WO2003057827A2 - Insulin-responsive dna binding protein-1 and methods to regulate insulin-responsive genes - Google Patents

Abstract

The present invention relates to the novel protein Insulin-Responsive DNA Binding Protein-1 (IRDBP-1) and nucleotide sequences that encode it. IRDBP-1 binds to nucleic acid regions of genes that respond when cells are exposed to insulin. IRDBP-1 regulates genes important in mediating the insulin response in mamals and in regulating conditions such as diabetes, obesity, insulin-resistant syndrome and cell proliferative disorders. The present invention provides nucleic acids useful as probes for detecting nucleic acids encoding regions of the IRDBP-1 protein. Within the scope of the present invention are recombinant cells, tissues and animals containing non-naturally occurring recombinant nucleic acid molecules encoding IRDBP-1, including expression vectors, antibodies specific for IRDBP-1, assays for IRDBP-1 polypeptide, and methods relating to all of the foregoing, the development of therapeutic and diagnostic agents that mimic, facilitate or inhibit the actionof IRDBP-1, and/or are based on relationships ot the structure and action of IRDBP-1.

Description

Title of the Invention

INSULIN-RESPONSIVE DNA BINDING PROTELN-1 AND METHODS TO

REGULATE INSULIN-RESPONSIVE GENES

Reference to Related Application

This application is a Continuation-in-Part of, and claims priority from, U.S. Patent Application Serial No.: 09/703,559 filed November 1, 2000, and also claims priority from U.S. Provisional Applications Serial Nos: 60/336,585 filed December 4, 2001 and 60/390,000 filed June 18, 2002.

Acknowledgment of Federal Research Support

This invention was made, at least in part, with funding from the National Institutes of Health (Grants NO. K08 DK022115 and RO1 DK52965). Accordingly, the United States Government has certain rights in this invention.

Field of the Invention

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.

ATLANTA 333137vl Background

It is estimated that there are more than 6.5 million people in the U.S. diagnosed as having diabetes mellitus . Of those diagnosed, more than 90% have

Type II diabetes mellitus. Although β-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

(diminished 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.

The action of insulin is initiated by binding to cell surface receptors. Autophosphorylation and activation of the intrinsic tyrosine kinase of the insulin receptor β-subunit leads to phosphorylation of several proximal interacting proteins, including insulin receptor substrate-1 (IRS-1), IRS-2, and She. IRS-1 interacts with several proteins that contain Src homology 2 (SH2) domains, including the p85 subunits of PI3' -kinase, GRB-2, Syp and Nek. Activation of these proteins and the subsequent cascade activation of other intracellular signaling molecules, such as p21^ras, raf-1, MAP kinases, and S6 kinase, account for many of insulin's pleiotropic

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).

Genetic factors also contribute to the development of non-insulin dependent Type II diabetes mellitus (NTDDM). The concordance rate for NTDDM in identical twins approaches 100%, while the risk to other siblings of a diabetic proband is between 30 and 40%. Despite considerable investigative efforts, the genetic heterogeneity of diabetes and the contribution of environmental factors in the development of the phenotype make the identification of specific diabetes-related genes difficult. Methods used in the study of the genetics of NIDDM include association of case control studies, positional searches, parametric linkage, and molecular screening using single-strand conformation polymorphism analysis . In addition, cloned genes, including genes important for both insulin secretion and insulin action, have been examined for sequence abnormalities. Specific mutations associated with insulin resistance and the development of diabetes have been identified for the - and β- subunits ofthe insulin receptor. Rad (Ras-associated with diabetes), and the glucokinase gene implicated in MODY (maturity onset diabetes of the young), as well as HNF-1 and HNF-4. Such mutations, however, appear to account for less than 5% of patients with Type II diabetes.

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.

ATLANTA 333137vl "} Each ofthe proteins in the signaling cascade is a potential candidate for an acquired or genetic defect contributing to insulin resistance. Thus, characterization ofthe insulin- responsive binding proteins (IRBPs) that may bind to gene transcriptional regulatory sequences essential for insulin-regulated expression of target genes, and delineation of the pattern of signal transduction to the IRBPs constitutes an important strategy to identify genes important in mediating insulin resistance.

Insulin-like growth factors I and II (IGF-I and -II) 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.

The actions of IGFs are modulated by a family of six IGF-binding proteins (IGFBPs) that have different tissue distribution and production sites. One binding protein, IGFBP- 1, has a molecular weight of approximately 30-40 kd in the human and the rat. Most of the circulating plasma IGF-I and IGF-IJ, however, are associated with IGFBP-3 and an acid-labile subunit thereof that serve as reservoirs for IGFs. Diabetes mellitus in humans and animal models is associated with decreased levels of serum IGFBP-3. Hepatic expression of IGFBP-3 is correlated with circulating IGFBP-3 levels in streptozotocin-diabetic and BB/W rats. Thus, hepatic expression of IGFBP-3 appears to determine systemic IGFBP-3 levels; and the study of the mechanisms by which insulin stimulates hepatic synthesis of IGFBP-3 is critical for understanding the regulation of systemic IGFBP-3.

Most evidence indicates that IGFBP-3 is inhibitory to IGF action. Furthermore, IGFBP-3 can: (a) mediate the growth inhibitory actions of transforming growth factor-β (TGF-β), retinoic acid, anti-estrogens and fibroblast growth factor, (b) mediate the induction of apoptosis by the tumor suppressor gene p53, and (c) travel to

ATLANTA 333137vl A the cell nucleus, potentially directly regulating the transcription of critical growth inhibitory genes independent of IGF-I.

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. These processes have been shown to alter the binding of IGFBP-3 to the acid-labile subunit, cell surfaces and to affect the affinity of IGFBP-3 for IGFs. 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 (base positions- 1148 to -1139) 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.

ATLANTA 333137vl Summary of the Invention

Briefly described, 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.

Also within the scope of the present invention are 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

ATLANTA 333137vl f. host cell. 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. In such cells, 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.

ATLANTA 333137vl 7 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.

Additional objects and aspects of the present invention will become more apparent upon review of the detailed description set forth below when taken in conjunction with the accompanying figures, which are briefly described as follows.

Brief Description of the Figures

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.

ATLANTA 333137vl 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 5¹ 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-

1 protein translated from the coding region (SEQ ID NO: 9) of the human ERDBP-1 cDNA SEQ ED NO: 7 depicted in Fig. 6B.

ATLANTA 333137vl 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. A

Northern blot of RNAs from hepatic nonparenchymal cells (50 μg/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

(+2270 to +2440 base position) of 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.

ATLANTA 333137vl 12 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 β 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.

ATLANTA 333137vl 15 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.

Detailed Description of the Preferred Embodiments

Reference now will be made in detail to the presently preferred embodiments of the invention, one or more examples of which are illustrated in the accompanying drawings. Each example is provided by way of explanation of the invention, not limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications, combination, additions, deletions and variations can be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment can be used in another embodiment to yield a still further embodiment. It is intended that the present invention covers such modifications, combinations, additions, deletions and variations as come within the scope ofthe appended claims and their equivalents.

Definitions

This description uses gene nomenclature accepted by the Cucurbit Genetics Cooperative as it appears in the Cucurbit Genetics Cooperative Report 18:85 (1995); herein incoφorated by reference in its entirety. Using this gene nomenclature, genes are symbolized by italicized Roman letters. If a mutant gene is recessive to the

ATLANTA 333137vl \η normal type, then the symbol and name of the mutant gene appear in italicized lower case letters.

For convenience, certain terms employed in the specification, examples, and appended claims are collected here. The term "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. The term

"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.

The term "mammalian" as used herein 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.

As used herein, the term "IRDBP-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.

ATLANTA 333137vl 18 The term "nucleic acid" as used herein 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. For ease of discussion, such 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. The term "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. The term "isolated nucleic acid" as used herein 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 incoφorated 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 not naturally occurring. Isolated nucleic acid molecules of the present invention can include, for example, natural

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. By the use of the term "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. The term significant as used herein is used to indicate that the level of increase is useful to the person making such an increase.

It is advantageous for some puφoses that a nucleotide sequence is in purified form. The term "purified" in reference to nucleic acid represents that the sequence has increased purity relative to the natural environment.

As used herein the terms "polypeptide" and "protein" refer to a polymer of amino acids of three or more amino acids in a serial array, linked through peptide bonds. The term "polypeptide" includes proteins, protein fragments, protein analogues, oligopeptides and the like. The term "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. The term "polypeptide" further contemplates polypeptides as defined above that include chemically modified amino acids or amino acids covalently or noncovalently linked to labeling ligands.

The term "fragment" as used herein 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

ATLANTA 333137vl 20 known in the art, or expressed in a host cell by recombinant nucleic acid technology well known to one of skill in the art. The term "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.

The term "modulates" as used herein 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 infracelular 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. The term "gene" or "genes" as used herein refers to nucleic acid sequences

(including both RNA or DNA) that encode genetic information for the synthesis of a whole RNA, a whole protein, or any portion of such whole RNA or whole protein. Genes that are not naturally part of a particular organism's genome are referred to as "foreign genes", "heterologous genes" or "exogenous genes" and genes that are naturally a part of a particular organism's genome are referred to as "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.

The term "expressed" or "expression" as used herein 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. The term "expressed" or

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.

As used herein, the term "locus" or "loci" 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. 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.

As used herein "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.

The term "transcription regulatory sequences" as used herein 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.

The term "coding region" as used herein 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.

The term "complementary" as used herein refers to two nucleic acid molecules that can form specific interactions with one another. In the specific interactions, 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. Also in the specific interactions, 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 term "probe" as used herein, when referring to a nucleic acid, refers to a nucleotide sequence that can be used to hybridize with and thereby identify the presence of a complementary sequence, or a complementary sequence differing from the probe sequence but not to a degree that prevents hybridization under the hybridization stringency conditions used. 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.

The term "capable of hybridizing under stringent conditions" as used herein refers to annealing a first nucleic acid to a second nucleic acid under stringent conditions as defined below. 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. The term "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

For example, the first nucleic acid may be a test sample or probe, and 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. Alternatively, hybridization of the first and second nucleic acid may be conducted under reduced stringency conditions, e.g. low temperature and/or high salt content that tend to favor hybridization of dissimilar nucleotide sequences. 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. A suitable hybridization protocol may, for example, involve hybridization in 6X SSC (wherein IX SSC comprises 0.015 M sodium cifrate and 0.15 M sodium chloride), at 65° Celsius in aqueous solution, followed by washing with IX SSC at 65° Celsius. Formulae to calculate appropriate hybridization and wash conditions to achieve hybridization permitting 30% or less mismatch between two nucleic acid molecules are disclosed, for example, in Meinkoth et al. (1984) Anal. Biochem. 138: 267-284; the content of which is herein incoφorated by reference in its entirety. Protocols for hybridization techniques are well known to those of skill in the art and standard molecular biology manuals may be consulted to select a suitable hybridization protocol without undue experimentation. See, for example, Sambrook

ATLANTA 333137vl 24 et al (1989) Molecular Cloning: A Laboratory Manual 2nd ed. Cold Spring Harbor Press, the contents of which are herein incoφorated by reference in their entirety.

Typically, 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.

The term "unique" nucleic acid region as used herein refers to a sequence present in a nucleic acid that is not present in any other nucleic acid sequence. The term "conserved nucleic acid region" as referred to herein 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.

The terms "percent sequence identity" or "percent sequence similarity" as used herein refer to the degree of sequence identity between two nucleic acid sequences or two amino acid sequences as determined using the algorithm of Karlin & Attschul (1990) Proc. Natl. Acad. Sci. 87: 2264-2268, modified as in Karlin & Attschul (1993) Proc. Natl. Acad. Sci. 90: 5873-5877. Such an algorithm is incoφorated into the NBLAST and XBLAST programs of Attschul et al. (1990) T. Mol. Biol. Q15: 403- 410. BLAST nucleotide searches are performed with the NBLAST program, score =

ATLANTA 333137vl 25 100, wordlength = 12, to obtain nucleotide sequences homologous to a nucleic acid molecule ofthe invention. BLAST protein searches are performed with the XBLAST program, score = 50, wordlength = 3, to obtain amino acid sequences homologous to a reference polypeptide. To obtain gapped alignments for comparison puφoses, Gapped BLAST is utilized as described in Attschul et al. (1997) Nuc. Acids Res. 25: 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g. XBLAST and NBLAST) are used. See http://www.ncbi.nlm.nih.gov.

Other algorithms, programs and default settings may also be suitable such as, but not only, the GCG-Sequence Analysis Package of the U.K. Human Genome Mapping Project Resource Centre that includes programs for nucleotide or amino acid sequence comparisons.

The term "sense strand" as used herein 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. The term "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.

The term "antisense DNA" as used herein 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. The term "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. For example, 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".

ATLANTA 333137vl 26 The term "antisense therapy" as used herein 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. In general, "antisense" therapy refers to the range of techniques generally employed in the art, and includes any therapy that relies on specific binding to oligonucleotide sequences.

The terms "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.

The term "expression vector" as used herein 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. As used herein, the term "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.

ATLANTA 333137vl 27 The terms "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.

The term "recombinant nucleic acid" as used herein 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. The term "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.

The term "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 exfragenomic. An exfragenomic 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 infragenomic. The term infragenomic defines a nucleic acid construct incoφorated within the recombinant cell's genome.

The term "antibody" as used herein 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.

Described herein are methods for the production of antibodies capable of specifically recognizing one or more differentially expressed or pathway gene epitopes. 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. Such antibodies may be used, for example, in the detection of a fingeφrint, target, or pathway gene in a biological sample, or, alternatively, as a method for the inhibition of normal or abnormal target gene activity. Thus, 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 fingeφrint, target, or pathway gene proteins, or for the presence of abnormal forms of the such proteins.

For the production of antibodies to a differentially expressed or pathway gene, various host animals may be immunized by injection with a differentially expressed or pathway gene protein, or a portion thereof. Such host animals may include but are not limited to rabbits, mice, and rats, to name but a few. Various 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.

ATLANTA 333137vl 29 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. For the production of polyclonal antibodies, 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. 77-96). Briefly, spleen cells are harvested from an immunized mouse and fused with immortalizing cells (i.e., myeloma cells) to yield antibody-producing hybridomas. 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. P. Gerhardt, (1994) (ASM Press, Washington, DC) the contents of which are incoφorated herein by reference in their entirety. Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridoma producing the mAb of this

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.

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.

Alternatively, techniques described for the production of single chain antibodies such as, but not only U.S. Patent No. 4,946,778; Bird (1988) Science 242: 423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. 85: 5879-5883; and Ward et al. (1989) Nature 334: 544-546 can be adapted to produce differentially expressed or pathway gene-single chain antibodies. 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. For example, 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. Alternatively, 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. The term "pharmaceutical compositions" as used herein 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.

ATLANTA 333137vl 31 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. Pharmaceutical compositions may be administered in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, or the like. 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 LD₅₀ 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.

As used herein, 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. In the typical transgenic animals ofthe present invention, the fransgene causes

ATLANTA 333137vl 32 cells to express a recombinant form of the subject ERDBP-1 protein, e.g. either agonistic or antagonistic forms, or in which the endogenous IRDBP-1 gene has been disrupted. However, transgenic animals in which the recombinant IRDBP-1 gene is silent are also contemplated, as for example, the FLP or CRE recombinase dependent constructs described below. The "non-human animals" of the invention 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. The term "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. The term "tissue- specific chimeric animal" indicates that the recombinant IRDBP-1 gene is present and/or expressed in some tissues but not others.

As used herein, the term "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.

As used herein, 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.

ATLANTA 333137vl 33 In the present context, 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. As used herein, the term "epitope" refers to a part of the protein that can specifically bind to an antibody by fitting into the antigen-binding site ofthe antibody. The term "thiazolidinedione" as used herein refers to "insulin sensitizers" that can bind to peroxisome proliferator-activated receptors such as, but not limited to, PPARγ) resulting in increased glucose production. Exemplary thiazolidinediones include, but are not limited to, troglitazone, rosiglitazone and pioglitazone.

The terms "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 incoφorated by reference in its entirety.

Abbreviations

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 cifrate ; DMSO, dimethyl sulfoxide.

ATLANTA 333137vl 34 Rat and Human IRDBP-1 nucleic acids

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). As used herein, 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.

A 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, incoφorated 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

ATLANTA 333137vl 35 nucleic acids of sequences unrelated to the ERE, as shown in Example 1. The interaction between the ERDBP-l -related polypeptide (SEQ ED NO: 3) and the IGFBP-3 ERE nucleic acid (SEQ ED NO: 1) was specific.

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. While not being bound by any theory, 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. While not wishing to be held to any one theory, it is believed that th enaturally occurring 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.

The abundance of a rat RNA transcript capable of hybridizing to the probe having a nucleic acid sequence of the clone 52 (SEQ ID NO: 4) was increased with

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

(GenBank Accession Nos: AF 439715, AF439716, AF439719 and AF respectively) and shown in Figs. 4A-4B, 5A-5B, 12A-12B, and 13 respectively. 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). The nucleotides at positions 68-349 of clone 52 (SEQ ID NO: 2) correspond to the nucleotide positions 2123-2404 of SEQ ID NO: 5 as shown in Fig. 4A-4B. 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.

ATLANTA 333137vl 37 It is contemplated that 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. 4A) of 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. 5A and 5B, thereof; c) an 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. 6B, or the coding region of SEQ ED NO: 45; and/or 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. Such 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, therefore, 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. For example, 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. In addition, 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.

For example, 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. However, it is expected that DNA sequence polymoφhisms that do lead to changes in the amino acid sequences of the present ERDBP-1 protein ofthe present invention will exist from one human or animal subject to the next ofthe same species. One skilled in the art will appreciate that these variations in one or more nucleotides (up to about 3-4% of the nucleotides) of the 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 polymoφhisms 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. As used herein, 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. For example, the cDNA clone 52 (SEQ ED NO: 2) could be used to detect ERDBP-1 homologs in other vertebrate species, such as, but not only, human, mice, rats, chickens. Thus SEQ ED NO:2 was used to identify a hybridizing human ERDBP-1 -encoding cDNA SEQ ED NO: 7 under medium stringency hybridization conditions.

One embodiment of the present invention, therefore, provides a 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.

In another embodiment of the present invention, a nucleic acid is provided 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.

In yet another embodiment ofthe present invention, a nucleic acid is provided 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.

In yet another embodiment of the present invention, an isolated nucleic acid is provided that comprises the nucleic acid sequence conesponding to a human ERDBP- 1 sequence SEQ ED NO: 7 as shown in Figs. 6A-6C.

ATLANTA 333137vl 41 In another embodiment of the present invention, an isolated nucleic acid is provided that comprises the nucleic acid sequence conesponding to a variant human ERDBP-1 (SEQ ED NO: 10) as shown in Figs. 8A and 8B.

Ln still another embodiment of the present invention, a mammalian IRDBP-1 gene or nucleic acid molecule can be allelic variants of SEQ ED NOS: 2, 5-10 and 44-

45. An 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.

In one embodiment ofthe present invention, 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.

In another embodiment of the present invention, 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.

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.

Such 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. Examples of 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. Similarly, 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).

IRDBP-1 Polypeptides

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

ED NO: 3, as shown in Fig 2) encoded by the rat cDNA clone 52 nucleic acid sequence SEQ ED NO: 2.

In another embodiment of the present invention, a rat ERDBP-1 protein is provided 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.

In still yet another embodiment ofthe present invention, 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).

ATLANTA 333137vl 43 In yet another embodiment ofthe present invention, 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. In still yet another embodiment of the present invention, 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).

In still other embodiments of the present invention, 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.

In one embodiment ofthe present invention, 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.

In another embodiment, 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).

It is further contemplated to be within the scope of the present invention for proteins having substantial similarity to the rat or human protein amino acid sequences SEQ ED NOS: 11, 12, 47 and 48 wherein the proteins retain the capacity to bind to the IGFBP-3 ERE SEQ ED NO: 1. Isolated peptides and polypeptides of the present invention may also include any protein fragments thereof, a protein analogue, or any immunologic fragments thereof.

In another embodiment of the present invention, 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.

In an embodiment of the present invention, 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. In addition, fragments can be chemically synthesized using techniques known in the art such as conventional Merrifield solid phase f-Moc or t-Boc chemistry. For example, 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. In an illustrative embodiment, 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) incoφorated herein by reference in their entireties.

Furthermore, it is also possible to modify the structure of an ERDBP-1 polypeptide for such puφoses as enhancing therapeutic or prophylactic efficacy, or stability (e.g., shelf life ex vivo and resistance to proteolytic degradation in vivo). Such 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.

For example, it is reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid (i.e. conservative mutations) will not have a major effect on the biological activity of the resulting molecule. Conservative replacements are those that take place within a family of amino acids that are related in their side chains. Genetically encoded amino acids can be divided into four families: (1) acidic = aspartate, glutamate; (2) basic = lysine, arginine, histidine; (3) nonpolar = alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar = glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids. In similar fashion, the amino acid repertoire can be grouped as (1) acidic = aspartate, glutamate; (2) basic = lysine, arginine histidine, (3) aliphatic = glycine, alanine, valine, leucine, isoleucine, serine, threonine, with serine and threonine optionally be grouped separately as aliphatic-hydroxyl; (4) aromatic = phenylalanine, tyrosine, tryptophan; (5) amide = asparagine, glutamine; and (6) sulfur-containing = cysteine and methionine. (see, for example, Biochemistry, 2nd ed, Ed. by L. Stryer, WH Freeman and Co.: 1981). 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

ATLANTA 333137vl 46 ERDBP-1. Peptides in which more than one replacement has taken place can readily be tested in the same manner.

In one embodiment of the present invention, therefore, 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. For example, 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.

Recombinant nucleic acids including IRDBP-1 -related sequences and insertion into vectors and mammalian cells

The DNA nucleic acid molecules of the present invention can be incoφorated 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.

For expression in heterologous systems, 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.

U.S. Patent No. 4,237,224 to Cohen & Boyer, which is hereby incoφorated by reference, describes the production of expression systems in the form of recombinant plasmids using restriction enzyme cleavage and ligation with DNA ligase. These recombinant plasmids are then infroduced by means of transformation and replicated in unicellular cultures including prokaryotic organisms and eukaryotic cells grown in tissue culture.

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, ρACYC177, 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 incoφorated by reference), pQE, pEH821, PGEX, pET series (see Studier, F. W. et. al. (1990) "Use of T7 RNA Polymerase to Direct Expression of Cloned Genes" Gene Expression Technology, vol. 185, which is hereby incoφorated by reference) and any derivatives thereof. 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

ATLANTA 333137vl 48 cloning procedures in the art, as described by Maniatis et al. Molecular Cloning: A Laboratory Manual, Cold Springs Laboratory, Cold Springs Harbor, N.Y. (1982), which is hereby incoφorated by reference.

A variety of host-vector systems may be utilized to express the protein- encoding sequence(s). Primarily, 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.

Different genetic signals and processing events confrol many levels of gene expression (e.g., DNA franscription and messenger RNA (mRNA) translation). 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. Furthermore, 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.

Similarly, translation of mRNA in prokaryotes depends upon the presence of the proper prokaryotic signals that differ from those of eukaryotes. Efficient translation of mRNA in prokaryotes requires a ribosome binding site called the Shine- Dalgarno (SD) sequence on the mRNA. This sequence is a short nucleotide sequence of mRNA that is located before the start codon, usually AUG, which encodes the amino-terminal methionine of the protein. The SD sequences are complementary to the 3 '-end of the 16S rRNA (ribosomal RNA) and probably promote binding of mRNA to ribosomes by duplexing with the rRNA to allow conect positioning of the ribosome. For a review on maximizing gene expression, see Roberts & Lauer (1979)

ATLANTA 333137vl 49 Methods in Enzymology 68: 473, which is hereby incoφorated by reference in its entirety.

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. coli, its bacteriophages, or plasmids, 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. Additionally, 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. In certain operons, the addition of specific inducers is necessary for efficient franscription of the inserted DNA. For example, the lac operon is induced by the addition of lactose or EPTG (isopropylthio- beta-D-galactoside). A variety of other operons, such as frp, pro, etc., are under different controls.

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. For instance, 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. Thus, any 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 incoφoration of synthetic nucleotides maybe used.

Once the isolated DNA molecule ofthe present invention has been cloned into an expression system, it is ready to be incoφorated into a host cell. Such incoφoration 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. With 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. Additionally, 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. Once the fusion protein has been purified, 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 Coφ., 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.

Expression of unfused foreign genes in E. coli may be accomplished with recombinant vectors including, but not limited to, the E. coli expression vector pUR278 as described in Ruther et al. (1983) E.M.B.O.J. 2: 1791, incoφorated herein

ATLANTA 333137vl 5 by reference in its entirety. Using the pUR278 vector, 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.

Expression of a foreign gene can also be obtained using 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 Coφ., San Diego, CA), incoφorated 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 incoφorated herein by reference in their entirety.

Other 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. 93: 11307-11302; Frolov et al. (1996) Proc. Natl. Acad. Sci. 93: 11371- 11377; Grunhaus et al. (1993) Seminars in Virology 3: 237-252 and U.S. Patent Nos. 5,591,639; 5,589,466; and 5,580,859 relating to DNA expression vectors, inter alia; the contents of which are incoφorated herein by reference in their entireties.

One embodiment of the present invention, therefore, 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. The

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.

In one embodiment of this aspect of the present invention, the recombinant adenoviral vector (or other suitable 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. For example, 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. For example, radioactive elements such as ³²P, ³H, and ³⁵S 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 Coφ., Madison, WI; the content of which is incoφorated herein in its entirety) may be consulted to select an appropriate labeling protocol without undue experimentation.

IRDBP-1 specific antibodies

It is further contemplated to be within the scope of the present invention to produce and use antibodies specifically reactive with an ERDBP-1 protein or a region thereof. 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. For example, a mammal (i.e., a mouse, rabbit, horse, sheep, or goat) may be immunized with a ERDBP-l protein of the present invention, an immunogenic fragment thereof, or an IRDBP-1 fusion protein or fragment thereof, using an immunization protocol conducive to producing antibodies reactive with the IRDBP-1 protein or a fragment thereof. Following completion of the immunization steps, antiserum reactive with the jointed protein may be collected and, if desired, polyclonal anti-ERDBP-1 antibodies isolated.

One embodiment ofthe present invention, therefore, 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. Exemplary sequences

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. Likewise, 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 hypeφlastic 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.

Another application of anti-ERDBP-1 antibodies is in the immunological screening of cDNA libraries constructed in expression vectors, such as λgtl l, λgtlδ- 23, λZAP, and λORF8. Messenger libraries of this type, having coding sequences inserted in the conect reading frame and orientation, can produce fusion proteins. For instance, λgtl 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.

It is further contemplated to be within the scope ofthe present invention for an assay to detect natural serum antibodies specific for the ERDBP-1 protein. These antibodies 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.

IRDBP-1 -specific oligonucleotide probes

Moreover, the 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. In addition the present invention contemplates 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. Similar to the diagnostic uses of anti-ERDBP-1 antibodies, 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 hypeφlastic disorders such as, but not limited to, unwanted cell growth. Used in conjunction with anti-ERDBP-1 antibody immunoassays, 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. For instance, 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.

In a diagnostic embodiment of the present invention, therefore the 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. Acad. Sci. 72: 3961-65, which are hereby incoφorated by reference). Alternatively, 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 incoφorated by reference) or in restriction fragment length polymoφhism (RFLP) diagnostic techniques, as described in Watson et al, (2d ed. 1992), Recombinant DNA, Scientific American Books, 519-522, 545-547, which is hereby incoφorated by reference.

Specifically, for example, 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.

For example, 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. In prefened embodiments, 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. To illustrate, 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. In one aspect of the invention there is provided 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. In certain embodiments, 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 incoφorated herein in their entirety. Alternatively, or additionally, the level of ERDBP-1 protein can be detected in an immunoassay.

ATLANTA 333137vl 58 IRDBP-1 and its role in the onset and maintenance of obesity

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, hernias, colon cancer; (7) Psychosocial: work disability, depression; (8) Genitourinary: urinary stress incontinence, hypogonadism, breast and uterine cancer; (9) Neurologic: stroke, meralgia paresthetica, idiopathic interacranial hypertension. Any of the above conditions, when associated with obesity, could be used as indications for the effective use of ERDBP-1 agonists or antagonists.

Using in-situ hybridization to localize IRDBP-1 mRNA in the brain, 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.

Gene Therapy Modulation of IRDBP-1 Activity

The 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

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"). Of course, in most instances, 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.

Methods for gene therapy are described in U.S. Patent No. 5,399,346, issued to Anderson et al. and U.S. Patent No. 5,766,899, issued to Kuo et al. describes methods for gene delivery into liver cells. The use of amphipathic compounds to deliver DNA is described in U.S. Patent No. 5,744,335 issued to Wolf et al. and which are incoφorated herein in their entirety.

It is further contemplated to be within the scope of the present invention for 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. For instance, 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 heφes 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 CaPO₄ precipitation carried out in vivo. It will be appreciated that because transduction of appropriate target cells represents the critical first step in gene therapy, choice of the particular gene delivery system will depend on such factors as the phenotype of the intended target and the route of administration, e.g. locally or systemically.

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. Infection of cells with a viral vector has the advantage that a large proportion of the targeted cells can receive the nucleic acid. Additionally, 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. The development of specialized cell lines (termed "packaging cells") that produce only replication-defective refroviruses has increased the utility of refroviruses for gene therapy, and defective refroviruses are well characterized for use in gene fransfer for gene therapy puφoses (for a review see Miller, A. D. (1990) Blood 76: 271). Thus, 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. (1991) Proc. Natl. Acad. Sci. 88: 8039-8043; Fe y et al. (1991) Proc. Natl. Acad. Sci. 88: 8377-8381; Chowdhury et al. (1991) Science 254: 1802-1805; van Beusechem et al. (1992) Proc. Natl. Acad. Sci. 89: 7640-7644; Kay et al. (1992) Human Gene Therapy 3: 641-647; Dai et al. (1992) Proc. Natl. Acad. Sci. 89: 10892-10895; Hwu et al. (1993) J. Immunol. 150: 4104-4115; U.S. Patent No. 4,868,116; U.S. Patent No. 4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345; and PCT Application WO 92/07573), and which are incoφorated herein in their entireties.

Furthermore, it has also been shown that it is possible to limit the infection spectrum of refroviruses and consequently of retroviral-based vectors, by modifying the viral packaging proteins on the surface of the viral particle (see, for example PCT publications WO93/25234, WO94/06920, and W094/11524). For instance, 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 incoφorated 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. Moreover, 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 incoφorated herein in their entirety. Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 dl324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) 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. Natl. Acad. Sci. 90: 2812-2816) and muscle cells (Quantin et al. (1992) Proc. Natl. Acad. Sci. 89: 2581-2584), and which are incoφorated herein in their entireties. Furthermore, the 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

(and foreign DNA contained therein) is not integrated into the genome of a host cell

^"but remains episomal, thereby avoiding potential problems that can occur as a result of insertional mutagenesis in situations where infroduced DNA becomes integrated into the host genome (e.g., retroviral DNA). Moreover, the carrying capacity of the adenoviral genome for foreign DNA is large (up to 8 kilobases) relative to other gene

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. 109- 127), and which are incoφorated herein in their entirety. 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.

Yet another viral vector system useful for delivery of, for example, the subject ERDBP-1 -gene, is the adeno-associated virus (AAV). Adeno-associated virus is a naturally occuning defective virus that requires another virus, such as an adenovirus or a heφes virus, as a helper virus for efficient replication and a productive life cycle. (For a review see Muzyczka et al. (1992) Cun. Topics in Micro, and Immunol. 158:97-129). It is also one of the few viruses that may integrate its DNA into non- dividing cells, and exhibits a high frequency of stable integration (see for example Flotte et al. (1992) Am. J. Respir. Cell. Mol. Biol. 7:349-356; Samulski et al. (1989) J. Virol. 63:3822-3828; and McLaughlin et al. (1989) J. Virol. 62:1963-1973). 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. 81:6466-6470; Tratschin et al. (1985) Mol. Cell. Biol. 4:2072-2081; Wondisford et al. (1988) Mol. Endocrinol. 2:32-39; Tratschin et al. (1984) J. Virol. 51:611-619; and Flotte et al. (1993) J. Biol. Chem. 268:3781-3790), and which are incoφorated herein in their entirety.

Other viral vector systems that may have application in gene therapy have been derived from such as, but not limited to, heφes virus, vaccinia virus, and several RNA

ATLANTA 333137vl 64 viruses. In particular, heφes virus vectors may provide a unique strategy for persistence ofthe recombinant IRDBP-1 gene in cells ofthe central nervous system.

In addition to viral fransfer methods, such as those illustrated above, 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. In prefened embodiments, 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.

In a representative embodiment, 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 incoφorated herein in their entireties. For example, lipofection of papilloma-virus infected epithelial cells can be carried out using liposomes tagged with monoclonal antibodies against, for example, squamous cells.

In similar fashion, 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 incoφorated herein in their entireties. For example, an IRDBP-1 gene construct encoding an antagonistic form of the protein, e.g. a dominant negative mutant, can be used to fransfect HPV-infected squamous cells in vivo using 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). It will also be appreciated that 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. For instance, 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 incoφorated herein in their entirety.

In clinical settings, 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. For instance, 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. Ln other embodiments, initial delivery of the recombinant gene is more limited with introduction into the animal being quite localized. For example, 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 incoφorated herein in their entireties.

Moreover, 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. Alternatively, where the complete gene delivery system can be produced intact from recombinant cells, e.g. retroviral packages, the pharmaceutical preparation can comprise one or more cells which produce the gene delivery system. In the case ofthe latter, 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. 4,883,666; Aebischer et al. U.S. Patent No. 4,892,538; Aebischer et al. U.S. Patent No. 5,106,627; Lim U.S. Patent No. 4,391,909; Sefton U.S. Patent No. 4,353,888; and

ATLANTA 333137vl 66 Aebischer et al. (1991) Biomaterials 12:50-55), and which are incoφorated herein in their entireties.

Further, 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.

Antisense/snse Nucleic Acid Modulation of IRDBP-1 Gene Expression

Another aspect ofthe invention relates to the use ofthe isolated nucleic acid in "antisense" therapy. 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. Alternatively, 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

ATLANTA 333137vl 67 ofthe subject IRDBP-1 proteins. Such 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. (1988) Biotechniques 6: 958-976; and Stein et al. (1988) Cancer Res. 48: 2659-2668 and which are incoφorated herein in their entirety. Accordingly, 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. An increase in cell regulation may result, but this condition may be desirable where, for example, a deterioration or deficiency in the number of cells results in a pathological condition such as, but not limited to, a progressive decrease in neural cells, or muscular atrophy. In therapeutic applications, the oligomers are utilized in a manner appropriate for antisense therapy in general. For such therapy, 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. For systemic administration, injection is prefened, including intramuscular, intravenous, infraperitoneal, and subcutaneous for injection, 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. In addition, 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. For fransmucosal or fransdermal

ATLANTA 333137vl 68 administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for fransmucosal adminisfration bile salts and fiisidic acid derivatives. In addition, detergents may be used to facilitate permeation. Transmucosal administration may be through nasal sprays or using suppositories. For oral administration, the oligomers are formulated into conventional oral adminisfration forms such as capsules, tablets, and tonics. For topical adminisfration, the oligomers of the invention are formulated into ointments, salves, gels, or creams as generally known in the art. In addition to use in therapy, 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.

Transgen ic An imals

Another aspect of the present invention concerns 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. In embodiments of the present invention, therefore, 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. In the present invention, 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. Toward this end, tissue-specific regulatory sequences and conditional regulatory sequences can be used to control expression of the fransgene in certain spatial patterns. Moreover, temporal patterns of expression

ATLANTA 333137vl 9 can be provided by, for example, conditional recombination systems or prokaryotic transcriptional regulatory sequences.

Genetic techniques that allow for the expression of transgenes can be regulated via site-specific genetic manipulation in vivo are well known to those skilled in the art. For instance, genetic systems are available which allow for the regulated expression of a recombinase that catalyzes the genetic recombination a target sequence. As used herein, the phrase "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. For example, 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. Moreover, 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.

In an illusfrative embodiment, either the 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 incoφorated 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.

Accordingly, 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. Thus, the activation of expression of the recombinant UBC9 gene can be regulated via regulation of recombinase expression.

Use of the these recombinase system to regulate expression of, for example, a dominant negative IRDBP-1 gene, or an antisense gene, requires the construction of 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. In such an instance, 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,

ATLANTA 333137vl 71 or in a developmentally restricted pattern. Thus, the creation of a founder population in which, for example, an antagonistic IRDBP-1 fransgene is silent will allow the study of progeny from that founder in which disruption of ERDBP-1 -mediated insulin responsiveness in a particular tissue or at certain developmental stages could result in, for example, a lethal phenotype.

Similar conditional transgenes can be provided using 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. Thus, as described above for the recombinase-mediated activation, 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. Moreover, 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. By this method, the ERDBP-1 transgene could remain silent into adulthood until "turned on" by the introduction of the trans-activator.

Additionally, 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;

ATLANTA 333137vl 72 and Walden et al. (1987) Gene 61:317-327), and which are incoφorated herein in their entirety.

Methods of making knock-out or disruption fransgenic animals are also generally known. See, for example, Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Recombinase dependent knockouts can also be generated, e.g. by homologous recombination to insert recombinase target sequences flanking portions of an endogenous IRDBP-1 gene, such that tissue specific and/or temporal confrol of inactivation of an ERDBP-1 allele can be confrolled as above. Furthermore, 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.

Screening for IRDBP-1 agonists/antagonists 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. Moreover, 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. 46:49-54; Froimowitz (1990) Biotechniques 8:640-644; Burbam et al. (1990) Proteins 7:99-111; Pedersen (1985) Environ Health Perspect. 61:185-190; and Kini et al. (1991) J. Biomol. Struct. Dyn. 9:475-488), and which are incoφorated herein in their entirety.

Moreover, a number of programs are presently available for virtual design of ERDBP-1 protein inhibitors. For instance, the increasing availability of biomacromolecule structures of potential pharmacophoric molecules that have been solved crystallographically has prompted the development of a variety of direct

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. 29: 2149-2153), and which are incoφorated herein in their entireties. Most algorithms of this type provide a method for finding a wide assortment of chemical structures that are complementary to the shape of a binding site ofthe subject protein. Each of a set of small molecules from a particular data-base, such as the Cambridge Crystallographic Data Bank (CCDB) (Allen et al. (1973) J. Chem. Doc. 13: 119), is individually docked to a nucleic acid or other ligand binding site of the ERDBP-1 protein in a number of geometrically permissible orientations with use of a docking algorithm. In an illusfrative embodiment, a set of computer algorithms called DOCK, can be used to characterize the shape of invaginations and grooves that form the active sites and recognition surfaces of the subject protein (Kuntz et al. (1982) J. Mol. Biol. 161 : 269-288). 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). However, the program has been shown to position accurately known cofactors for inhibitors based on shape constraints alone.

Other 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 incoφorated herein in their entireties.

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 incoφorated 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 ³²P as the radiolabel. The radioactive probe was added at a concentration of 1-2 x 10⁶ 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) incoφorated 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, incoφorated 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. A nucleic acid

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.

Seventy-nine clones were found which grew on His^'Leu^" plates containing 15 mM 3-amino-l,2,4-triazole (Sigma Chemical Co., St. Louis, MO). The plasmids from those clones were isolated and transformed into E. coli. The isolated cDNAs were sequenced using automated sequencing. The sequences were identified by an NCBI BLAST search for similarity to sequences reported in GenBank.

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 incoφorated 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).

Gel mobility shift assays were done essentially as described in Villafuerte et al, (1997) J. Biol. Chem. 272: 5024-5030, incoφorated herein by reference in its entirety. ³²P-ATP -labeled oligonucleotides conesponding to nucleotide positions - 1150 to -1117 bp-fragment (SEQ ED NO: 1) ofthe rat IGFBP-3 gene were incubated

ATLANTA 333137vl 77 with the proteins derived from the cDNA clones at concentrations of approximately 20 ng protein per lane in 25 μl 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 μg of bovine serum albumin, 36 μg 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 (SEQ ID NO: 2) 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. 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 OD₆oo0.6. EPTG (isopropyl-β-thiogalactopyranoside) was added to a final concenfration of 1 mM three hours before harvest. 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

ATLANTA 333137vl 7 particular theory, 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.

Example 2: Sequencing of cDNA clones

Single-strand sequencing of cDNA clones use an Applied Biosystems Automated DNA Sequencer and a PCR-based fluorescent dideoxy method, according to the recommendations ofthe manufacturer. The partial rat IRDBP-1 cDNA clone 52

(SEQ ID NO: 2) is shown in Fig. 1. 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

Total 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 [³²P] 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 incoφorated herein by reference in its entirety, as shown in Fig. 20.

Northern analysis showed that hepatic cells expressed an mRNA species of at least 5.0 kb in length. Densitometric analysis of ERDBP-1 expression normalized to β-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

Laboratories, Inc (Palo Alto, CA) contained 2 μg of polyadenylayed RNA per lane isolated from various human tissues. The 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.

Use of a 250 bp β-actin and a 170 bp ERDBP-1 probe for ribonuclease protection studies demonstrated expression of ERDBP-1 in multiple organs (Fig. 21 A). 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,

Austin, TX) according to the manufacturer's protocol.

Example 4: Interactions with other insulin-response binding proteins

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 incoφorated 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 luciferase levels still further.

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). To develop the N-segment specific antibody nAb, 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.

In addition, 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 Ni²⁺-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 OD₆₅o 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.

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. In western blotting experiments using rat liver nuclear extracts, 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 southwestern blots by an IGFBP-3 ERE

ATLANTA 333137vl 81 probe. Detectable ERDBP-1 -specific band levels were reduced in the livers of streptozotocin-diabetic rats compared to normal rats, indicating insulin-sensitive expression.

The 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

IGFBP-3 IRE, but not by the unrelated NFKB oligonucleotide (Fig. 26, left panel). In addition, the gel-shift band was supershifted with the anti-ERDBP-1 cAb antibody, but not with preimmune serum (Fig. 26, right panel).

Example 6: Screening of bacteriophage human and rat cDNA libraries

Because the 952 bp IRDBP-1 -encoding clone 52 cDNA (SEQ ED NO: 2) encodes a protein (SEQ ED NO: 3) that contains a binding domain with limited transactivating properties, 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 10⁸ 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. Subsequent rescreening of the library combined with 5' RACE extensions yielded isolated nucleic acids comprising the nucleotide sequence of SEQ ID NO: 5, as shown in Figs. 4A and 4B, or fragments, variants or derivatives thereof, such as SEQ ID NO: 6 (Figs 5A and 5B), SEQ ID NO: 14 (Figs, 12A and 12B) and SEQ ID NO: 44 (Fig. 13). Automated sequencing confirmed regions within SEQ ED NOS: 5,

ATLANTA 333137vl 2 6 and 44 having substantially similarity to the nucleotide sequence of rat clone 52 (SEQ ID NO: 2).

To obtain an isolated nucleic acid encoding a region of the human ERDBP-1, 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. Subsequent 5' extensions were obtained by 5' RACE techniques, using the SMART RACE cDNA amplification system from Clontech, Inc, and polyA mRNA isolated from the human cerebellum, until the 4584 bp sequence (SEQ ED NO: 7, shown in Fig 7) and the 6331 bp sequence (SEQ ED NO: 45, shown in Fig. 14) were obtained. Overlapping regions of the human IRDBP-1 nucleic acid sequence SEQ ED NO: 45 and the rat ERDBP-1 sequence SEQ ED NO: 44 showed at least 75% similarity when aligned.

Comparison of the human ERDBP-1 nucleic acid sequence SEQ ED NO: 45 with the human genomic DNA sequence Accession Nos. XM059482 and AKO74062 and sequences obtained from Celera Discovery Inc. showed that the human cDNA sequence SEQ ID NO: 45 was derived from at least AIC074062 exons (SEQ ED NOS: 57-62 and 16 - 41, as shown in Table 1).

Table 1. Exon positions of the human ERDBP-1 nucleic acid sequence SEQ

ID NO: 45

ATLANTA 333137vl 83

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) incoφorated herein by reference in its entirety.

Example 7: IRE specificity of HtDBP-l

As shown in Figs. 19 and 27, 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 ED NO: 56). A farwestern blot showed that the shifted bands formed between the nuclear extracts and the IREs of IGFBP-3, IGF-I and IGFBP-1 reacted strongly with anti- IRDBP-1 cAb antibody (Fig. 27, right panel). Specificity of this reaction was shown with an anti-SpI antibody that recognized Spl reacting with the IGF-I ERE, and a PPAR gamma antibody used as a negative control.

ATLANTA 333137vl 85 The shifted band seen with the ERE of the prolactin gene was unreactive with IRDBP-1 cAb. In the farwestem technique, denaturation separates individual proteins and it is possible for ERDBP-1 to bind to cofactors that can interfere with antibody epitope binding, or that the binding of IRDBP-1 to the ERE limits reaction with the antibody. These interactions with multiple genes indicate that ERDBP-1 is involved in coordinating a variety of metabolic responses to insulin.

Example 8: Metabolic activity of the IRDBP-1 protein

To investigate the biological effects of ERDBP-1, 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 [³H] 2-deoxyglucose uptake (Fig. 28B). The insulin induction of the expression of the IRDBP-1 gene was shown by

Northern analysis wherein from about 10^"9 M to about 10^"7 M insulin increased the formation of IRDBP-1 -specific mRNA of about 5.0 kb, as shown in Fig. 20. Normal liver parenchymal cells have elevated levels of the ERSBP1 -specific mRNA compared to cells from a rat having diabetes mellitus, also as shown in Fig. 20. In addition, the 3.4 kb cDNA region SEQ TD NO: 14 (as shown in Figs. 12A,

12B) (Genbank Accession NO. AF439719) and obtained from the lambda bacteriophage cDNA library screening, was also tested for the ability to induce a luciferase reporter gene expressed under the regulatory control of the IGFBP-3 ERE (Fig. 23). The cDNAs found to produce proteins that bound to ERE sequences were subcloned into plasmid pTARGET, a mammalian expression vector containing Kozak initiation sequences (Promega, Madison, WI). 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, incoφorated 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 μg to 2.5 μg 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.

The cDNA was also subcloned into the mammalian expression vector pCR 3.1 (Invitrogen Coφ., 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).

As shown in Fig. 29, the truncated 3.4 kb ERDBP-1 cDNA, including 1503 bp of translated sequence and 1899 bp of 3'- untranslated sequence, 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.

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 To examine the ability of the ERDBP-l polypeptide to allow storage of substrates related to insulin action, 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. As shown in Fig. 30, 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. Thus, 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.

Following oral glucose load, tracer and forearm catheterization techniques showed that 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

ATLANTA 333137vl δδ transcribed in vitro to give a riboprobe (antisense probe). The assay was carried out using the Hybspeed RPA Kit (Ambion, Inc., Austin, TX) according to the manufacturer's directions. RNA extracted from various tissues of the rat was hybridized with the [³²P] 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.

Using an ribonuclease protection assay, both insulin-dependent (muscles, adipose tissue, liver) and non-insulin dependent tissues or organs in which insulin were shown not to be required for utilization of glucose (brain, kidney, gut) expressed

ERDBP-1 mRNA (Figs. 21A and 22). Thus, the common denominator for ERDBP-1 expression is the dependence ofthe organ or tissues on glucose for energy utilization.

At the tissue level, studies have shown that when comparing the glucose metabolic rates of different adipose regions, measured as the sum of glucose converted to CO₂, triglycerides and lactate, the mesenteric fat cells metabolized significantly more glucose per cell than other fat depots. The hierarchy ofthe glucose metabolic rate in the different adipose depot is as follows: mesenteric > refroperitoneal > epididymal > subcutaneous fat.

Cultured beta cells express ERDBP-1, shown in Fig. 40. In cultured adipocytes, IRDBP-1 fransfection results in increased Glut4 and Glutl expression and franslocation of Glut 4 to the plasma membrane, as shown in Fig. 41.

As shown in Figs. 21 A and 22, IRDBP-1 expression is highest in mesenteric fat, followed by refroperitoneal, epididymal and subcutaneous fat. Thus, 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.

ATLANTA 333137vl 89 Example 11: IRDBP-1 expression is detected in many areas of the brain, including areas associated with feedings and satiety.

In-situ hybridization of rat brains using S-labeled IRDBP-1 riboprobe.

Rat brains were obtained and fixed by immersion in 4% paraformaldehyde in 0.1 M NaPO₄, sectioned on a cryostat to 5-10 μM thickness, and mounted on slides.

Sense and antisense ³⁵S-labeled ERDBP-1 riboprobes were generated by in vitro transcription with ³⁵S-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

(600,000 cpm/slide) and tRNA (200 μg/ml) for hybridization. Hybridization was done overnight at 55 C; slides were then washed, freated with RNAse A, dehydrated, and coated with photographic emulsion. The slides were exposed and developed after 4-12 weeks. Both sagittal (Fig. 31) and coronal (Fig. 32) sections of brains from normal Sprague-Dawley rats, obese fa/ fa Zucker rats and lean fa/+ Zucker rats were compared.

Example 12: IRDBP-1 is expressed in the hypothalamus and nucleus of the solitary tract

Information about the qualities of food are relayed by the primary senses of smell, sight, and taste to the nucleus of the solitary tract in the medulla. The nucleus of the solitary tract (NTS) 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.

ATLANTA 333137vl 9Q Brain lesions ofthe venfromedial hypothalamus produced hypeφhagic obesity. Lesions of the lateral hypothalamus caused hypophagia and weight loss. The central administration of insulin also changed the level of defended body weight rather than a simple suppression of food intake. Thus, the 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.

The results from in-situ hybridization with a IRDBP-1 riboprobe indicated that IRDBP-1 mRNA is highly expressed in multiple areas of the hypothalamus. As shown in Figs. 31-34, 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). Furthermore, comparison of the brain sections from obese and lean Zucker rats showed that the number of silver grains, representing ERDBP-1 mRNA, is higher in the obese than the lean rats (Fig. 33), showing that IRDBP-1 is regulated in the feeding center of the brain, and has a potential role in regulating weight of animals.

Since all of the information from the neural pathways related to feeding and satiety are integrated in the nucleus of the solitary tract (NTS) of the hindbrain, the expression of ERDBP-1 in the NTS was investigated. As shown in Fig. 34 a sagittal cut through the medullary section ofthe brain showed that ERDBP-1 mRNA is higher in the NTS of the lean rats, compared to the obese Zucker rats. This further shows that IRDBP-1 is involved not only in altering sympathoadrenal and vagal activities throughout the hypothalamus, but that it has also a potential role in integrating the limbic and autonomic systems involved in maintenance of energy balance.

Differential ERDBP-1 expression was also seen in the in situ hybridization, with labeled antisense IRDBP-1 probe, of the pyramidal tract and decussations of the pyramidal tract of obese and lean Zucker rats (Figs. 35 and 36).

ATLANTA 333137vl 91 Example 13: Role of IRDBP-1 in the glucose and insulin regulation of food intake and body weight

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.

Since 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.

In addition to the roles of ERDBP-1 in affecting glucose metabolism, synthesis of IRDBP-1 is stimulated by addition of insulin; therefore we also need to consider the effect of insulin action on glucose utilization of the brain. 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 β-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

ATLANTA 333137vl 92 and coφus callosum. Lesions ofthe posterodorsal aspects of the amygdala have been associated with hyperinsulinemia, hypeφhagia, and obesity without the preference for particular food that characterized other brain lesion-induced obesity. The involvement ofthe olfactory system with high levels of expressed ERDBP-1 (Figs. 37 and 38) in the primary sense of smell, and the affective component associated with eating under the confrol of the limbic system, including the amygdala and coφus callosum. 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

Langerhans

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.

A polyclonal antibody raised against the epitopic region SEQ ID NO: 15 ofthe rat IRDBP-1 polypeptide SEQ ED NO: 11 and capable of detecting the presence ofthe IRDBP-1 protein from rat or human, was used in immunohistochemical staining of the pancreas to detect the cellular location of ERDBP-1. 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 μg/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. Color development was performed with the ABC-vector Red complex from an alkaline phosphatase standard kit. The slide was counterstained with Gill's hematoxylin, dehydrated and mounted. As shown in Fig. 39, immunostain of pancreas showed intense accumulation of IRDBP-1 is the cytoplasm of the islet of Langerhans. In particular, the insulin- secreting β cells ofthe pancreas, which comprised 74% ofthe islet mass and is cenfral in location, expressed abundant ERDBP-1, as described in Examples 13 and 14 above. IRDBP-1 expression was also detected by immunodetection in the mesangium of the glomerulus of the kidney (Fig. 42A), in vascular endothelial cells (Fig. 42B) and in neuronal cells (Fig. 42C).

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. Mesangial cell proliferation and expansion is the initial event in the development of diabetic nephropathy. Thus, 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.

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).

In immunodepletion experiments, prior exposure of COS 7 exfracts to an agarose-coupled anti-ERDBP-1 antibody reduced the amount of ERDBP-1 immunoreactivity that coprecipitated with anti-Erk antibody . Cell lysates from L6 myoblasts stably overexpressing Flag-tagged ERDBP-1

(detected as a 70 kDa protein using anti-Flag antibody)) were also exposed to antibodies specific for Akt and Erk. IRDBP-1 was coprecipitated by anti-Akt and anti-Erk antibodies (as detected by anti-flag antibody), but was not coprecipitated by control IgG. In the confrol, Akt and Erk were each precipitated by their respective antibodies.

B. 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 μM) and LY 294002 (50 μM), and insulin (10^"7 M) was added 30 mins later as indicated. Coprecipitated proteins were

ATLANTA 333137vl 95 probed with anti-ERDBP-1 and anti-phospho Erk antibodies using western immunoblots, as shown in Fig. 45A. When 3T3-L1 adipocytes were freated with insulin, nuclear extracts showed increases in ERDBP-1 and Erkl/2 (Fig. 45B) shown with anti-Erk coimmunoprecipitation of cell extracts. Addition of PD 98059 to inhibit Mek 1 that specifically phosphorylates Erkl and Erk2, reduced insulin-stimulated IRDBP-1 and Erkl levels compared to insulin-stimulated controls. Inhibition of PI3- kinase with LY 294002 increased Erk-associated ERDBP-1. Insulin signaling, therefore, can regulate IRDBP-1 via both the Mek 1 and Erkl pathways, since blockade of the PI3 -kinase- Akt pathway would otherwise be expected to enhance signaling through the Mek-Erk pathway.

C 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 [γ-³²P] ATP for 20 mins., and analyzed by SDS-PAGE. E. cø/z^'-expressed NFKB p50 was used as negative control. Akt phosphorylated the RPRAATF substrate of glycogen synthase kinase 3β, 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.

D. The effects of Aktl on basal and IRDBP-1 -induced IRE activity in COS7 cells. COS 7 cells were fransfected with either 1 μg of ERDBP-1 (pCR ERDBP-1) or confrol vector (pCR vector), plus 0.5 μg 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). Substitution of methionine for lysine at residue 179 of Akt abolishes the kinase activity (Akt K179M). As shown in Fig. 45C, the expression of ERDBP-1 increased transcription of the IRE by 6-fold as compared to the control vector (lane 5

ATLANTA 333137vl 96 vs. lane 1); insulin treatment of these cells for 24 hours further increased ERDBP-1- activated transcription by 107.7 ± 2% (lane 6 vs. 5). The effect of Akt myr on ERDBP-1 activation was similar to that observed for insulin (lane 7 vs. 6). Akt K179M did not increase IRDBP-1 -activated franscription above the level induced by ERDBP-1 alone (lane 8 vs. 5). Control studies without the ERDBP-1 expression construct revealed that insulin and Akt myr stimulated the reporter gene, whereas Akt K179M had no effect (lanes 2, 3, 4 vs. lane 1).

E. Differences between the signaling output from Akt and Erk on IRDBP-1 activation. 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). 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). When Erk2 and Aktl were added together, the inhibitory effect of Erk2 predominated over the stimulatory effect of Akt (lane 9 vs lane 7). Therefore, Erk2 stimulates phosphorylation of ERDBP-1 and inhibits its function, while Aktl stimulates phosphorylation of IRDBP-1 and ERDBP-1 function. The effect of Akt equals the effect of insulin on activation of IRDBP-1.

Example 17: Proteolysis and cell distribution of IRDBP-1 in adipocytes,

COS 7 and HepG 2 cells

Akt mediates the metabolic actions of insulin by phosporylating regulatory proteins at the serine or threonine residue. Both anti-phosphotyrosine and anti-

ATLANTA 333137vl 97 phosphoserine/threonine antibodies 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. Therefore, IRDBP-1 can be useful to screen for a specific protease able to cleave ERDBP-1. A. Proteolytic cleavage of IRDBP-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.

Whole-cell exfracts (500 μg/lane) were obtained from 3T3-L1 adipocytes (day 5 after differentiation), COS 7 and HepG 2 cells and immunoprecipitated with anti- Erk antibodies as shown in Fig. 46A. Samples were subjected to western blots and probed with the anti-ERDBP-1 -cAb anti-ERDBP-1 antibody. The Erkl level was measured to show equal loading ofthe protein.

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.

The cytoplasmic and the nuclear proteins were separated from HepG2 cells, and the fractionated exfracts analyzed by western blotting. As shown in Fig. 46B, the anti-IRDBP-1 nAb antibody recognized an approximately 120 kDa band in the cytoplasmic extracts, but reacted poorly with the nuclear exfracts. In confrast, the

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. B. Confocal microscopy. Confocal microscopy of HepG 2 cells was done on cells grown in the absence or presence of 10^"8 M insulin. Cells were permeabilized, incubated with anti-IRDBP-1 nAb or cAb antibody, and with Oregon green 488 goat anti-rabbit IgG (Molecular Probe) as the secondary antibody. 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. However, 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) is restricted mostly to the cytoplasm, whereas the truncated portion retaining the carboxyl ends (i.e. 70 and 50 kDa bands) was localized predominantly to the nucleus.

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. Phosphorylation of ERDBP-1 at the serine/threonine residue indicates that 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.

Example 19: The physiologic relevance of IRDBP-1

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).

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 μM [H³] 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.

ATLANTA 333137vl 1QQ The effects of IRDBP-1 in the absence and presence of pioglitazone were compared. 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.

Example 20: IRDBP-1 function in intact animals

The 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 10⁷ 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. Glucose was measured in blood samples from the tail vein of Ad- IRDBP-1 -treated ZDF rats (n=6, black bars) and Ad-GFP-treated ZDF rats (n=6, white bars) before and after treatment with the recombinant adenovirus ERDBP-1 or GFP vector, as shown in Fig. 53B. Values are means ± SEM. Body weight ofthe Ad- IRDBP-1 transfected ZDF rats was monitored in ad libitum-fed ERDBP-1 -treated (n=6, shown as solid line) and GFP-freated rats (n=6, shown as broken line, Fig. 53C). GTT on 12 week and 18 week old rats freated with or without Ad-ERDBP-1 is shown in Figs. 53D and 53E. Two gms of dextrose/KBW was injected infraperitoneally into Ad-IRDBP-1 and Ad-GFP-treated rats (n=6 in each group), and blood glucose was measured every hour for 3 hours. The results are shown in in Figs. 53D, 53E. Solid line: Ad-ERDBP-1 -treated rats; broken line: Ad-GFP-treated rats. Each bar represents mean ± SEM. There was no difference between the effects of normal saline (vehicle) vs. Ad-GFP (viral vector) infusions on glucose levels in the ZDF rats.

ATLANTA 333137vl 1Q1 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. 51 A) and insulin-sensitizing activity (Fig. 53 A). As shown in 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). However, 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). In confrast, glucose levels remained high in the Ad-GFP rats (baseline of 258 ± 25 vs. 282 ± 48 mg/dl at 12 days, p = NS). The Ad-GFP recipient rats gained weight continuously through the study, while weight was stable in Ad-IRDBP-1 animals for the first 3-5 days (associated with some decrease in food intake), with steady weight gain thereafter (Figs. 53C, 53D).

On day 11, both groups of animals were fasted overnight. A glucose tolerance test (GTT) was performed with intraperitoneal injection of 2 gm/kg body weight of 50%) dextrose. Glycogen was measured by the amyloglucosidase/hexokinase enzymatic assay after acid hydrolysis. Rat insulin was measured by RIA (kit from Linco Research Inc.) Interassay CV = 0.20, infraassay CV = 0.06.

As shown in Figs. 53E and 53F, fasting glucose levels were slightly lower in the Ad-IRDBP-1 compared to the Control Ad GDF rats (101 ± 5 vs. 116 ± 6 mg/dl). During the GTT, glucose levels in the Ad-IRDBP-1 rats were 166 + 6 mg/dl at 1 hour and 113 + 3 mg/dl at 2 hours, whereas glucose levels in the Ad-GFP rats were 296 ± 31 mg/dl and 181 ± 22 mg/dl, respectively, which was significantly higher than the

ATLANTA 333137vl Q2 respective glucose levels in the Ad-ERDBP-1 rats (both p <0.05). Despite lower fasting glucose levels, improved glucose tolerance, and higher liver glycogen (Fig. 54), mean fasting insulin levels were also slightly lower in Ad-ERDBP-1 compared to the Ad-GFP rats (0.81 ± 0.06 vs. 0.91 ± 0.12 ng/ml, p = 0.5), consistent with increased insulin sensitivity.

The GTTs were repeated with 18 week old ZDF rats with more severe diabetes. Before gene therapy, glucose levels at 3 pm during ad libitum feeding were 395 + 18 mg/dl in Ad-IRDBP-1, and 390 + 18 mg/dl in Ad-GFP rats. Fourteen days after therapy, despite comparable weight (435 + 8 gms in Ad-ERDBP-1 rats and 421 ± 14 gms in Ad-GFP rats), the fasting glucose level was significantly lower in Ad- ERDBP-1 rats (125 ± 6 mg/dl vs. 223 ± 10 mg/dl, p <0.001 in Ad-GFP rats) (Fig. 53E). During the GTT, glucose levels in the Ad-ERDBP-1 rats rose to 165 ± 12 and 150 + 3 mg/dl at 2- and 4-hrs post-challenge, whereas glucose levels in the Ad-GFP rats were 417 ± 38 and 375 ± 38 mg/dl (both p <0.05 vs. values in Ad-ERDBP-1 animals), demonstrating that ERDBP-1 adminisfration decreases fasting glucose and insulin levels, and improves the response to a glucose challenge. Treatment with ERDBP-1 appears to be sufficient to ameliorate hyperglycemia in ZDF rats.

Example 21: Administration of IRDBP-1 sense and antisense oligonucleotides to an animal

Methods & Materials. The methodology was adapted from that of Apostolakis et al, J. Neurosci. 16: 4823-4834) incoφorated herein by reference in its entirety. Briefly, ovariectomized (OVX) female rats (200-250 gm) were housed individually and maintained on a 12: 12 hour lightidark cycle (lights on at 0700 CST) with rat chow and water in excess ad libitum. After acclimation (7 days), females underwent stereotaxic implantation of third ventricle cannula guides (26 gauge, Plastics One, Roanoke VA). The experiment started 7 days after surgery. Females were randomly assigned identification numbers and weighed daily between 0900 and 1000 CST for 5 days immediately before experimental treatment, allowing each animal to serve as its own confrol. Each animal (n = 5) received a single infraventricular (icv) injection of

ATLANTA 333137vl 1Q3 antisense (AS, sequence 5'-CTAACTCACAGGTGATGATGTAGAG-3', SEQ ID NO: 42) oligos (4 nM in 1 μl vol. over 2 min) after weighing on the^* fifth day. Another group of animals (n = 4) served as positive controls and received sense (S, sequence 5'-CTCTACATCATCACCTGTGAGTTAG-3', SEQ ED NO: 43) oligos (4 nM in 1 μl vol. over 2 min). Animals were euthanized under deep anesthesia 8 days after oligonucleotide freatment.

There was a difference in mean weight loss between those animals following treatment with antisense DNA and sense DNA (Fig. 55A). For the five days prior to oligonucleotide treatment, the animals demonstrated stable weight. Within 24 hours after freatment, animals that received sense DNA began to lose weight with the greatest loss (54+6.4 gm) being attained at 72-96 hours after freatment as shown in Fig. 55 A. Antisense DNA had no significant effect on weight (251+2 gm) while sense DNA-treated animals lost weight (54.3+6.2 gm). Individually, the females receiving sense DNA lost 27% of their body weight as compared with either their initial weight, their mean weight during the prefreatment time or mean weight of antisense-freated animals as shown in Fig. 55B.

Example 22: Inhibitory effect of IRDBP-1 on cell proliferation

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.

Matched tumor/normal expression anay analysis showed that in certain cancer types, including breast, rectal, colon, lung and kidney, ERDBP-1 is expressed at higher levels than is found in normal tissues, as shown in Fig. 57A.

RNA was obtained from various normal and tumor tissues, and was converted into cDNA. PCR amplified the cDNA and the samples were blotted onto nylon membranes. cDNAs originating from 68 human tumors and conesponding normal

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.

Expression of 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

Figs. 60 and 61.

Flow cytometry analysis of the cell cycle compartments of L6 cells with or without over-expression ofthe IRDBP-1 nucleic acid sequence. Flow cytometry was conducted to analyze DNA content and progression through the cell cycle. Stable transfectants were fixed in ice cold 100% ethanol and debris was removed by centrifugation through a cushion of fetal bovine serum. Cell pellets were freated with RNAse solution (500 units/ml of 1.12% (w/v) sodium citrate) at 37° C for 15 mins, and 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

To investigate the function of ERDBP-1, 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. Finally, the Heφes Simplex Virus thymidine kinase (HSV-tk) gene will be incoφorated into each end ofthe DNA construct, adjacent to the ERDBP-1 sequences, to allow for selection of homologous recombinants.

Following linearization, 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.

If inactivation of ERDBP-1 proves to be lethal, we will produce tissue-specific knockouts. A fransgenic line will be created encoding Cre recombinase in selected tissues using the strategy of targeted fransgene expression. A responder "knock-in"

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").

Once 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. With 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.

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.

ATLANTA 333137vl Qη Although prefened embodiments of the invention have been described using specific terms, devices, and methods, such description is for illusfrative puφoses only. The words used are words of description rather than of limitation. It is to be understood that changes and variations may be made by those of ordinary skill in the art without departing from the spirit or the scope ofthe present invention, which is set forth in the following claims. In addition, it should be understood that aspects of the various embodiments may be interchanged both in whole or in part.

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Claims

What is Claimed is:

1. An isolated mammalian nucleic acid capable of regulating the expression of a mammalian gene responsive to insulin, wherein the isolated nucleic acid is capable of hybridizing a hybridization probe, the nucleotide sequence of which comprises SEQ ED NO: 44 or the complement of SEQ ID NO: 44.

2. The isolated nucleic acid according to Claim 1, comprising a nucleotide sequence selected from any of the group consisting of SEQ ED NOS: 2, 5-10, 14, 16-41 and 42-45, or degenerate variants thereof.

3. The isolated nucleic acid according to Claim 1 , comprising SEQ ED NO: 2.

4. The isolated nucleic acid according to Claim 1, comprising SEQ ED NO: 5.

5. The isolated nucleic acid according to Claim 1, comprising SEQ ED NO: 44.

6. The isolated nucleic acid according to Claim 1, comprising SEQ ED NO: 45.

7. The isolated nucleic acid according to Claim 1, the nucleic acid encoding an epitope of an ERDP-1 polypeptide suitable for inducing the formation of an antibody specific for IRDBP-1.

8. The isolated nucleic acid according to Claim 7, the nucleic acid encoding the amino sequence SEQ ID NO: 15 or 46.

9. The isolated nucleic acid according to Claim 1, comprising the nucleic acid sequence selected from the group consisting of SEQ ED NOS: 42 and 43.

10. The isolated nucleic acid according to Claim 1, wherein the nucleic acid is an antisense nucleic acid comprising the nucleic acid sequence SEQ ED NO: 42.

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11. A polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ED NOS: 3, 11-13, 15, and 46-48.

12. The polypeptide according to Claim 11, comprising the sequence SEQ ED NO: 15 or 46.

13. The polypeptide according to Claim 11, comprising the sequence SEQ ED NO: 47 or 48.

14. The polypeptide according to Claim 11, comprising the sequence SEQ ED NO:

47.

15. The polypeptide according to Claim 11, comprising the sequence SEQ ED NO: 48.

16. A vector comprising the isolated nucleic acid according to Claim 1.

17. The vector of Claim 16, wherein the vector is an expression vector.

18. The vector of Claim 16, wherein the vector is a viral vector.

19. The vector of Claim 16, wherein the vector is an adenovirus vector.

20. An antibody capable of selectively binding to a mammalian ERDBP-1 polypeptide, the polypeptide having an amino acid sequence selected from the group consisting of SEQ ED NOS: 3, 11-13, 15, and 46-48.

21. The antibody according to Claim 20, wherein the antibody selectively binds to the peptide comprising the amino acid sequence SEQ ED NO: 47.

22. The antibody according to Claim 20, wherein the antibody selectively binds to the peptide comprising the amino acid sequence SEQ ID NO: 15.

23. The antibody according to Claim 20, wherein the antibody selectively binds to the peptide comprising the amino acid sequence SEQ ED NO: 46.

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24. A method of regulating the expression of a nucleic acid encoding an IRDBP-1 polypeptide in a mammalian cell, comprising the steps of:

(a) obtaining a mammalian cell;

(b) delivering to the cell a composition comprising a nucleic acid capable of regulating the level of expression in the cell of a mammalian gene encoding a mammalian ERDBP-1 polypeptide; and

(c) allowing the nucleic acid to regulate the level of the ERDBP-l polypeptide in the cell.

25. The method according to Claim 24, further comprising the step of:

allowing the ERDBP-1 polypeptide to regulate the expression of at least one insulin-responsive gene.

26. The method according to Claim 24, wherein the nucleic acid is an antisense nucleic acid comprising the sequence SEQ ED NO: 42.

27. The method according to Claim 24, wherein the- nucleic acid is expressed in the cell and encodes a mammalian ERDBP-1 polypeptide capable of binding to an insulin-responsive element of an insulin-responsive gene, and wherein the nucleic acid comprises the sequence selected from the group consisting of SEQ ED NOS: 2, 5-10, 14, and 44-45.

28. The method according to Claim 24, wherein the nucleic acid comprises the sequence further selected from the group consisting of SEQ ED NOS: 2, 5, 6, 8,

14 and 44.

29. The method according to Claim 24, wherein the nucleic acid comprises the sequence selected from the group consisting of SEQ ED NOS: 45.

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30. The method according to Claim 24, wherein the nucleic acid regulates the expression of an ERDBP-1 gene in the cell.

31. The method according to Claim 24, wherein the nucleic acid is an antisense nucleic acid capable of regulating the expression of an ERDBP-1 gene in the cell and comprising the sequence SEQ ED NO: 42.

32. The method according to Claim 24, wherein regulating the level ofthe ERDBP- 1 polypeptide in the cell regulates glucose metabolism by the cell, and wherein regulating the level of the ERDBP-1 polypeptide in the cell increases glucose transport into the cell.

33. The method according to Claim 24, wherein regulating the level of the IRDBP- 1 polypeptide in the cell regulates proliferation ofthe cell.

34. The method according to Claim 24, wherein the nucleic acid is delivered to the cell by transfection, by a liposome, by transformation or by elecfroporation.

3.5. The method according to Claim 24, wherein the nucleic acid comprises an expression vector capable of expressing the nucleic acid as a single-strand nucleic acid.

36. The method according to Claim 24, wherein the nucleic acid comprises an expression vector capable of expressing the nucleic acid as a polypeptide selected from the group consisting of SEQ ED NOS : 3 , 11 - 13 , and 47-48

37. The method according to Claim 24, wherein the expression vector is a viral vector.

38. The method according to Claim 24, wherein the viral vector is an adenovirus.

39. The method according to Claim 24, wherein the cell is an adipocyte.

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40. The method according to Claim 24, wherein the mammalian cell is in animal, and wherein the animal undergoes a loss of weight.

41. The method according to Claim 24, wherein the mammalian cell is in animal, wherein the appetite ofthe animal is reduced.

42. The method according to Claim 24, wherein regulating the level ofthe IRDBP- 1 polypeptide in the cell reduces the serum glucose level in an animal or human having the cell therein.

43. The method according to Claim 24, wherein regulating the level of the ERDBP- 1 polypeptide in the cell reduces obesity in an animal or human having the cell therein.

44. The method according to Claim 24, wherein regulating the level of the ERDBP-1 polypeptide in the cell results in weight loss in an animal or human having the cell therein.

45. The method according to Claim 24, wherein regulating the level of the ERDBP-1 polypeptide in the cell reduces diabetes in an animal or human having the cell therein.

46. A method of regulating the .serum glucose level in an animal or human, comprising the steps of:

(a) administering to an animal or human an effective amount of a pharmaceutically acceptable composition comprising a compound capable of modulating the activity of ERDBP-1; and

(b) modulating the activity of ERDBP-1, thereby regulating the serum glucose level in the animal or human.

47. The method according to Claim 46, wherein the compound capable of modulating the activity of ERDBP-1 is an agonist or an antagonist.

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48. The method according to Claim 46, wherein the compound capable of modulating the activity of ERDBP-1 is a thiazolidinedione.

49. The method according to Claim 46, wherein the thiazolidinedione is selected from the group consisting of troglitazone, rosiglitazone and pioglitazone.

50. The method according to Claim 46, wherein the thiazolidinedione is pioglitazone.

51. The method according to Claim 46, wherein the effective amount of the pharmaceutically effective composition increases the phosphorylation of IRDBP-1.

52. The method according to Claim 46, wherein modulating ERDBP-1 activity increases glucose transport into a cell of the animal or human, thereby modulating the serum glucose level in the animal or human.

53. A method of inhibiting the proliferation of a mammalian cell, comprising the steps of:

(a) delivering to a mammalian cell selected from a tumor cell, an endothelial cell and an adipocyte cell, a nucleic acid comprising a nucleic acid sequence selected from the group consisting of SEQ ED NOS: 2, 5-10, 14-41 and 42-46, or degenerate variants thereof;

(b) expressing the nucleic acid as a polypeptide capable of binding to an ERE of at least one gene ofthe mammalian cell; and

(c) allowing the expressed polypeptide to modulate the activity of an insulin-responsive gene, thereby inhibiting the proliferation ofthe mammalian cell.

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54. The method according to Claim 53, wherein the cell is a tumor cell in a cancer selected from the group consisting of lung, colon, breast and kidney.

55 The method according to Claim 53, wherein nucleic acid is delivered to the mammalian cell by a vector, wherein the vector is a viral vector.

56. A method of detecting a nucleic acid encoding an ERDBP-1 polypeptide in a cell, comprising the steps of:

(a) obtaining a mammalian cell having target nucleic acid therein;

(b) contacting the target nucleic acid with a nucleic acid probe capable of hybridizing to an ERDBP-1 -encoding nucleic acid SEQ TD NO: 44, or the complement thereof;

(c) detecting the hybridization of the nucleic acid probe to the target nucleic acid; and

(d) identifying the presence of an ERDBP-1 -encoding nucleic acid in the mammalian cell.

57. A method of detecting an IRDBP-1 polypeptide in a cell, comprising the steps of:

(a) obtaining a mammalian cell having target polypeptide therein;

(b) contacting the target polypeptide with an antibody capable of selectively binding to an epitope of a mammalian ERDBP-1 - polypeptide;

(c) detecting the binding of the antibody to the target nucleic acid; and

ATLANTA 333137vl U5 (d) identifying the presence of an ERDBP-1 polypeptide in the mammalian cell.

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