may not function in a prokaryotic system, and, further, prokaryotic promoters are not recognized and do not function in eukaryotic cells.
  • SD Shine- Dalgarno
  • Promoters vary in their "strength" (i.e. their ability to promote transcription). For the pmposes of expressing a cloned gene, it is desirable to use sfrong promoters in order to obtain a high level of franscription and, hence, expression of the gene. Depending upon the host cell system utilized, any one of a number of suitable promoters may be used. For instance, when cloning in E.
  • promoters such as the T7 phage promoter, lac promotor, frp promotor, recA promotor, ribosomal RNA promotor, the P R and P promoters of coliphage lambda and others, including but not limited, to lacUV5, ompF, bla, lpp, and the like, may be used to direct high levels of franscription of adjacent DNA segments.
  • a hybrid frp-lacUV5 (tac) promotor or other E. coli promoters produced by recombinant DNA or other synthetic DNA techniques may be used to provide for franscription of the inserted gene.
  • Bacterial host cell strains and expression vectors may be chosen which inhibit the action ofthe promotor unless specifically induced.
  • the addition of specific inducers is necessary for efficient franscription of the inserted DNA.
  • the lac operon is induced by the addition of lactose or EPTG (isopropylthio- beta-D-galactoside).
  • lactose or EPTG isopropylthio- beta-D-galactoside
  • Specific initiation signals are also required for efficient gene franscription and translation in prokaryotic cells. These franscription and translation initiation signals may vary in "strength" as measured by the quantity of gene specific messenger RNA and protein synthesized, respectively.
  • the DNA expression vector which contains a promoter, may also contain any combination of various "strong" franscription and/or translation initiation signals.
  • efficient translation in E. coli requires a Shine-Dalgarno (SD) sequence about 7-9 bases 5' to the initiation codon (ATG) to provide a ribosome binding site.
  • SD-ATG combination that can be utilized by host cell ribosomes may be employed. Such combinations include but are not limited to the SD-ATG combination from the cro gene or the N gene of coliphage
  • ATLANTA 333137vl 5Q lambda or from the E. coli tryptophan E, D, C, B or A genes. Additionally, any SD- ATG combination produced by recombinant DNA or other techniques involving inco ⁇ oration of synthetic nucleotides maybe used.
  • the isolated DNA molecule ofthe present invention Once the isolated DNA molecule ofthe present invention has been cloned into an expression system, it is ready to be inco ⁇ orated into a host cell. Such inco ⁇ oration can be carried out by the various forms of transformation noted above, depending upon the vector/host cell system.
  • Suitable host cells include, but are not limited to, bacteria, virus, yeast, mammalian cells, and the like.
  • Recombinant expression vectors can be designed for the expression of the encoded proteins in prokaryotic or eukaryotic cells.
  • the prokaryotic expression system may comprise the host bacterial species E. coli, B. subtilis or any other host cell known to one of skill in the art.
  • Useful vectors may comprise constitutive or inducible promoters to direct expression of either fusion or non-fusion proteins.
  • fusion vectors a number of amino acids are usually added to the expressed target gene sequence such as, but not limited to, a protein sequence for thioredoxin.
  • a proteolytic cleavage site may further be introduced at a site between the target recombinant protein and the fusion sequence.
  • a region of amino acids such as a polymeric histidine region may be infroduced to allow binding to the fusion protein by metallic ions such as nickel bonded to a solid support, and thereby allow purification of the fusion protein.
  • the cleavage site allows the target recombinant protein to be separated from the fusion sequence.
  • Enzymes suitable for use in cleaving the proteolytic cleavage site includes, but are not limited to, Factor Xa and thrombin.
  • Fusion expression vectors that may be useful in the present invention include pGex (Amrad Co ⁇ ., Melbourne, Australia), pRIT5 (Pharmacia, Piscataway, NJ) and pMAL (New England Biolabs, Beverly, MA), that fuse glutathione S-transferase, protein A, or maltose E binding protein, respectively, to the target recombinant protein.
  • E. coli expression vector pUR278 as described in Ruther et al. (1983) E.M.B.O.J. 2: 1791, inco ⁇ orated herein
  • the nucleotide sequence coding for the IRDBP-1 gene product may be ligated in frame with the lacV coding region to produce a fusion protein.
  • eukaryotic vectors such as mammalian, yeast or insect cells.
  • the use of eukaryotic vectors permits partial or complete post-translational modification such as, but not only, glycosylation and/or the formation ofthe relevant inter- or infra-chain disulfide bonds.
  • Examples of vectors useful for expression in the yeast Saccharomyces cerevisiae include pYepSecl as in Baldari et al, (1987), E.M.B.O.J., 6: 229-234 and pYES2 (Invitrogen Co ⁇ ., San Diego, CA), inco ⁇ orated herein by reference in their entirety.
  • Baculovirus vectors are also available for the expression of proteins in cultured insect cells (F9 cells).
  • the use of recombinant Baculovirus vectors can be, or is, analogous to the methods disclosed in Richardson CD. ed., (1995), "Baculovirus Expression Protocol” Humana Press Inc.; Smith et al. (1983) Mol. Cell. Biol. 3: 2156- 2165; Pennock et al. (1984) Mol. Cell. Biol. 4: 399-406 and inco ⁇ orated herein by reference in their entirety.
  • vectors useful for expressing the IRDBP-1 protein, or an epitope of a ERDBP-1 protein include viral vectors.
  • Methods for making a viral recombinant vector useful for expressing the ERDBP-1 protein are analogous to the methods disclosed in U.S. Patent Nos. 4,603,112; 4,769,330; 5,174,993; 5,505,941; 5,338,683; 5,494,807; 4,722,848; Paoletti, E. (1996) Proc. Natl. Acad. Sci. 93: 11349-11353; Moss (1996) Proc. Natl. Acad. Sci. 93: 11341-11348; Roizman (1996) Proc. Natl. Acad. Sci.
  • One embodiment of the present invention is a recombinant viral vector comprising an adenovirus vector capable of expressing in a suitable host cell a polypeptide encoded by at least a region of the nucleic acids SEQ ED NO: 44.
  • ATLANTA 333137vl 52 expressed polypeptide is capable of binding to an IRE, wherein the binding can be modulated by insulin, as described in Example 20.
  • the recombinant adenoviral vector may express the ERDBP-1 nucleic acid as an antisense nucleic acid that is not translated but, by hybridizing to a region of the
  • ERDB-1 gene or a transcript thereof can modulate the level of ERDBP-1 activity in a cell.
  • Probes, primers and sense/antisense oligonucleotides specific for IRDBP-1 Another aspect of the present invention pertains to the use of an isolated nucleic acid molecule for constructing nucleotide probes and primers useful for a variety of functions.
  • synthetic oligonucleotide probes are useful for detecting complementary nucleotide sequences in biological materials such as cells, cell extracts or tissues (as well as in an in situ hybridization technique).
  • Isolated nucleic acids synthesized according to the present invention can determine whether a cell expresses an mRNA transcript encoding the ERDBP-1 protein.
  • the present invention also contemplates the use of antisense nucleic acid molecules, which are designed to be complementary to a coding strand of a nucleic acid (i.e., complementary to an mRNA sequence) or, alternatively, complimentary to a 5' or 3' unfranslated region ofthe mRNA.
  • Another use of synthetic nucleotides is as primers (DNA or RNA) for a polymerase chain reaction (PCR), ligase chain reaction (LCR), or the like.
  • Synthesized nucleotides can be produced in variable lengths - the number of bases synthesized will depend upon a variety of factors, including the desired use for the probes or primers. Additionally, sense or anti-sense nucleic acids or oligonucleotides can be chemically synthesized using modified nucleotides to increase the biological stability of the molecule or ofthe binding complex formed between the anti-sense and sense nucleic acids. For example, acridine substituted nucleotides can be synthesized. Protocols for designing isolated nucleotides, nucleotide probes, and/or nucleotide primers are well-known to those of ordinary skill, and can be
  • ATLANTA 333137vl 53 purchased commercially from a variety of sources (e.g., Sigma Genosys, The Woodlands, TX or The Great American Gene Co., Ramona, CA).
  • Nucleotides constructed in accordance with the present invention can be labeled to provide a signal as a means of detection.
  • radioactive elements such as 32 P, 3 H, and 35 S or the like provide sufficient half-life to be useful as radioactive labels.
  • Other materials useful for labeling synthetic nucleotides include fluorescent compounds, enzymes and chemiluminescent moieties. Methods useful in selecting appropriate labels and binding protocols for binding the labels to the synthetic nucleotides are well known to those of skill in the art. Standard immunology manuals such as Promega: Protocol and Applications Guide. 2nd Edition, 1991 (Promega Co ⁇ ., Madison, WI; the content of which is inco ⁇ orated herein in its entirety) may be consulted to select an appropriate labeling protocol without undue experimentation.
  • the antibody may be monoclonal or polyclonal and may be produced by conventional methodology using the ERDBP-1 protein, or an immunologic fragment thereof, as an immunogen.
  • a mammal i.e., a mouse, rabbit, horse, sheep, or goat
  • a mammal i.e., a mouse, rabbit, horse, sheep, or goat
  • an immunization protocol conducive to producing antibodies reactive with the IRDBP-1 protein or a fragment thereof.
  • antiserum reactive with the jointed protein may be collected and, if desired, polyclonal anti-ERDBP-1 antibodies isolated.
  • One embodiment ofthe present invention is a fragment of an amino acid sequence of the rat ERDBP-1 protein of SEQ TD NOS: 3, 11 or 47, or human ERDBP-1 protein (SEQ ED NOS: 12, 13 or 48) that may be synthesized and used as an immunogen to produce an anti-IRDBP-1 polyclonal antibody.
  • SEQ ED NOS: 12, 13 or 48 human ERDBP-1 protein
  • ATLANTA 333137vl 54 of the immunogenic peptide synthesized are: AcetylatedCys-Thr-Ser-Gln-Asn-Thr- Lys-Ser-Arg-Tyr-Ile-Pro-Asn-Gly-Lys-Leu (SEQ ED NO: 15) at amino acid positions 786-800 ofthe rat LRDBP-1 amino acid sequence SEQ ED NO: 47 and AcetylatedCys- Arg-Asn-Gly-Gly-Thr-Tyr-Lys-Glu-Thr-Gly-Asp-Glu-Tyr-Arg (SEQ ID NO: 46).
  • the polyclonal antibody raised against the peptide SEQ ED NO: 15 was specific for the carboxy region rat ERDBP-1 protein and cross-reacted with the human ERDBP-1 protein.
  • the polyclonal antibody raised against the peptide SEQ ED NO: 46 is specific for the N-region ofthe rat or human ERDBP-1.
  • Antibodies that specifically bind, for example, ERDBP-1 epitopes can also be used in immunohistochemical staining of tissue samples in order to evaluate the abundance and pattern of expression of ERDBP-1.
  • Anti-ERDBP-1 antibodies can be used diagnostically in immuno-precipitation and immuno-blotting to detect and evaluate IRDBP-1 levels in tissue or bodily fluid as part of a clinical testing procedure. For instance, such measurements can be useful in predictive valuations of the onset or progression of diabetes or cell proliferation disorders.
  • the ability to monitor IRDBP-1 levels in an individual can allow determination of the efficacy of a given treatment regimen for an individual afflicted with such a disorder.
  • the level of ERDBP-1 can be measured in cells isolated from bodily fluid, such as in samples of cerebral spinal fluid or blood, or can be measured in tissue, such as produced by biopsy.
  • Diagnostic assays using anti-ERDBP-1 antibodies can include, for example, immunoassays designed to aid in early diagnosis of a diabetic, neoplastic or hype ⁇ lastic disorder, e.g. the presence of insulin-responsive negative cells in the sample, e.g. to detect cells in which a lesion ofthe IRDBP-1 gene has occuned.
  • anti-ERDBP-1 antibodies are in the immunological screening of cDNA libraries constructed in expression vectors, such as ⁇ gtl l, ⁇ gtl ⁇ - 23, ⁇ ZAP, and ⁇ ORF8.
  • Messenger libraries of this type having coding sequences inserted in the conect reading frame and orientation, can produce fusion proteins. For instance, ⁇ gtl 1 will produce fusion proteins whose amino termini consist of .beta.- galactosidase amino acid sequences and whose carboxy termini consist of a foreign polypeptide.
  • Antigenic epitopes of ERDBP-1 can then be detected with antibodies, as,
  • ATLANTA 333137vl 55 for example, reacting nitrocellulose filters lifted from infected plates with anti- ERDBP-1 antibodies. Phage, scored by this assay, can then be isolated from the infected plate. Thus, the presence of ERDBP-l homologs can be detected and cloned from other human sources, i.e. to identified other closely homologous human isoforms, as well as to identify IRDBP-1 homologs in other mammals.
  • an assay to detect natural serum antibodies specific for the ERDBP-1 protein may be induced as a result of the release of ERDBP-1 or fragments thereof, during the onset of deterioration and destruction ofthe cells ofthe islets of Langerhan. The detection of the antibodies will provide a diagnostic indication of the onset of diabetes, cancer and the progressive loss of pancreatic activity.
  • nucleotide sequence determined from the cloning of subject IRDBP-1 from a human or animal cell line will further allow for the generation of probes designed for use in identifying IRDBP-1 homologs in other animal cell-types, particularly cells associated with the onset and maintenance of diabetes and obesity, cancer or other transformed or immortalized cells, as well as IRDBP-1 homologs from other non-human mammals.
  • nucleotide probes can be generated from a cloned nucleic acid sequence of the ERDBP-1 protein, which allow for histological screening of intact tissue and tissue samples for the presence of IRDBP-1 mRNA.
  • the use of probes directed to ERDBP-1 mRNA, or to genomic ERDBP-1 sequences can be used for both predictive and therapeutic evaluation of allelic mutations which might be manifest in, for example, diabetes or other metabolic disorders directly or indirectly attributed to a failure of the cells to respond or over-respond to insulin as well as neoplastic or hype ⁇ lastic disorders such as, but not limited to, unwanted cell growth.
  • the nucleotide probes can help facilitate the determination of the molecular basis for a disorder or
  • ATLANTA 333137vl 56 ailment that may involve some abnormality associated with expression (or lack thereof) of an ERDBP-1 protein and perturbation of insulin regulation of a gene expression or activity.
  • nucleic acid molecules complementary to an ERDBP-l coding sequence can be used to determine if cells contain ERDBP-1 coding sequences using Southern hybridization analysis.
  • Nucleic acid molecules can also be used to determine the level of expression of ERDBP-1 mRNA in cells using Northern analysis as discussed in Example 8.
  • nucleotide sequence of the isolated DNA molecule of the present invention may be used as a probe in nucleic acid hybridization assays for the detection of the IRDBP-1 gene in various patient body fluids.
  • the nucleotide sequence of the present invention may be used in any nucleic acid hybridization assay system known in the art, including, but not limited to, Southern blots (Southern, E.M. (1975) J. Mol. Biol. 98: 508; Northern blots (Thomas et al. (1980) Proc. Natl. Acad. Sci. 77: 5201-05); Colony blots (Grunstein et al, (1975) Proc. Natl.
  • the isolated DNA molecule of the present invention can be used in a gene amplification detection procedure such as a polymerase chain reaction (Erlich et al. (1991) "Recent Advances in the Polymerase Chain Reaction” Science 252: 1643-51, which is hereby inco ⁇ orated by reference) or in restriction fragment length polymo ⁇ hism (RFLP) diagnostic techniques, as described in Watson et al, (2d ed. 1992), Recombinant DNA, Scientific American Books, 519-522, 545-547, which is hereby inco ⁇ orated by reference.
  • a polymerase chain reaction Erlich et al. (1991) "Recent Advances in the Polymerase Chain Reaction” Science 252: 1643-51, which is hereby inco ⁇ orated by reference
  • RFLP restriction fragment length polymo ⁇ hism
  • the DNA molecules of the invention can be used in prenatal or postnatal diagnosis of the human diseases associated with defects in response to variation in the level of insulin.
  • a probe for the DNA encoding ERDBP-1 can be designed using the DNA molecule ofthe invention, and used to probe the DNA obtained from amniotic fluid or chorionic tissue and amplified by PCR, LCR or any other known amplification technique for the presence of the IRDBP-1 gene or a variant thereof, as noted above. Similar procedures can be used in postnatal
  • ATLANTA 333137vl 57 diagnostic work as, for example, to diagnose the source of an ERDBP-1 deficiency in a person who is diabetic.
  • the present method provides a method for determining if a subject is at risk for a disorder characterized by unwanted insulin non-responsiveness or cell proliferation.
  • the subject method can be generally characterized as comprising: detecting in a tissue of a subject (e.g. a human patient), the presence or absence of a genetic lesion characterized by at least one of (i) a mutation of a gene encoding IRDBP-1 or (ii) the mis-expression ofthe IRDBP-1 gene.
  • such genetic lesions can be detected by ascertaining the existence of at least one of (i) a deletion of one or more nucleotides from the IRDBP-1 gene, (ii) an addition of one or more nucleotides to the IRDBP-1 gene, (iii) a substitution of one or more nucleotides of the IRDBP-1 gene, (iv) a gross chromosomal reanangement of the IRDBP-1 gene, (v) a gross alteration in the level of a messenger RNA transcript of the IRDBP-1 gene, (vi) the presence of a non-wild type splicing pattern of a messenger RNA transcript ofthe IRDBP-1 gene, and (vii) a non- wild type level ofthe IRDBP-1 protein.
  • a probe/primer comprising an oligonucleotide containing a region of nucleotide sequence which is capable of hybridizing to a sense or antisense sequence derived from nay of the rat or human ERDBP-1 sequences SEQ ED NOS: 2, 5-10, 14 or 44-45, or naturally occurring mutants thereof, or 5' or 3' flanking sequences or intronic sequences naturally associated with the IRDBP-1 gene.
  • the probe is exposed to nucleic acid of a tissue sample; and the hybridization of the probe to the sample nucleic acid is detected.
  • detection of the lesion comprises utilizing the probe/primer in, for example, a polymerase chain reaction (PCR) (see, e.g., U.S. Patent Nos. 4,683,195 and 4,683,202), or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al. (1988) Science 241: 1077-1080; and Nakazawa et al. (1994) Proc. Natl. Acad. Sci. 91: 360-364), the later of which can be particularly useful for detecting even point mutations in the IRDBP-1 gene and which are inco ⁇ orated herein in their entirety.
  • the level of ERDBP-1 protein can be detected in an immunoassay.
  • ERDBP-1 can also be used for the treatment of obesity and complications associated with obesity.
  • the organ systems and the specific diseases associated with obesity include the following: (1) cardiovascular system: hypertension, congestive heart failure, cor pulmonale, varicose veins, pulmonary embolism, coronary heart disease; (2) Endocrine: insulin resistance, glucose intolerance, type II diabetes mellitus, dyslipidemia, polycystic ovary syndrome, infertility, amenonhea; (3) Musculoskeletal: immobility, degenerative arthritis, low back pain; (4) Integument: venous stasis of legs, cellulitis, intertrigo, carbuncles; (5) Respiratory system: dyspnea and fatigue, obstructive sleep apnea, hypoventilation (pickwickian) syndrome; (6) Gastrointestinal: gastroesophageal reflux disease, hepatic steatosis, nonalcoholic steatohepatitis, cholelithiasis,
  • ERDBP-1 expression was detected in the areas of the brain known to be involved in ingestive, autonomic and neuroendocrine functions of feeding and satiety, as described in the Examples 10 - 12 below.
  • Regulation of body weight requires a balance among energy intake, expenditure, and storage. The brain appears to define the set point around which body weight is regulated.
  • the levels of ERDBP-1 mRNA in the lateral hypothalamus and the nucleus of the solitary tract are differentially regulated in obese as compared to lean Zucker rats, showing a significant interactive role of IRDBP-1 in modulating body weight.
  • IRDBP-1 polypeptides of the invention can be used in therapeutic applications. Since IRDBP-1 increases the transcription of IGFBP-3, ERDBP-1 can be used.
  • ATLANTA 333137vl 59 used to treat diseases (e.g., diabetes) associated with low levels of IGFBP-3. Further, many diseases are associated with an excess of circulating IGF-1 or IGF-EI, for example, some cancers and type II diabetes. IRDBP-1 can be used in patients with low levels of IGFBP-3 or high levels of IGF. Introduction ofthe gene encoding ERDBP-1 (or a functional derivative) into cells using either retroviral vectors or liposomes results in increased production of IGFBP-3. Many methods of delivering expressible coding sequences to cells are known in the art.
  • a useful application of the DNA molecules of the present invention is the possibility of increasing the amount of IRDBP-1 protein present in a mammal by gene transfer (so-called "gene therapy").
  • gene therapy gene transfer
  • this gene would be transfened into the animal host along with promoters, inducers, and the like (which are well known and recognized techniques in the field of genetic engineering, as noted supra) to allow the cell to initiate and continue production of the genetic product protein.
  • the DNA molecule ofthe present invention can be transfened into the extra- chromosomal or genomic DNA of the host.
  • IRDBP-1 -expression vectors to be used as a part of a gene therapy protocol to reconstitute ERDBP-1 function in a cell in which ERDBP-1 is mis-expressed, or alternatively, to provide an antagonist of the naturally-occurring ERDBP-1 or an antisense construct.
  • expression constructs of the subject ERDBP-1- proteins may be administered in any biologically effective carrier, e.g. any formulation or composition capable of effectively fransfecting cells in vivo with a recombinant ERDBP-1 -gene.
  • Approaches include insertion of the subject gene in viral vectors including recombinant retroviruses, adenovirus, adeno-associated virus, and he ⁇ es simplex virus- 1, or recombinant bacterial or eukaryotic plasmids.
  • viral vectors including recombinant retroviruses, adenovirus, adeno-associated virus, and he ⁇ es simplex virus- 1, or recombinant bacterial or eukaryotic plasmids.
  • Viral vectors can be
  • ATLANTA 333137vl 0 used to fransfect cells directly; plasmid DNA can be delivered with the help of, for example, cationic liposomes (lipofectin) or derivatized (e.g. antibody conjugated), polylysine conjugates, gramacidin S, artificial viral envelopes or other such intracellular carriers, as well as direct injection of the gene construct or CaPO 4 precipitation carried out in vivo.
  • lipofectin lipofectin
  • derivatized e.g. antibody conjugated
  • a prefened approach for in vivo infroduction of nucleic acid encoding one of the subject proteins into a cell is by use of a viral vector containing nucleic acid, e.g. a cDNA, encoding the gene product.
  • a viral vector containing nucleic acid e.g. a cDNA
  • Infection of cells with a viral vector has the advantage that a large proportion of the targeted cells can receive the nucleic acid.
  • molecules encoded within the viral vector e.g., by a cDNA contained in the viral vector, are expressed efficiently in cells that have taken up viral vector nucleic acid.
  • Retrovirus vectors and adeno-associated virus vectors are generally understood to be the recombinant gene delivery system of choice for the fransfer of exogenous genes in vivo, particularly into humans. These vectors provide efficient delivery of genes into cells, and the transfened nucleic acids are stably integrated into the chromosomal DNA of the host. A major prerequisite for the use of refroviruses is to ensure the safety of their use, particularly with regard to the possibility of the spread of wild-type virus in the cell population.
  • recombinant retrovirus can be constructed in which part of the retroviral coding sequence (gag, pol, env) has been replaced by nucleic acid encoding an IRDBP-1 proteins, thereby rendering the retrovirus replication defective.
  • the replication defective retrovirus is then packaged into virions that can be used to infect a target cell through the use of a helper virus by
  • ATLANTA 333137vl 1 standard techniques. Protocols for producing recombinant refroviruses and for infecting cells in vitro or in vivo with such viruses can be found in Cunent Protocols in Molecular Biology, Ausubel et al. (1989) (eds.) Greene Publishing Associates, Sections 9.10-9.14 and other standard laboratory manuals. Examples of suitable refroviruses include pLJ, pZEP, pWE and pEM which are well known to those skilled in the art. Examples of suitable packaging virus lines for preparing both ecofropic and amphotropic retroviral systems include psiCrip, psiCre, psi2 and psiAm.
  • Refroviruses have been used to introduce a variety of genes into many different cell types, including neural cells, epithelial cells, endothelial cells, lymphocytes, myoblasts, hepatocytes, bone manow cells, in vitro and/or in vivo (see for example Eglitis, et al. (1985) Science 230: 1395-1398; Danos & Mulligan (1988) Proc. Natl. Acad. Sci. 85: 6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci. 85: 3014-3018; Armentano et al. (1990) Proc. Natl. Acad. Sci. 87: 6141-6145; Huber et al.
  • retroviral-based vectors by modifying the viral packaging proteins on the surface of the viral particle.
  • strategies for the modification of the infection spectrum of retroviral vectors include: coupling antibodies specific for cell surface antigens to the viral env protein (Roux et al. (1989) Proc. Natl. Acad. Sci. 86: 9079-9083; Julan et al. (1992) J. Gen. Virol. 73: 3251- 3255; and Goud et al. (1983) Virology 163: 251-254); or coupling cell surface ligands to the viral env proteins (Neda et al. (1991) J. Biol. Chem. 266: 14143-14146), and
  • ATLANTA 333137vl 62 which are inco ⁇ orated herein in their entireties.
  • Coupling can be in the form of the chemical cross-linking with a protein or other variety (e.g. lactose to convert the env protein to an asialoglycoprotein), as well as by generating fusion proteins (e.g. single- chain antibody/env fusion proteins).
  • This technique while useful to limit or otherwise direct the infection to certain tissue types, and can also be used to convert an ecofropic vector into an amphofropic vector.
  • use of retroviral gene delivery can be further enhanced by the use of tissue- or cell-specific transcriptional regulatory sequences that control expression ofthe ERDBP-1 -gene ofthe retroviral vector.
  • Another viral gene delivery system useful in the present invention utilizes adenovirus-derived vectors.
  • the genome of an adenovirus can be manipulated such that it encodes a gene product of interest, but is inactivated in terms of its ability to replicate in a normal lytic viral life cycle (see, for example, Berkner et al. (1988) BioTechniques 6: 616; Rosenfeld et al. (1991) Science 252: 43 1434; and Rosenfeld et al. (1992) Cell 68: 143-155), and which are inco ⁇ orated herein in their entirety.
  • adenoviral vectors derived from the adenovirus strain Ad type 5 dl324 or other strains of adenovirus are well known to those skilled in the art.
  • Recombinant adenoviruses can be advantageous in certain circumstances in that they are not capable of infecting nondividing cells and can be used to infect a wide variety of cell types, including airway epithelium (Rosenfeld et al. (1992) cited supra), endothelial cells (Lemarchand et al. (1992) Proc. Natl. Acad. Sci. 89: 6482- 6486), hepatocytes (Herz & Gerard (1993) Proc.
  • virus particle is relatively stable and amenable to purification and concentration, and as above, can be modified so as to affect the spectrum of infectivity. Additionally, infroduced adenoviral DNA
  • ATLANTA 333137vl 63 delivery vectors (Berkner et al. supra; Haj-Ahmand & Graham (1986) J. Virol. 57:267).
  • Most replication-defective adenoviral vectors cunently in use and therefore favored by the present invention are deleted for all or parts of the viral El and E3 genes but retain as much as 80% of the adenoviral genetic material (see, e.g., Jones et al. (1979) Cell 16:683; Berkner et ah, supra; and Graham et al. in Methods in Molecular Biology, E. J. Murray, (1991) Ed. (Humana, Clifton, N.J.) vol. 7. pp.
  • IRDBP-1-gene expression of the inserted IRDBP-1-gene can be under confrol of, for example, the E1A promoter, the major late promoter (MLP) and associated leader sequences, the E3 promoter, or exogenously added promoter sequences.
  • MLP major late promoter
  • E3 promoter E3 promoter
  • Adeno-associated virus is a naturally occuning defective virus that requires another virus, such as an adenovirus or a he ⁇ es virus, as a helper virus for efficient replication and a productive life cycle.
  • AAV adeno-associated virus
  • Vectors containing as little as 300 base pairs of AAV can be packaged and can integrate. Space for exogenous DNA is limited to about 4.5 kb.
  • An AAV vector such as that described in Tratschin et al. (1985) Mol. Cell. Biol. 5:3251-3260 can be used to introduce DNA into cells.
  • a variety of nucleic acids have been introduced into different cell types using AAV vectors (see for example Hermonat et al. (1984) Proc. Natl. Acad. Sci.
  • viral vector systems that may have application in gene therapy have been derived from such as, but not limited to, he ⁇ es virus, vaccinia virus, and several RNA
  • ATLANTA 333137vl 64 viruses may provide a unique strategy for persistence ofthe recombinant IRDBP-1 gene in cells ofthe central nervous system.
  • non-viral methods can also be employed to cause expression of an ERDBP-1 -protein, or an ERDBP-1 antisense molecule, in the tissue of an animal.
  • Most non- viral methods of gene transfer rely on normal mechanisms used by mammalian cells for the uptake and intracellular transport of macromolecules.
  • non-viral gene delivery systems ofthe present invention rely on endocytic pathways for the uptake of the subject IRDBP-1 gene by the targeted cell.
  • Exemplary gene delivery systems of this type include liposomal derived systems, poly-lysine conjugates, and artificial viral envelopes.
  • a gene encoding one of the subject ERDBP-1 proteins can be entrapped in liposomes bearing positive charges on their surface (e.g., lipofectins) and (optionally) which are tagged with antibodies against cell surface antigens of the target tissue (Mizuno et al. (1992) NO Shinkei Geka 20:547-551 ; PCT publication WO91/06309; Japanese patent application 1047381; and European patent publication EP-A-43075), and which are inco ⁇ orated herein in their entireties.
  • lipofection of papilloma-virus infected epithelial cells can be carried out using liposomes tagged with monoclonal antibodies against, for example, squamous cells.
  • the gene delivery system comprises an antibody or cell surface ligand that is cross-linked with a gene binding agent such as polylysine (see, for example, PCT publications WO93/04701, WO92/22635, WO92/20316, WO92/19749, and WO92/06180), and which are inco ⁇ orated herein in their entireties.
  • a gene binding agent such as polylysine
  • an IRDBP-1 gene construct encoding an antagonistic form of the protein e.g. a dominant negative mutant
  • a soluble polynucleotide carrier comprising an HPV viral coat protein conjugated to a polycation, e.g. poly-lysine (see U.S. Patent No. 5,166,320).
  • effective delivery of the subject nucleic acid constructs via receptor-mediated endocytosis can be improved using agents
  • ATLANTA 333137vl 65 which enhance escape of gene from the endosomal structures.
  • whole adenovirus or fusogenic peptides ofthe influenza HA gene product can be used as part of the delivery system to induce efficient disruption of DNA-containing endosomes (Mulligan et al. (1993) Science 260-926; Wagner et al. (1992) Proc. Natl. Acad. Sci. 89:7934; and Christiano et al. (1993) Proc. Natl. Acad. Sci. 90:2122), and which are inco ⁇ orated herein in their entirety.
  • the gene delivery systems can be infroduced into a patient by any of a number of methods, each of which is familiar in the art.
  • a pharmaceutical preparation of the gene delivery system can be infroduced systemically, e.g. by intravenous injection, and specific transduction of the gene into the target cells relies predominantly on the specificity of transfection provided by the gene delivery vehicle, cell-type or tissue-type expression due to the transcriptional regulatory sequences controlling expression of the gene, or a combination thereof.
  • initial delivery of the recombinant gene is more limited with introduction into the animal being quite localized.
  • the gene delivery vehicle can be introduced by catheter (see U.S. Patent No. 5,328,470) or by stereotactic injection (e.g. Chen et al. (1994) Proc. Natl. Acad. Sci. 91 : 3054-3057), both of which references are inco ⁇ orated herein in their entireties.
  • the pharmaceutical preparation can consist essentially of the gene delivery system in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded.
  • the pharmaceutical preparation can comprise one or more cells which produce the gene delivery system.
  • methods of introducing the viral packaging cells may be provided by, for example, rechargeable or biodegradable devices. The generation of such implants is generally known in the art. See, for example, Concise Encyclopedia of Medical & Dental Materials, ed. by David Williams (MIT Press: Cambridge, Mass., 1990); Sabel et al. U.S. Patent No.
  • ERDBP-1 encoding sequences ofthe invention are useful in increasing production of recombinant IGFBP-3 for treatment of the aforementioned diseases, including GH deficiencies and complications caused by increased unbound IGF, can be accomplished by adminisfration of recombinant IGFBP-3 (for example, produced in cell culture) via pharmaceutical compositions. Production of IGFBP-3 from recombinant cells can be increased by transfecting such cells with an ERDBP-1 encoding sequence either under the confrol of its own or a heterologous promoter. IRDBP-1 polypeptides ofthe present invention are also useful in the treatment of growth hormone disorders, especially those where IGFBP-3 levels are below normal.
  • IRDBP-1 is formulated into a pharmaceutical composition for parenteral administration, and a therapeutical dose is administered, with the result of raising IGFBP-3 and ERDBP-1 levels in the treated patient.
  • the presence of micro-satellite DNA downsfream of the ERDBP-1 coding sequence is also further noted. Expression ofthe IRDBP-1 coding sequence is greater in the presence than absence of this micro-satellite DNA. Probes and/or primers for analysis of this region may allow the identification of genetic diseases associated with abenant ERDBP-1 expression.
  • An antisense construct ofthe present invention can be delivered, for example, as an expression plasmid which, when transcribed in the cell, produces RNA which is complementary to at least a unique portion of the cellular mRNA which encodes a IRDBP-1 -protein, e.g. the rat or human ERDBP-1 nucleic acid sequences represented in SEQ ED NOS: 2, 5-10, 14, and 44-45, as described in Example 21.
  • the antisense construct can be an oligonucleotide probe which is generated ex vivo and which, when infroduced into the cell causes inhibition of expression by hybridizing with the mRNA and/or genomic sequences encoding one
  • oligonucleotide probes are preferably modified oligonucleotides that are resistant to endogenous nucleases, e.g. exonucleases and/or endonucleases, and are therefore stable in vivo.
  • Exemplary nucleic acid molecules for use as antisense oligonucleotides are phosphoramidate, phosphorothioate and methylphosphonate analogs of DNA (see also U.S. Patent Nos. 5,176,996; 5,264,564; and 5,256,775). Additionally, general approaches to constructing oligomers useful in antisense therapy have been reviewed, for example, by van der Krol et al.
  • the modified oligomers ofthe invention are useful in therapeutic, diagnostic, and research contexts. Inhibition of cell proliferation may result, but this condition may be desirable where, for example, proliferation may lead to a pathological condition such as, but not limited to a blockage of a blood vessel after angioplasty, or proliferation of endothelial cells for angiogenesis in tumor formation.
  • the oligomers are utilized in a manner appropriate for antisense therapy in general.
  • the oligomers of the invention can be formulated for a variety of loads of adminisfration, including systemic and topical or localized administration. Techniques and formulations generally may be found in Remmington's Pharmaceutical Sciences, Meade Publishing Co., Easton, Pa.
  • the oligomers of the invention can be formulated in liquid solutions, preferably in physiologically compatible buffers such as Hank's solution or Ringer's solution.
  • the oligomers may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included.
  • Systemic administration can also be by fransmucosal or transdermal means, or the compounds can be administered orally.
  • fransmucosal or fransdermal For fransmucosal or fransdermal
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for fransmucosal adminisfration bile salts and fiisidic acid derivatives.
  • detergents may be used to facilitate permeation.
  • Transmucosal administration may be through nasal sprays or using suppositories.
  • the oligomers are formulated into conventional oral adminisfration forms such as capsules, tablets, and tonics.
  • the oligomers of the invention are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the oligomers of the invention may be used as diagnostic reagents to detect the presence or absence of the target DNA or RNA sequences to which they specifically bind. Such diagnostic tests are described in further detail below.
  • transgenic animals such as, but not limited to animal models for diabetes, obesity, mood disorders, developmental and, proliferative diseases, that are comprised of cells (of that animal) which contain a transgene of the present invention and which preferably (though optionally) express the subject IRDBP-1 in one or more cells in the animal.
  • the expression of the transgene is restricted to specific subsets of cells, tissues or developmental stages utilizing, for example, cis-acting sequences that confrol expression in the desired pattern.
  • such mosaic expression ofthe subject ERDBP-1 proteins can be essential for many forms of lineage analysis and can additionally provide a means to assess the effects of ERDBP-1 mutations or overexpression that might grossly alter development in small patches of tissue within an otherwise normal embryo.
  • tissue-specific regulatory sequences and conditional regulatory sequences can be used to control expression of the fransgene in certain spatial patterns.
  • temporal patterns of expression can be used to control expression of the fransgene in certain spatial patterns.
  • ATLANTA 333137vl 9 can be provided by, for example, conditional recombination systems or prokaryotic transcriptional regulatory sequences.
  • target sequence refers to a nucleotide sequence that is genetically recombined by a recombinase.
  • the target sequence is flanked by recombinase recognition sequences and is generally either excised or inverted in cells expressing recombinase activity.
  • Recombinase catalyzed recombination events can be designed such that recombination of the target sequence results in either the activation or repression of expression ofthe subject receptor.
  • excision of a target sequence that interferes with the expression of the receptor can be designed to activate expression of that protein.
  • This interference with expression of the subject protein can result from a variety of mechanisms, such as spatial separation of the IRDBP-1 gene from the promoter element or an internal stop codon.
  • the fransgene can be made wherein the coding sequence of the IRDBP-1 gene is flanked by recombinase recognition sequences and is initially transfected into cells in a 3' to 5' orientation with respect to the promoter element. In such an instance, inversion of the target sequence will reorient the subject IRDBP-1 gene by placing the 5' end ofthe coding sequence in an orientation with respect to the promoter element that allow for promoter driven transcriptional activation.
  • cre/loxP recombinase system of bacteriophage PI (Lakso et al. (1992) Proc. Natl. Acad. Sci. 89:6232-6236; Orban et al. (1992) Proc. Natl. Acad. Sci. 89:6861-6865) or the FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al. (1991) Science 251:1351-1355; PCT publication WO 92/15694), and which are inco ⁇ orated herein in their entireties, can be used to generate in vivo site-specific genetic recombination systems. Cre recombinase catalyzes the site-specific recombination of an intervening target sequence located between loxP sequences. loxP sequences are 34 base pair nucleotide
  • ATLANTA 333137vl 70 repeat sequences to which the Cre recombinase binds and are required for Cre recombinase mediated genetic recombination.
  • the orientation of loxP sequences determines whether the intervening target sequence is excised or inverted when Cre recombinase is present (Abremski et al. (1984) J. Biol. Chem. 259:1509-1514); catalyzing the excision ofthe target sequence when the loxP sequences are oriented as direct repeats and catalyzes inversion of the target sequence when loxP sequences are oriented as inverted repeats.
  • genetic recombination of the target sequence is dependent on expression of the Cre recombinase.
  • Expression of the recombinase can be regulated by promoter elements which are subject to regulatory control, e.g., tissue-specific, developmental stage-specific, inducible or repressible by externally added agents. This regulated control will result in genetic recombination of the target sequence only in cells where recombinase expression is mediated by the promoter element.
  • the activation of expression of the recombinant UBC9 gene can be regulated via regulation of recombinase expression.
  • a transgenic animal containing transgenes encoding both the Cre recombinase and the subject gene.
  • Animals containing both the Cre recombinase and the IRDBP-1 genes can be provided through the construction of "double" fransgenic animals.
  • a convenient method for providing such animals is to mate two transgenic animals each containing a fransgene, e.g., one harboring the IRDBP-1 gene, and the other harboring the recombinase gene.
  • One advantage derived from initially constructing fransgenic animals containing a IRDBP-1 transgene in a recombinase-mediated expressible format derives from the likelihood that the subject ERDBP-1 protein, whether antagonistic or agonistic, will be deleterious upon expression in the fransgenic animal.
  • a founder population in which the subject transgene is silent in all tissues, can be propagated and maintained. Individuals of this founder population can be crossed with animals expressing the recombinase in, for example, one or more tissues,
  • prokaryotic promoter sequences which require prokaryotic proteins to be simultaneous expressed in order to facilitate expression of the fransgene.
  • Operators present in prokaryotic cells have been extensively characterized in vivo and in vitro and can be readily manipulated to place them in any position upstream from or within a gene by standard techniques. Such operators comprise promoter regions and regions which specifically bind proteins such as activators and repressors.
  • One example is the operator region of the lexA gene of E. coli to which the Lex A polypeptide binds.
  • Other exemplary prokaryotic regulatory sequences and the conesponding trans-activating prokaryotic proteins are given in U.S. Patent No. 4,833,080.
  • silent fransgenic animals can be created which harbor the subject transgene under transcriptional confrol of a prokaryotic sequence that is not appreciably activated by eukaryotic proteins. Breeding of this transgenic animal with another animal that is fransgenic for the conesponding prokaryotic frans- activator, can permit activation of the ERDBP-1 fransgene.
  • expression of the conditional transgenes can be induced by gene therapy-like methods (such as described above) wherein a gene encoding the trans-activating protein, e.g.
  • a recombinase or a prokaryotic protein is delivered to the tissue and caused to be expressed, such as in a cell-type specific manner.
  • the ERDBP-1 transgene could remain silent into adulthood until "turned on” by the introduction of the trans-activator.
  • inducible promoters can be employed, such as the tet operator and the metallothionein promoter which can be induced by treatment with tefracycline and zinc ions, respectively (Gossen et al. (1992) Proc. Natl. Acad. Sci. 89:5547-5551;
  • the present invention by making available purified and recombinant forms of the subject IRDBP-1 proteins, will allow the development of assays which can be used to screen for drugs which either agonize or antagonize the function of ERDBP-1 in vivo.
  • Assays for the measurement of ERDBP-l can be generated in many different forms, and include assays based on cell-free systems, e.g. purified proteins or cell lysates, as well as cell-based assays which utilize intact cells. Such agents can be used, for example, in the treatment of diabetic or feeding disorders, proliferative and/or differentiative disorders, and to modulate cellular metabolism. In many drug screening programs which test libraries of compounds and natural exfracts, high throughput assays are desirable in order to maximize the number of compounds surveyed in a given period of time.
  • Assays which are performed in cell-free systems such as may be derived with purified or semi-purified proteins or with lysates, are often prefened as "primary" screens in that they can be generated to permit rapid development and relatively easy detection of an alteration in a molecular target which is mediated by a test compound.
  • the effects of cellular toxicity and/or bioavailability of the test compound can be generally ignored in the in vitro system, the assay instead being focused primarily on the effect ofthe drug on the molecular target as may be manifest in an alteration of binding affinity with other proteins or change in enzymatic properties of the molecular target. Accordingly,
  • ATLANTA 333137vl 73 potential inhibitors of ERDBP-1 function can be detected in a cell-free assay generated by constitution of a functional ERDBP-1 /target nucleic acid sequence in a cell lysate.
  • Another aspect of the present invention concerns three-dimensional molecular models of the subject ERDBP-1 proteins, and their use as templates for the design of agents able to inhibit at least one biological activity of the ERDBP-1 protein.
  • An integral step to designing inhibitors of the subject IRDBP-1 involves construction of computer graphics models ofthe IRDBP-1 that can be used to design pharmacophores by rational drug design. For instance, for an inhibitor to interact optimally with the subject protein, it will generally be desirable that it have a shape which is at least partly complimentary to that of a particular binding site ofthe protein, as for example those portions of the human IRDBP-1 that are involved in recognition of a particular region of a nucleic acid sequence. Additionally, other factors, including electrostatic interactions, hydrogen bonding, hydrophobic interactions, desolvation effects, and cooperative motions of ligand and enzyme, all influence the binding effect and should be taken into account in attempts to design bioactive inhibitors.
  • a computer-generated molecular model of the subject protein can be created by homology modeling, and then calculate the structure of the protein and velocities of each atom at a simulation temperature.
  • Computer programs for performing energy minimization routines are commonly used to generate molecular models. For example, both the CHARMM (Brooks et al. (1983) J. Comput. Chem. 4:187-217) and AMBER (Werner et al (1981) J. Comput. Chem. 106: 765) algorithms handle all of the molecular system setup, force field calculation, and analysis (see also, Eisenfield et al. (1991) Am. J. Physiol. 26LC376-386; Lybrand (1991) J Pharm. Belg.
  • ATLANTA 333137vl 74 computational methods for molecular design, in which the steric and electronic properties of substrate binding sites are used to guide the design of potential inhibitors (Cohen et al. (1990) J. Med. Cam. 33: 883-894; Kuntz et al. (1982) J. Mol. Biol. 161: 269-288; Desjarlais (1988) J. Med. Cam. 31: 722-729; Bartlett et al. (1989) Spec. Publ., Roy. Soc. Chem. 78: 182-196; Goodford et al. (1985) J. Med. Cam. 28: 849- 857; Desjarlais et al. J. Med. Cam.
  • DOCK a set of computer algorithms called DOCK
  • DOCK can be used to characterize the shape of invaginations and grooves that form the active sites and recognition surfaces of the subject protein
  • the program can also search a database of small molecules for templates whose shapes are complementary to particular binding sites of the protein (Desjarlais et al. (1988) J. Med. Chem. 31: 722-729). These templates normally require modification to achieve good chemical and elecfrostatic interactions (Desjarlais et al. (1989) ACS Symp. Ser. 413: 60-69).
  • the program has been shown to position accurately known cofactors for inhibitors based on shape constraints alone.
  • exemplary virtual drug design programs include GRID (Goodford (1985, J. Med. Chem. 28:849-857); Boobbyer et al. (1989) J. Med. Chem. 32:1083- 1094), CLIX Lawrence et al. (1992) Proteins 12:31-41), GROW (Moon et al. (1991) Proteins 11:314-328), the multiple copy simultaneous search method (MCSS) (described by Miranker et al. (1991) Proteins 11: 29-34), and NEWLEAD (Tschinke et al. (1993) J. Med. Chem. 36: 3863,3870), which are inco ⁇ orated herein in their entireties.
  • GRID Goodford (1985, J. Med. Chem. 28:849-857
  • Boobbyer et al. (1989) J. Med. Chem. 32:1083- 1094 CLIX Lawrence et al. (1992) Proteins 12:31-41
  • GROW
  • ATLANTA 333137vl 75 The present invention is further illusfrated by the following examples, which are provided by way of illustration and should not be construed as limiting. The contents of all references, published patents and patents cited throughout the present application are hereby inco ⁇ orated by reference in their entirety.
  • Example 1 Cloning of cDNAs encoding IRSBP An isolated nucleic acid with the IRE associated with the IGFBP-3 gene and comprising the nucleotide sequence SEQ ED NO: 1 was multimerized as follows. Two antiparallel oligonucleotides, one representing the sense strand of SEQ ED NO: 1, and the other its antisense complement were annealed. The resulting double-sfranded DNA was phosphorylated with T4 polynucleotide kinase, concatemerized with T4 DNA ligase at 22°C for 5 minutes, and electrophoretically fractionated on a polyacrylamide gel.
  • a fragment containing three contiguous copies of the annealed oligonucleotides was inserted into the pHISi reporter vector (Clontech, Palo Alto, CA), and transformed into the yeast Saccharomyces cerevisiae strain YM4271 (Clontech, Palo Alto, CA).
  • Southern blotting confirmed the integration of the multimerized ERE nucleic acid sequence into the yeast genome. Southern blotting was carried out using prehybridization and hybridization buffers containing 1% w/v BSA, 1 mM EDTA. 0.5 M NaHPO , pH 7.2, and 7% w/v SDS. Hybridization was done with the radiolabeled multimerized ERE of IGFBP-3 with 32 P as the radiolabel. The radioactive probe was added at a concentration of 1-2 x 10 6 cpm/ml. After hybridization, blots were washed twice with 2X SSC, 0.1% SDS for 30 mins., followed by a 30 min. wash with 0.1X SSC, 0.1%) SDS at 50°C, and autoradiography. The procedure was as described in Ausubel et al. (1993) inco ⁇ orated herein by reference in its entirety
  • a rat liver cDNA library was screened using a yeast one-hybrid system, as described by Chong et al, (1995) Cell 80: 949-957 and Li & Herskowitz, (1993) Science 1252: 1870-1873, inco ⁇ orated herein by reference in their entireties.
  • the yeast one-hybrid system is an in vivo genetic assay that uses growth selection based on reconstruction and activation of the nutritional reporter gene HIS3.
  • ATLANTA 333137vl 76 fragment comprising three contiguous repeats of the IGFBP-3 ERE (SEQ ED NO: 1) was inserted in the region 5' upstream of a HIS3 reporter under the confrol of a GAL4- responsive promoter. The construct was transformed into yeast cells.
  • Yeast containing the ERE target nucleic acid sequence SEQ ED NO: 1 were transformed with DNA purified from an activation domain (AD) library that contained fusions between a target-independent activation domain (GAL4 AD) and cDNA derived from a normal rat liver. Colonies of yeast were selected on His ' Leu " plates and their plasmid contents were isolated. Positive clones were confirmed by retransformation ofthe cDNA into yeast containing a Lac Z reporter gene with tandem repeats of the IRE target nucleic acid, and tested for transcriptional activation of the GAL4 promoter.
  • AD activation domain
  • GAL4 AD target-independent activation domain
  • Two of the clones contained cDNAs encoding the known transcription factors NFkB p65 and HBP1. Eleven out of seventy-nine clones contained novel sequences that were selected for further characterization. Gel shift mobility assays. cDNAs from the 11 novel clones were subcloned into the plasmid pSPUTK (Stratagene). The coding regions of the cDNA clones were translated into protein using coupled transcriptional in vitro translation as described by Hook et al. (1996) Peptide Research 9: 183-187 and inco ⁇ orated herein in its entirety. After translation, the proteins were tested for their ability to bind to the ERE element of IGFBP-3 (SEQ ID NO: 1) by gel mobility shift analysis (Figs. 17A and 17B).
  • ATLANTA 333137vl 77 with the proteins derived from the cDNA clones at concentrations of approximately 20 ng protein per lane in 25 ⁇ l of binding buffer containing 10 mM Tris, pH 7.6, 50 mM KCl, ImM EDTA, 0.5 mM dithiothreitol, 0.2% Nonidet P-40, 20 ⁇ g of bovine serum albumin, 36 ⁇ g of salmon sperm DNA, and 10% glycerol at 25°C for 20 mins. Incubations were carried out with or without unlabeled competitor DNA. Protein- DNA complexes were separated from free probe on 6% polyacrylamide gels in 0.25.x TBE at 12 V/cm for 2-3 hours, and visualized by autoradiography.
  • One clone of the eleven, clone 52 contained a 952 bp cDNA insert (SEQ ID NO: 2, shown in Fig. 1) that encoded a polypeptide (SEQ ID NO: 3, shown in Fig 2) that formed a DNA-protein complex.
  • the clone 52 nucleic acid sequence comprises 952 bp of sequence capable of hybridizing to a second ERDBP-1 nucleic acid sequences SEQ ED NOS: 5, 14 and 44 shown in Figs. 4A-4B, and 12A- 12B and 6 respectively, and to a region of the human genomic sequence having the GenBank Accession number AC005237.
  • the specificity of binding was established by using 25 pmoles of labeled or unlabeled IGFBP-3 ERE (SEQ ID NO: 1) as shown in Fig. 18. Excesses of unlabeled IGFBP-3 ERE and NFKB were incubated with about 20 ng protein per lane. The double-sfranded DNA competitors were added at molar concentrations between about 10-fold and about 100-fold greater than labeled oligonucleotides, or 2.5 pmole, 12.5 pmole and 25 pmole, and electrophoresed on a 6% polyacrylamide gel. cDNA coding sequences were also expressed as thioredoxin (Trx) fusion proteins in E. coli.
  • Trx thioredoxin
  • the clone 52 cDNA (SEQ ID NO: 2) was subcloned in-frame into a prokaryotic expression vector (pET-32a from Novagen, Madison, WI), transformed into the AD494(DE3) strain of E. coli, and grown in culture until OD 6 oo0.6.
  • EPTG isopropyl- ⁇ -thiogalactopyranoside
  • the thioredoxin-clone 52 fusion protein was purified by affinity chromatography on immobilized His-bound metal chelation resin (Novagen), and used in a gel-shift assays, as shown in Figs. 17A, 17B and 18.
  • the fusion proteins were tested in additional gel mobility shift experiments with the IGFBP-3 ERE nucleic acid (SEQ ED NO: 1) as describe above. While not wishing to be bound by any
  • the ERE of IGFBP-3 (SEQ ED NO: 1) includes an AGGAAAGTCTCCTT palindrome, and the leucine zipper encourages dimerization, and gel shift bands seen in Figs. 17A-18 reflect binding of ERDBP-1 to the IGFBP-3 ERE as a homodimer and monomer, respectively.
  • Competiton assays demonstrate that EREs associated with other insulin-responsive genes compete with the ERE of IGFBP- 3 (SEQ ID NO: 1) for binding to ERDBP-1, as shown in Fig. 19.
  • SEQ ID NO: 2 is shown in Fig. 1.
  • SEQ JD NO: 3 The longest open-reading frame amino acid sequence derived from SEQ ID NO: 2 is SEQ JD NO: 3, as shown in Fig. 2.
  • Example 3 Expression of clone 52 mRNAs using Northern blot analysis
  • RNA was isolated from cultured hepatic non-parenchymal cells using a Tri-Reagent Kit (Molecular Research Center, Cincinnati, OH) according to the manufacturer's protocol.
  • Clone 52 cDNA (SEQ ED NO: 2) containing the 952 bp cDNA nucleic acid obtained from the yeast one-hybrid screen as described in Example 1, was random primer labeled with [ 32 P] dCTP and used as a probe to hybridize with RNA electrophoresed on 1.2% formaldehyde-agarose gel using protocols described by Ausubel et al. (1993) and inco ⁇ orated herein by reference in its entirety, as shown in Fig. 20.
  • hepatic cells expressed an mRNA species of at least 5.0 kb in length.
  • Densitometric analysis of ERDBP-1 expression normalized to ⁇ -actin expression showed that ERDBP-1 expression levels were highest in the brain and muscle, followed by liver, small intestine, kidney, subcutaneous fat, and spleen, as shown in Fig. 21B. Tissue distribution in adipose tissues is shown in Fig. 21C. IRDBP-1 is, therefore, distributed to target tissues known to be critical for the
  • ATLANTA 333137vl 79 peripheral and central actions of insulin, and ERDBP-1 expression is responsive to the insulin/diabetes status ofthe RNA source.
  • the membrane was hybridized with a radiolabelled human ERDBP-l probe.
  • IRDBP-1 is highly expressed in the jejenum, ascending colon, descending colon, transverse colon, cecum and rectum.
  • a multiple tissue Northern blot from Clontech is highly expressed in the jejenum, ascending colon, descending colon, transverse colon, cecum and rectum.
  • RNA was analyzed on a formaldehyde- agarose gel, and transfened to anylon membrane.
  • the mRNA was expressed as two transcripts of about 9 kb and 6 kb. While not wishing to be bound by any one theory, this result may indicate alternative splicing or differential adenylation.
  • the high expression of the IRDBP-1 in the gastro-intestinal tract shows its importance in the physiologic functioning of this system.
  • the RNase Protection assay utilized a Kpnl/Xhol fragment of the 3.4 kb ERDBP-1 cDNA that was inserted into pGEM 7Z and transcribed in vitro to produce a 170 nt antisense probe.
  • the assays were carried out using a Hybspeed RPA kit (Ambion,
  • CHO cells were co-transfected with a IGFBP-1 IRE reporter construct and combinations of vectors containing an IRDBP-1 cDNA (SEQ ED NO: 2) or DNA encoding for HBP1.
  • Cells were prepared and transfected as described in Example 7. Luciferase activity was measured as described in Example 7.
  • the ERE identified from the IGFBP-1 is described in Cichy et al. (1998) J. Biol. Chem. 273: 6483-6487; OBrien et al. (1994) J. Biol. Chem. 269: 30419-30428 and inco ⁇ orated herein in their entireties.
  • HBP-1 induced the formation of luciferase in the absence of ERDBP-1 or insulin, (as shown in Fig. 23).
  • Cotransfection with the ERDBP-1 -encoding cDNA reduced the activation of the IGFBP-1 ERE and decreased the luciferase expression. Induction of cellular IRDBP-1 by insulin reduced luci
  • ATLANTA 333137vl 80 The IREs were used to construct multimers as described in Example 1, inserted into the pGL3 promoter vector (Promega., Madison, WI) and transfected into COS7 cells. Such cells showed an increase in luciferase reporter activity augmented by the addition of insulin to the media, as shown in Fig. 24.
  • Example 5 Anti-IRDBP-1 antibodies Antibodies were developed to oligopeptides conesponding to the carboxy- terminal segment (cAb) and the amino-segment (nAb) of rat ERDBP-1.
  • the C- segment antibody cAb was raised against an epitope of the rat IRDBP-1 protein between amino acids 786-800 of the protein having sequence SEQ ED NO: 47, and had the following sequence: AcetylatedCys-Thr-Ser-Gln-Asn-Thr-Lys-Ser-Arg-Tyr- Ile-Pro-Asn-Gly-Lys-Leu (SEQ ID NO: 15).
  • the peptide fragment between ERDBP-1 amino acids 233-247 which had the following sequence: AcetylatedCys-Arg-Asn-Gly-Gly-Thr-Tyr-Lys-Glu-Thr- Gly-Asp-Glu-Tyr-Arg (SEQ ED NO: 46) was used.
  • the 1503 bp coding region of the cDNA SEQ ED NO: 44 was expressed in E. coli as a His6-tagged thioredoxin (Trx) fusion protein (pET-32, Novagen, Madison, WI), and purified with Ni 2+ -nitriloacetate (Qiagen, Valencia, CA).
  • the 1.5 kb cDNA was, subcloned in-frame into the prokaryotic expression vector pET- 32a, transformed into the E.coli AD494(DE3) and grown at 37°C until it reached an OD 65 o of 0.6. EPTG was added to a final concenfration of 1 mM three hours before harvest.
  • the fusion protein was purified by affinity chromatography on immobilized His-bind metal chelation resin, and used for gel shift and western analyses.
  • FIG. 25A A western blot of the induction control (Trx) and the fusion protein (Trx- IRDBP-1) is shown in Fig. 25A.
  • Both anti-ERDBP-1 cAb and anti-histidine antibodies recognized a 65-70 kDa polypeptide, the size of which is consistent with the predicted size of the fusion protein containing 20 kDa Trx.
  • the anti-ERDBP-1 cAb antibody recognized a 70 kDa protein, as shown in Fig. 25B, consistent with the size of the insulin-responsive protein recognized in soiled blots by an IGFBP-3 ERE
  • Trx-ERDBP-1 fusion protein also produced a gel shift band with an IGFBP-3 ERE probe, as shown in Figs. 26 and 27. This band was competed by the Trx-ERDBP-1 fusion protein
  • cDNA sequences (SEQ ID NOS: 5, 6, 14 and 44) were obtained by screening a lambda bacteriophage rat brain cDNA library (Uni-Zap XP library, Stratagene, La Jolla, CA) and then extending the cDNAs thus obtained by 5' RACE.
  • the 952 bp clone 52 cDNA of SEQ ID NO: 2 was used as the nucleic acid probe to screen about 10 8 plaques.
  • the phage and host E. coli were spread on agarose plates and incubated to form plaques, nitrocellulose filters were applied, and the phage particles and unpackaged DNA were adsorbed to the filter to produce a replica of the plate surface.
  • the filters were treated with NaOH to denature the phage DNA, which was then hybridized with the cDNA probe. After isolation of positive plaques that hybridized to the probe, the pBluescript phagemid was rescued with VCSM12 helper phage.
  • the final product is a double-sfranded pBluescript phagemid with an inserted DNA.
  • ATLANTA 333137vl 2 6 and 44 having substantially similarity to the nucleotide sequence of rat clone 52 (SEQ ID NO: 2).
  • the 3404 bp rat cDNA SEQ ED NO: 14 was used to screen a human lambda phage cDNA library (Uni-Zap XR human liver cDNA library, Sfratagene, La Jolla, CA). Two clones hybridizing to the 3404 bp rat IRDBP-1 -specific probe were obtained. One was about 2480 bp long and another clone was about 1700 bp long.
  • the N-terminus of the ERDBP-1 of SEQ ID NO: 45 extends 175 amino acids beyond a genomic sequence isolated by the Human Genome Project. Alignmant of the SEQ ED NO: 45 with the Celera, Inc sequence gave four differences at the amino acid positions (of SEQ ID NO: 48) 250 (SS to DD), 369 (P to R), 127 (V deleted) and
  • ATLANTA 333137vl 84 1324 (P deleted). Sequences were compared by the "Gapped Blat and PSI-Blast as described by Zhang et al. in Nuc. Acid Res. 25, 3389-3402 (1997) inco ⁇ orated herein by reference in its entirety.
  • IREs isolated from GAPDH, IGFBP-1, IGF-1 and amylase genes competed for ERDBP-1 binding to the IGFBP-3 ERE. Competition was weaker with the IREs from the PEPCK and TAT genes, and much weaker with the prolactin IRE.
  • Double-stranded oligonucleotides conesponding to the published sequences of the IREs were used for competition, including the IREs identified from the following genes: IGFBP-3 (5'-AATTCAAGGGTATCCAGGAAAGTCTCCTTCAAG-3') (SEQ ID NO: 63), Glyceraldehyde-6-phosphate dehydrogenase (5'- AAGTTCCCCAACTTTCCCGCCTCTCAGCCTTTGAAAG-3') (SEQ ED NO: 49).
  • Insulin-like growth factor binding protein- 1 or IGFBP-1 (5'-GTTTGTTTTGCTAGT- 3') (SEQ ID NO: 50), Insulin-like growth factor-1 or IGF-I (5'- GCCTCATTATTCCTGCCCACCAAT-3') (SEQ ID NO: 51) amylase (5'- TATTTTGCGTGAGAGTTTCTAAAAGTCCAT-3') (SEQ ED NO: 52), phosphoenolpyruvate carboxykinase or PEPCK (5'-TGGTGTTTTGACAAC-3') (SEQ ED NO: 53), tyrosine aminotransferase or TAT (5'- GACTAGAACAAACAAGTCCTGCGTA-3') (SEQ ED NO: 54), prolactin (5'- ATCTATTTCCGTCATTAAGATA-3') (SEQ ID NO: 55), and the consensus sequence for NFKB binding (5'-GGGACTTTCCGGGACTTTCC-3') (SEQ ID NO: 56).
  • the partial cDNA sequence encoding IRDBP-1 (SEQ ED NO: 14 shown in Fig. 12) was subcloned into the pCMV-Tag epitope tagging mammalian expression vector (Stratagene, La Jolla, CA) and transfected into L6 myoblasts to establish ERDBP-1 -stably fransfected cell lines. G418-resistant clones were isolated and tested for [ 3 H] 2-deoxyglucose uptake (Fig. 28B). The insulin induction of the expression of the IRDBP-1 gene was shown by
  • the cDNA-containing constructs and a chimeric construct comprising the IGFBP-3 promoter region SEQ ED NO: 1 attached to the firefly luciferase reporter gene of pGL2-Basic (Promega) as described in Villafuerte et al, (1997) J. Biol. Chem. 272: 5024-5030, inco ⁇ orated herein by reference in its entirety, were co-fransfected into Chinese hamster ovary (CHO) cells.
  • ATLANTA 333 37vl 86 A chimeric construct also contained three tandem copies of the ERE region of the IGFBP-3 promoter sequence (SEQ ID NO: 1) attached to the pGL3 promoter vector (Promega, Madison, WI).
  • Transient transfections of the IGFBP-3 IRE-luciferase construct together with the cDNA clones in pTARGET were undertaken with CHO cells when the cells reached 60-70% confluence. Lipofectin (Life Technologies, Rockvillc, MD) and DNA complexes were mixed at a 15 ⁇ g to 2.5 ⁇ g ratio and incubated with the cells overnight. Medium was replaced with serum-free DMEM medium, with or without the addition of 10 "6 M human recombinant insulin (Life Technologies, Rockville, MD) for 24 hours, and cell exfracts were assayed for gene activity using a luciferase assay system (Promega, Madison WI, and following the manufacturer's recommended protocol) and measured using a luminometer.
  • luciferase assay system Promega, Madison WI, and following the manufacturer's recommended protocol
  • the cDNA was also subcloned into the mammalian expression vector pCR 3.1 (Invitrogen Co ⁇ ., Carlsbad, CA), and transiently cotransfected into COS 7 cells already containing target IGFBP-3 IRE nucleic acid linked to a luciferase reporter in the pGL3 promoter (Promega, Madison, WI).
  • the truncated 3.4 kb ERDBP-1 cDNA increased ERE-linked reporter activity 15-fold, but had only a 2-fold effect on the confrol reporter vector.
  • the addition of 10 "8 M insulin had little effect on the confrol vector but provided 3- fold stimulation of the ERE reporter in the absence or presence of added IRDBP-1.
  • the impact of ERDBP-1 alone was greater than that of insulin alone, and ERDBP-1 and insulin combined were more than additive. Similar findings were obtained with an IRE reporter gene transfected into primary cultures of hepatic nonparenchymal cells.
  • ERDBP-1 ofthe IRE reporter Extending the 5' end of the expressed 3.4kb ERDBP-1 cDNA reduced stimulation by ERDBP-1 ofthe IRE reporter as shown in Fig. 29. Since the additional sequence contained mostly the EGF-like repeats, the EGF-like repeats may have a silencing effect. The truncated carboxyl-half of the protein of about 50 kDa appears to be sufficient for transcriptional stimulation.
  • ATLANTA 333137vl 87 The expressed 3.4 kb cDNA region SEQ ED NO: 14 increased IGFBP-3 ERE- induced reporter activity 14-fold, and addition of 10 "6 M insulin increased the activity further by 3 -fold (Fig. 24). There was a 110% increase in glucose uptake in cells stably fransfected with nucleic acid SEQ ED NO 14, as compared to wild type cells, as shown in Fig. 28. With the addition of 10 "6 M insulin, there was a 30% further increase in glucose uptake in wild type cells and a 16% further increase in clone 52- fransfected cells. IRDBP-1 is functionally an insulin-mimetic.
  • Example 9 Determination of ERDBP-1 biological activity
  • wild type L6 myoblasts and the 3.4 kb ERDBP-1 - encoding nucleic acid stably transfected cells were stained with the periodic acid- Schiff base stain specific for glycogen.
  • the ERDBP-1 -stably transfected cell line showed intense periodic acid Schiff base (+) materials in the cytoplasm, consistent with glycogen accumulation in the ERDBP-1 -transfected cells.
  • ERDBP-1 mediates storage of ingested substrates, and it can act as an insulin substitute at the target organ level.
  • Example 10 IRDBP-1 expression correlates to tissue-specific glucose utilization Northern blotting with a 1503 bp ERDBP-1 specific probe showed that hepatic nonparenchymal cells that exhibit insulin-responsive IGFBP-3 expression, also have insulin-responsive IRDBP-1 expression (Fig. 20, left panel). ERDBP-1 expression was decreased in the livers of sfreptozotocin-induced diabetic rats (DM) compared to normal rats (Fig. 20, right panel). In vivo disposition of glucose in various organs and tissues was studied.
  • DM sfreptozotocin-induced diabetic rats
  • glucose is taken up by splanchnic tissues, including liver and gut (29%), muscle (26%), brain (23%), kidney (7%). heart (4%), fat (3%) and others (8%).
  • the RNase protection assay used a Kpnl-Xhol fragment (SEQ ID NO: 4, shown in Fig. 3) from clone 52 cDNA (SEQ TD NO: 2) inserted in pGem7Z and
  • RNA extracted from various tissues of the rat was hybridized with the [ 32 P] UTP-labeled clone 52 probe-derived RNA at 45°C in the presence of 40 mM PIPES and 80% formamide.
  • the unhybridized probe was degraded with RNase A and T, and protected RNA was purified and resolved on a sequencing gel.
  • ERDBP-1 mRNA (Figs. 21A and 22).
  • the common denominator for ERDBP-1 expression is the dependence ofthe organ or tissues on glucose for energy utilization.
  • IRDBP-1 expression is highest in mesenteric fat, followed by refroperitoneal, epididymal and subcutaneous fat.
  • the mRNA abundance of IRDBP-1 in adipose tissues reflects the glucose utilization rates in those tissues. Since diabetes is characterized by resistance to insulin action on glucose uptake and utilization in adipocytes and skeletal muscle, the studies illustrated in Figs. 18-22, 28-29 indicate that over-expression of ERDBP-1 may overcome the problems of both poor glucose uptake and poor glucose utilization.
  • IRDBP-1 expression is detected in many areas of the brain, including areas associated with feedings and satiety.
  • Rat brains were obtained and fixed by immersion in 4% paraformaldehyde in 0.1 M NaPO 4 , sectioned on a cryostat to 5-10 ⁇ M thickness, and mounted on slides.
  • Sense and antisense 35 S-labeled ERDBP-1 riboprobes were generated by in vitro transcription with 35 S-UTP, and derived from the linearized fragment SEQ ID NO: 4 of IRDBP-1 cDNA. Following proteinase K treatment, prehybridization of the various brain sections was performed for 3 hrs at 42 C, in a buffer containing 10 mM DTT, 0.3 M NaCI, 20 mM Tris pH8, 5 mM EDTA, lx Denhardt's, 10% Dexfran sulfate, and 50% formamide. This was followed by addition of S-labeled probe
  • Example 12 IRDBP-1 is expressed in the hypothalamus and nucleus of the solitary tract
  • the nucleus of the solitary tract integrates afferent and efferent information and connects with nearby vagal and sympathetic centers that control metabolism in peripheral organs.
  • the NTS communicates rosfrally with the cenfral nucleus of the amygdala.
  • the central nucleus is integrated into the limbic and autonomic systems throughout the brain, including the hypothalamus (paraventricular nucleus-PVN, lateral hypothalamus-LH, venfromedial hypothalamus-VMH) and brainstem. Stimulation of PVN, VMH or LH alters sympathoadrenal and vagal activities.
  • hypothalamic pathways that are sensitive to adiposity signals have anatomical connections with caudal brainstem neurons (solitary tract nucleus) that respond to meal-related signals and regulate meal size.
  • IRDBP-1 mRNA is highly expressed in multiple areas of the hypothalamus.
  • a sagittal cut through the thalamic portion of the diencephalon of a normal Sprague Dawley rat showed that ERDBP-1 is expressed in the venfromedial and dorsomedial hypothalamus, arcuate nucleus and perivenfricular nucleus (Fig. 31).
  • a coronal cut through the thalamic region also showed that IRDBP-1 mRNA is expressed in the lateral hypothalamus (Fig. 32).
  • ERDBP-1 mRNA is highly expressed in the olfactory bulb and amygdala of normal rats, as shown in Figs. 37 and 38.
  • the glucostatic hypothesis proposed that short-term changes in plasma glucose levels can be detected by the brain and will lead to alterations in food intake. Although virtually all neurons require glucose, only select populations in various areas of the brain respond to changes in glucose concentration by changes in their firing rates. An increase in plasma glucose leads to increased plasma norepinephrine levels and sympathetic nervous system activation, and this is mediated by the glucoresponsive neurons in the hypothalamus.
  • ERDBP-1 -specific mRNA appears to be concenfrated in selected areas of the hypothalamus, and acts to increase glucose uptake and utilization, as shown in Example 12 above, it is likely that IRDBP-1 has a critical role in mediating the autonomic nervous system activation associated with food intake. With a diet high in fat and sucrose, the plasma norepinephrine response to glucose is predictive of later weight gain, with a high responder becoming obese and a low responder becoming resistant to obesity. IRDBP-1 appears to be a factor that determines basal glucose metabolism in the peripheral tissues, and is concomitantly expressed in regions of the brain that modulate food intake. The efficiency of cellular functions related to ERDBP-1 activity will have a significant impact on overall energy homeostasis.
  • IRDBP-1 insulin receptors
  • Insulin receptors have been localized to the olfactory bulb, hypothalamus, hippocampus, cerebellum, cerebral cortex, and hindbrain. At the cellular level, insulin modulates expression of hypothalamic neuropeptides, inhibits reuptake of norepinephrine, and enhances endogenous ⁇ -adrenergic activity. Central administration of insulin decreases food intake and body weight.
  • IRDBP-1 mRNA is highly expressed in the olfactory bulb and amygdala of normal rats (Figs. 37 and 38), and is also expressed in the cerebral cortex, cerebellum
  • ERDBP-1 therefore may affect multiple aspects of brain function associated with feeding and satiety.
  • Example 14 IRDBP-1 is targeted to pancreatic beta cells of the islets of
  • Glucose is the principal regulator of insulin secretion from pancreatic beta cells, and the kinetic response of insulin to glucose is biphasic in nature. A rapid secretory burst begins within 1 min and decreases over the next 3 to 5 mins. The second phase is characterized by a gradual increase in insulin levels over 5-10 mins, which continues for the next hour. Many type II diabetics have a marked reduction in first phase insulin secretion.
  • Insulin acts on beta cells to regulate insulin secretion, insulin synthesis, and glucose sensing/utilization.
  • Functional insulin receptor and IRS-1 have been identified in beta cells. As shown in Fig. 39, ERDBP-1 expression is strongest in the beta cells ofthe islets of Langerhans.
  • Example 15 Immunodetection of IRDBP-1 expresson in pancreatic renal, vascular and neural tissues Since glucose is the principal regulator of insulin secretion from pancreatic beta cells, and ERDBP-1 mimics insulin action on glucose transport and metabolism, we also determined the expression of IRDBP-1 in the pancreas.
  • ATLANTA 333137vl 93 Fixed and paraffin embedded tissue was deparaffinized, rehydrated, freated with proteinase K at 50 ⁇ g/ml for 10 min at room temperature, washed with PBS, and blocked with a l% gelatin/PBS mixture for 20 mins.
  • the primary antibody, anti-rat ERDBP-1 peptide cAb antibody was added at 1:200 dilution in 1% BSA / lx PBS, and the sample was incubated in a humid chamber for 1 hour at room temperature. After washing, a biotinylated secondary antibody was added at 1 :400 dilution, and incubated with the sample for 30 min.
  • a Western blot analysis of the expressed proteins from cultured human vascular endothelial cells, freated or untreated with insulin, probed with a polyclonal anti-rat ERDBP-1 antibody shows that insulin induces the formation of IRDBP-1 in such cells (Fig. 43).
  • This experiment further shows that a rabbit anti-rat IRDBP-1 antibody will cross-react with an IRDBP-1 of a different species.
  • the localization of IRDBP-1 to the endothelium, mesangium and neurons has implications on the development of vascular, renal and neuropathic complications of diabetes. Insulin causes endothelium-derived nitric oxide-dependent vasodilation and modulates vascular tone.
  • ERDBP-1 Mesangial cell proliferation and expansion is the initial event in the development of diabetic nephropathy.
  • the action of ERDBP-1 in mediating insulin action in the endothelium will increase the vasodilatory capacity of the blood vessels, and decrease blood pressure and the subsequent onset of atherosclerosis.
  • the antiproliferative action of ERDBP-1 will also decrease the capacity of mesangium to expand and delay the development of diabetic nephropathy.
  • ATLANTA 333137vl 94 Example 16: Insulin stimulates gene transcription through both the mitogen- activated protein (MAP) extracellular signal-regulated kinase (Erk) and the PI3- kinase/Akt pathways A. Physical interaction between Akt or Erk and endogenous IRDBP-1 (in COS 7 cells). A pull-down approach was used to establish the position of ERDBP-1 in the insulin signaling cascade, and to show physical contact between ERDBP-1 and signaling antecedents.
  • MAP mitogen- activated protein
  • Erk extracellular signal-regulated kinase
  • PI3- kinase/Akt pathways A. Physical interaction between Akt or Erk and endogenous IRDBP-1 (in COS 7 cells). A pull-down approach was used to establish the position of ERDBP-1 in the insulin signaling cascade, and to show physical contact between ERDBP-1 and signaling antecedents.
  • Fig. 44A As shown in Fig. 44A (left panel), exposure of COS 7 cells to 10 "6 M insulin for 24 hr increased the levels of IRDBP-1 and Erkl detected by immunoblotting of crude lysates (lane 6 vs. 5). ERDBP-1 was coprecipitated by anti-Akt-specific antibodies (lanes 1 and 2) or anti-Erkl/2-specific antibodies (lanes 3 and 4). Erkl was precipitated by anti Erk-antibody (lanes 3 and 4), but not by the anti-Akt antibody (lanes 1 and 2). Spl, that is not insulin responsive, was not precipitated by either antibody (Fig. 44A, right panel). Decreased Erk-mediated phosphorylation of ERDBP- 1 in diabetic and obese rats was also seen (Fig. 44B).
  • IRDBP-1 was coprecipitated by anti-Akt and anti-Erk antibodies (as detected by anti-flag antibody), but was not coprecipitated by control IgG.
  • Akt and Erk were each precipitated by their respective antibodies.
  • IRDBP-1 is situated downstream of the Ras-Mek-Erk kinase signaling enzymes.
  • ERDBP-1 is a target downsfream of insulin signaling cascades.
  • Agarose conjugated anti-Erkl/2 antibody was incubated with cell exfracts from differentiated 3T3-L1 adipocytes freated with or without PD 98059 (50 ⁇ M) and LY 294002 (50 ⁇ M), and insulin (10 "7 M) was added 30 mins later as indicated.
  • Coprecipitated proteins were
  • Akt and Erk phosphorylate IRDBP-1 in vitro Akt and Erk kinases were immunoprecipitated from insulin-treated COS 7 cells, and kinase reactions performed with the Trx fusion proteins described in Example 1 above.
  • Thioredoxin (Trx) and Trx-ERDBP-1 fusion protein were expressed in E. coli (using a pET32 vector), incubated with Akt or Erk in the presence of [ ⁇ - 32 P] ATP for 20 mins., and analyzed by SDS-PAGE. E. c ⁇ /z ' -expressed NFKB p50 was used as negative control.
  • Akt phosphorylated the RPRAATF substrate of glycogen synthase kinase 3 ⁇ , used as a positive control. As shown in Fig. 45B, Akt and Erk did not phosphorylate the controls of Trx alone, and the p50 subunit of NFKB. However, Akt and Erk Trx- ERDBP- 1 were capable of phosphorylating Trx-ERDBP- 1.
  • Aktl Basal and IRDBP-1 -induced IRE activity in COS7 cells.
  • COS 7 cells were fransfected with either 1 ⁇ g of ERDBP-1 (pCR ERDBP-1) or confrol vector (pCR vector), plus 0.5 ⁇ g of Aktl myr or Akt K179M or the confrol vector (pUSE amp) as indicated in Fig. 45C.
  • IGFBP-3 ERE-luc expression was normalized to total protein.
  • Activated Aktl mimics the stimulatory effect of insulin on ERDBP-1, indicating that insulin action on IRDBP-1 may be mediated through phosphorylation by Akt.
  • N-terminal myristoylation of Akt produces a constitutively activated enzyme (Akt myr).
  • ERDBP-1 -encoding nucleic acid was cotransfected with either wild-type Aktl and/or Erk2-encoding nucleic acid in pUSEamp expression vectors, and the effect on ERE- mediated transcription was determined. As shown in Fig. 45D, the effect of ERDBP-1 on the ERE was similar to that seen in previous experiments; ERDBP-1 increased ERE activity by 6.8 -fold (lane 3 vs. lane 1). Addition of 10 " M insulin overnight increased transcription 8.8-fold (lane 4 vs. lane 1).
  • Aktl Wild-type Aktl expression, like Akt myr, stimulated IRDBP-1 -induced IRE activity to the same extent as the addition of insulin (lane 7 vs. lane 4). Insulin treatment had no further additive effect on Akt-stimulated transcription (lane 8 vs. lane 7). Thus, Aktl is sufficient to mediate the effect of insulin on ERDBP-1. In contrast, Erk2 decreased ERDBP-1 -induced ERE transcription by 45 ⁇ 4% (lane 5 vs. lane 3), although it did not completely abolish the effect of IRDBP-1. The inhibitory effect of Erk on ERDBP-1 activation was partially reversed with addition of insulin (lane 6 vs. lane 5).
  • Example 17 Proteolysis and cell distribution of IRDBP-1 in adipocytes
  • Akt mediates the metabolic actions of insulin by phosporylating regulatory proteins at the serine or threonine residue. Both anti-phosphotyrosine and anti-
  • ERDBP-1 immunoprecipitated ERDBP-1 (shown as a about 120 kDa band) and treatment of the cells with insulin increased significantly serine and/or threonine phosporylation of ERDBP-l.
  • a truncated protein of about 50 kDa was detected in insulin-treated, but not in non-insulin-tretaed cells. This lower molecular weight form of ERDBP-1 was detected only in the serine/threonine phospohorylated proteins.
  • Evidence for the proteolytic cleavage of ERDBP-1 is shown below.
  • IRDBP-1 can be useful to screen for a specific protease able to cleave ERDBP-1.
  • ERDBP-1 contains the peptide sequences LSVLS (positions 374-378) and DRSR (positions 603-606) that have been identified as optimal substrates for cleavage of sterol regulatory element binding protein-2 (SREBP-2). Similar cleavage is required for release of SREBP-2 from the endoplasmic reticulum and transit into the nucleus where the truncated SREBP-2 protein modulates the transcription of genes involved in fatty acid and cholesterol synthesis. Similar proteolysis of IRDBP-1 would produce 74- and 49-kDa proteins. A 49 kDa protein is consistent with the size ofthe franscriptionally active polypeptide that was encoded by the recombinant expression vector.
  • HepG 2 cells were subfractionated into cytosolic and nuclear fractions by detergent disruption of cell membranes and high salt extraction of crude nuclei. Samples were subjected to western blot and probed with anti-ERDBP-1 nAb or cAb antibody, as shown in Fig. 46B.
  • cytoplasmic and the nuclear proteins were separated from HepG2 cells, and the fractionated exfracts analyzed by western blotting.
  • the anti-IRDBP-1 nAb antibody recognized an approximately 120 kDa band in the cytoplasmic extracts, but reacted poorly with the nuclear exfracts.
  • the nuclear exfracts were confrast.
  • ATLANTA 333137vl 98 anti-ERDBP-1 cAb antibody recognized both the approximately 120 kDa band in the cytoplasmic exfracts, and an approximately 50 kDa band in the nuclear extracts. Exposure of cells to 10 "7 M insulin for 16 hours decreased cytoplasmic ERDBP-l.
  • Optical sections in the center of the nuclei were performed with a Zeiss confocal microscope at a magnification of 630x, as shown in Fig. 47.
  • the nAb immunoreactivity was localized predominantly to the cytoplasm, and tended to aggregate in the perinuclear area.
  • cAb immunoreactivity was confined mainly to the nucleus, even in the absence of stimulation by insulin, which is consistent with constitutive basal expression shown by stimulation ofthe IRE reporter gene in fransfection studies.
  • Insulin increased the level of cAb staining in both the cytoplasm and the nucleus.
  • the full-length protein 120 kDa band
  • the truncated portion retaining the carboxyl ends i.e. 70 and 50 kDa bands
  • Example 18 Insulin-induced phosphorylation of ERDBP-1 at the serine and/or threonine residue is associated with proteolytic cleavage of IRDBP-1.
  • ERDBP-1 is phosphorylated by Akt at serine/threonine, thereby affecting proteolysis ofthe protein.
  • Phosphoproteins from COS7 cell extracts were precipitated with phospho-specific antibodies, and the phosphorylated fraction of ERDBP-1 was analyzed by immunoblot analysis. As shown in Fig. 48, both anti-phosphotyrosine and anti-phosphoserine/threonine antibodies immunoprecipitated a ERDBP-1 -specific 120 kDa band. Treatment of the cells with insulin significantly increased the serine and/or threonine phosphorylation level of ERDBP-1.
  • ERDBP-1 is a direct substrate for Aktl (Fig. 48). Also, a truncated protein of about 50 kDa size was detected in the insulin-treated, but not in non-insulin treated, cells (indicated by anow). This lower molecular weight
  • ATLANTA 333137vl 99 variant of ERDBP-1 was detected only in the serine/threonine-phosphorylated proteins, and not in the tyrosine-phosphorylated proteins.
  • the size of this truncated protein is similar to the predicted size of the protein encoded by the franscriptionally active expression vector; activation of this vector by insulin, therefore, could involve both nuclear translocation and/or post-translational modifications.
  • a schematic map ofthe predicted protein structure, the conesponding DNA segments used for functional studies, and a proposed pathway of proteolysis/activation of IRDBP-1 is shown in Fig. 49.
  • An adenoviral recombinant vector containing the franscriptionally active fragment of IRDBP-1 nucleotide positions 1641-3144) of SEQ ED NO: 44 was constructed by subcloning the IRDBP-1 -encoding cDNA into the pAdTrack-CMV vector.
  • a 1.5 kb franscriptionally active fragment of rat sensitin cDNA was subcloned into the Hind EQ7 EcoR V sites of the pAdTack-CMV vector plasmid (Clontech, Palo Alto, CA).
  • Recombinant adenovirus was generated, amplified and purified by Cesium gradient centrifugation. Purified virus was tittered, and the biological effects compared to that of an equivalent dose of confrol virus (AdGFP).
  • Ad-ERDBP-1 Recombinant adenovirus-encoding IRDBP-1 (Ad-ERDBP-1) and GFP (Ad- GFP) were fransfected into 3T3-L1 adipocytes, cell lysates were subjected to western blot analysis and probed with anti-ERDBP-1 cAb or nAb antibody to show that the recombinant Ad-IRDBP-1 vector could express the heterologous ERDBP-1 in the recipient 3T3-L1 cells (Fig. 50A). 2.8 ⁇ M [H 3 ] 2-deoxyglucose was added to human adipocytes with or without added 10 "8 M insulin for 15 mins (Fig. 50B).
  • Glucose uptake was compared between confrol adipocytes and cells transfected with Ad-GFP or Ad-ERDBP-1, and cells freated overnight with or without 10 "5 M pioglitazone. After addition of 10 "7 M insulin for 15 mins, there was a 30 ⁇ 3%> increase in glucose uptake in wild type cells, but only a 16 ⁇ 5% increase in IRDBP-1 -overexpressing cells. IRDBP-1 confers insulin-like glucose uptake enhancement . Thiazolidinedione modulates IRDBP-1 activity.
  • Pioglitazone is a thiazolidinedione used to increase insulin sensitivity and lower glucose levels in patients with diabetes. Glucose uptake rose by 183 ⁇ 14% in adipocytes treated with pioglitazone for 16 hours, which was comparable to the increased glucose uptake in the Ad-ERDBP-1 cells, as shown in Fig. 51 A. However, addition of pioglitazone produced no further increase in glucose uptake in the Ad- ERDBP-1 cells, as shown in Fig. 5 IB. Pioglitazone and insulin added individually can increase the interaction between Aktl and ERDBP-1 (Fig. 52). However, the effect of pioglitazone and insulin together is not additive.
  • adenoviral constructs were introduced into 10-week old male Zucker diabetic fatty (ZDF) rats via tail vein infusion at a dose of 5.0 x 10 7 plaque forming units (pfu)/gm body weight and resulting in an increase in IRDBP-1 levels in hepatic tissue (Fig. 53A).
  • Control studies included age-matched ZDF rats infused with virus encoding the green fluorescent protein (Ad-GFP), given at a dose equivalent to that of Ad-IRDBP-1.
  • Ad-GFP green fluorescent protein
  • Ad-ERDBP-1 The effects of Ad-ERDBP-1 were similar whether the adenovirus was introduced via the portal vein or tail vein.
  • the distribution of ERDBP-1 was traced using immunofluorescence microscopy detection of GFP.
  • Expression of Ad-ERDBP-1 was high in the liver, particularly in hepatocyte nuclei, and also high in mesenteric adipocytes and mesenteric veins.
  • Northern blotting showed that hepatic ERDBP-1 was increased by adminisfration of the transgene (Fig. 53A).
  • An associated increase in IGFBP-3 expression mimicked the ability of insulin to stimulate IGFBP-3 expression. Since ZDF rats are hyperinsulinemic, the findings indicate that ERDBP-1 can have both insulin-mimicking (Fig.
  • Fig. 53 A insulin-sensitizing activity
  • Fig. 53 A baseline ⁇ 3 pm capillary glucose levels of ad libitum- fed Ad-ERDBP-1 and Ad-GFP rats were not significantly different before treatment (245 ⁇ 17 vs. 258 ⁇ 25 mgfdl).
  • Fig. 53 A baseline ⁇ 3 pm capillary glucose levels of ad libitum- fed Ad-ERDBP-1 and Ad-GFP rats were not significantly different before treatment (245 ⁇ 17 vs. 258 ⁇ 25 mgfdl).
  • there was a transient increase in plasma glucose 24 hr after adminisfration of Ad-ERDBP-1 followed by a gradual decline over 10-12 days.
  • After 12 days there was a significant decrease in the glucose levels of Ad-IRDBP-1 rats (baseline of 245 ⁇ 17 vs. 151 ⁇ 9 mg/dl at 12 days, p ⁇ 0.05).
  • GTT glucose tolerance test
  • Example 21 Administration of IRDBP-1 sense and antisense oligonucleotides to an animal
  • ATLANTA 333137vl 1Q3 antisense (AS, sequence 5'-CTAACTCACAGGTGATGATGTAGAG-3', SEQ ID NO: 42) oligos (4 nM in 1 ⁇ l vol. over 2 min) after weighing on the * fifth day.
  • Transfected L6 cells are growth arrested when transfected with IRDBP-1 expressing nucleic acid.
  • L6 cells stably transfected with the ERDBP-1 -encoding cDNA (SEQ ID NO: 14) exhibited significantly reduced proliferation rates when cultured in soft agar, compared to fransfection with the vector alone, as shown in Fig. 56.
  • ATLANTA 333137vl 1 Q4 tissues from a single individual were anayed and immobilized as separate dots.
  • the membrane was hybridized with a human IRDBP-1 probe.
  • IRDBP-1 Intra-Refected IRDBP-1 in HepG2 cells showed that over-expression of the transfected IRDBP-1 induces apoptopic events (Fig. 58). ERDBP-1 further inhibits DNA synthesis in HCT-116 and SW620 cells (Fig. 59). Overexpression of ERDBP-1 will further induce proliferating cell lines to enter Go stationary phase, as shown in
  • RNAse solution 500 units/ml of 1.12% (w/v) sodium citrate
  • DNA was stained with propidium iodide (5 mg/100 ml of 1.12%) sodium citrate) for 30 minutes at room temperature before analyzing on the flow cytometer.
  • Stable cell lines fransfected with the expression vector pCMV-Tag with and without the IRDBP-1 nucleic acid sequence (SEQ ID NO: 14) were grown to confluence, fixed with ethanol, and stained with propidium iodide. Cell cycle compartment analysis was done by flow cytometry, measuring excitation at 488 nm with argon lasers. The histograms illustrated in Fig. 61 show that cells fransfected with the vector alone, 0.2% were in the Go phase, 58%) were in the Gl phase, 8%> in the S phase and 28% in the G2/M phases. With cells transfected with the plasmid comprising the ERDBP-l sequence (SEQ TD NO: 14), 60% were in the Go phase, 25% ofthe cells were in the G 1 phase, and only 7% ofthe cells were in S or G2/M phases.
  • Example 23 Generation of IRDBP-1 knockout mice using the ERDBP-1 cDNA clones of the present invention
  • fransgenic mice will be generated in which the IRDBP-1 gene is replaced by the neo r gene.
  • a DNA construct will first be
  • ATLANTA 333137vl 1 Q5 generated that contains neo r linked to a constitutive promoter.
  • This gene will be flanked on either side by at least lkb of genomic ERDBP-1 sequence, which will allow for homologous recombination and integration of neo r into the endogenous IRDBP-1 gene.
  • HSV-tk He ⁇ es Simplex Virus thymidine kinase
  • the DNA construct described above will be transfected into embryonic stem (ES) cells by elecfroporation, and these cells will be transfened to culture on gelatin-coated dishes.
  • the addition of G418 will permit selection for cells that contain integration of neo r > while the nucleoside analog gancyclovir will allow for selection of cells in which homologous recombination has occuned; homologous but not heterologous recombination results in removal of the HSV-tk genes from the transfected construct, thus preventing gancyclovir cytotoxicity.
  • ES cell colonies that are resistant to both G418 and gancyclovir will be screened by PCR or Southern analysis for presence and copy number ofthe neo r gene, and positive colonies will be subcultured and amplified.
  • ES cells that have successfully integrated neo r in place of the IRDBP-1 gene will be used for morula aggregation with 8-cell embryos; aggregates will subsequently be implanted into pseudopregnant female mice.
  • Chimeric mice will be identifiable by their coat color, since the aggregation of ES cells from the 129 strain of agouti mice with 8-cell embryos from an albino strand such as CDI will result in chimeric mice exhibiting white coats with brown splotches.
  • Chimeras will be bred in order to look for germline transmission of the transgene; fransgenic offspring will have completely brown coats and will be heterozygous for the fransgene. These heterozygous mice will be crossed, resulting in a homozygous line if mutation of the ERDBP-l is not lethal.
  • ATLANTA 333137vl JQ6 mouse will also be created containing a targeting vector that is nearly an exact copy of a segment of genomic ERDBP-1 DNA, except that a critical exon will be flanked by the sites binding the recombinanse (lox-P sites).
  • the mice will be intercrossed, and compound transgenic knockout mice will be produced in which the target gene is excised by Cre recombinase only in the desired cell type (Cre x ERDBP-1"'- or Cre +/" x IRDBP-1 7" ).
  • IRDBP-1 knockout mice (ERDBP-1 -/ ) are obtained that survive widespread tissue inactivation of ERDBP-1, we will look for tissue-specific, developmental and metabolic changes. Although it is possible that other gene products might be capable of functionally replacing IRDBP-1, our preliminary experiments with stably transfected L6 myoblasts suggest that IRDBP-1 acts at the distal end of the insulin activation pathway and is thus unlikely to be genetically redundant.
  • tissue-specific knockouts (muscle Cre + + x floxed ERDBP-l"'-' adipose Cre +/+ x floxed ERDBP-1 - - > and liver Cre +/+ x floxed ERDBP-1 " ' " )
  • serial crosses of mice will be conducted to allow concunent inactivation of ERDBP-1 in multiple organs.
  • IRDBP-1 knockout mice Once IRDBP-1 knockout mice are obtained , they will used to test insulin action, and investigate their susceptibility to diabetes and diabetes complications. Growth curves, including intrauterine growth, size and weight of the viscera, and necropsy at various ages to determine for gross histological differences will be determined. We will define the phenotype of the animals in terms of blood glucose, insulin, glucagon, cortisol and leptin measurements at fasted and fed levels. Immunohistochemical analysis of the pancreas, kidneys, intestine and organs targeted for knockout studies will be done to confirm the effectiveness of gene ablation and detect presence of changes associated with diabetes.
  • Glucose tolerance tests will be performed by intraperitoneal glucose injection (2 mg/gm body weight), and tail bleed will be at 0, 30, 60 and 120 mins, after injection to check for glucose and insulin levels. Finally, wild-type, ERDBP-1 -deficient and heterozygote littermates will be subjected to a diet high in fat (50% of calories from fat) and monitored for parameters of obesity-induced insulin resistance.

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Abstract

La présente invention concerne une nouvelle protéine appelée IRDBP-1 (protéine 1 de liaison à l'ADN sensible à l'insuline) ainsi que des séquence nucléotidiques codant pour celle-ci. L'IRDBP-1 se lie à des régions de l'acide nucléique de gènes produisant une réponse lorsque les cellules sont mises en contact avec de l'insuline. L'IRDBP-1 régule des gènes contribuant à induire une réponse à l'insuline chez les mammifères et à réguler des états pathologiques tels que le diabète, l'obésité, le syndrome d'insulino-résistance et les troubles de la prolifération cellulaire. La présente invention concerne des acides nucléiques utiles comme sondes destinées à détecter des acides nucléiques codant pour des régions de la protéine IRDBP-1. Cette invention concerne en outre des cellules recombinantes, des tissus et des animaux comprenant des molécules d'acides nucléiques recombinantes non naturelles codant pour IRDBP-1, telles que des vecteurs d'expression, ainsi que des anticorps spécifiques pour IRDBP-1, des analyses destinées au polypeptide IRDBP-1 et des méthodes correspondantes. Par ailleurs, ladite invention se rapporte au développement d'agents thérapeutiques et diagnostiques qui imitent, facilitent ou inhibent l'action d'IRDBP-1, et/ou qui présentent une activité en rapport avec la structure et l'action d'IRDBP-1.
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AU2002359580A1 (en) 2003-07-24
WO2003057827A3 (fr) 2004-10-28
CA2469177A1 (fr) 2003-07-17
EP1497305A4 (fr) 2005-11-30

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