WO2007097751A1

WO2007097751A1 - Methods for reducing glucose intolerance by inhibiting chop

Info

Publication number: WO2007097751A1
Application number: PCT/US2006/006345
Authority: WO
Inventors: Randal J. Kaufman; Benbo Song; Donalyn Scheuner
Original assignee: The Regents Of The University Of Michigan
Priority date: 2006-02-22
Filing date: 2006-02-22
Publication date: 2007-08-30

Abstract

Provided herein are methods for preventing and treating glucose intolerance related diseases in a subject or in cells thereof. A method may comprise administering to a subject or cells thereof an agent that inhibits the protein level or activity of a CHOP protein. A method may also comprise contacting pancreatic islet cells with such an agent and administering the cells to a subject in need thereof.

Description

UM 3329/UMS-329.25

METHODS FOR REDUCING GLUCOSE INTOLERANCE BY

INHIBITING CHOP

Government Support

This invention was made with Government support under grant number DK42394, awarded by the NIH to R. Kaufman. The Government has certain rights in the invention.

Background of the Invention

Diabetes is a world wide major cause of morbidity and mortality that is influenced by genetic factors, diet and exercise. Diabetes refers to a disease process derived from multiple causative factors and is characterized by elevated levels of plasma glucose (hyperglycemia) in the fasting state or following glucose administration during an oral glucose tolerance test. Diabetes may be classified broadly into two types: type 1 diabetes caused by insulin hyposecretion; and type 2 diabetes caused by glucose intolerance in peripheral tissues.

Patients with non-insulin dependent diabetes mellitus (type 2 diabetes mellitus), which comprise approximately 95% of patients with diabetes mellitus, frequently display elevated levels of serum lipids, such as cholesterol and triglycerides, and have poor blood- lipid profiles, with high levels of LDL-cholesterol and low levels of HDL-cholesterol. Those suffering from type 2 diabetes mellitus are thus at an increased risk of developing macrovascular and microvascular complications, including coronary heart disease, stroke, peripheral vascular disease, hypertension (for example, blood pressure greater than or equal to 130/80 mmHg in a resting state), nephropathy, neuropathy and retinopathy.

Patients having type 2 diabetes mellitus characteristically exhibit elevated plasma insulin levels compared with nondiabetic patients; these patients have developed a resistance to insulin stimulation of glucose and lipid metabolism in the main insulin- sensitive tissues (muscle, liver and adipose tissues). Type 2 diabetes results from a failure of beta cells to produce adequate amounts of insulin in response to insulin resistance that occurs frequently in response to a high-fat diet. Thus, type 2 diabetes, at least early in the natural progression of the disease is characterized primarily by insulin resistance or glucose intolerance rather than by a decrease in insulin production, resulting in insufficient uptake, oxidation and storage of glucose in muscle, inadequate repression of lipolysis in adipose

B3163748.3 tissue, and excess glucose production and secretion by the liver. The net effect of decreased sensitivity to insulin is high levels of insulin circulating in the blood without appropriate reduction in plasma glucose (hyperglycemia). Hyperinsulinemia is a risk factor for developing hypertension and may also contribute to vascular disease. Recently, the incidence of type 2 diabetes has increased rapidly due to environmental factors such as diet. Despite current treatment methods, the incidence of the disease has risen dramatically in recent years. Approximately 150 million people worldwide have been diagnosed with diabetes, and it is estimated that the number of people afflicted by the disease will increase to 300 million by the year 2025. Because current prevention and treatment methods have proved insufficient for decreasing the incidence of type 2 diabetes, new treatment methods are imperitave for combatting the disease.

Summary of the Invention

Provided herein are methods for treating or preventing glucose intolerance and related diseases or disorders ("glucose intolerance related diseases") in a subject or in a pancreatic islet or cells thereof. In one embodiment, a method comprises administering to a subject in need thereof a therapeutically effective amount of an agent that reduces the level of protein or activity of a C/EBP homologous protein (CHOP) protein, hi another embodiment, a method comprises contacting a pancreatic islet or cells thereof with an agent that reduces the level of protein or activity of a CHOP protein. A method may further comprise administering the pancreatic islet or cells thereof to a subject, such as a subject in need thereof, e.g., a subject in need of pancreatic beta cells producing insulin.

Also provided herein are methods, e.g., screening methods, for identifying agents that may be used for treating a glucose intolerance related disease. A method may comprise identifying an agent that decreases the level of expression of a CHOP gene or the level of a CHOP protein in a cell. A method may also comprise identifying an agent that inhibits or decreases the level of activity of a CHOP protein, such as by inhibiting its transcriptional activity and/or binding to a nucleic acid comprising a target nucleotide sequence. A method may further comprise subjecting the test agent to a glucose intolerance test or another test that may be used to determine the likelihood of the test agent to be effective in treating or preventing a glucose intolerance related disease. Brief Desription of the Drawings

FIG. 1 depicts the unfolded protein response (UPR) in diabetes. Accumulation of unfolded protein in the endoplasmic reticulum activates three signaling pathways. This activation is referred to as the unfolded protein response. During the unfolded protein response, phosphorylation of eukaryotic initiation factor 2 (eIF2) by the kinase PERK inhibits translation and reduces the load upon the endoplasmic reticulum (ER). Preferential translation of at least one transcription factor and activation of a subset of UPR genes also occurs. The PERK/eIF2 signaling pathway is required for Beta cell survival upon ER stress. FIG. 2 illustrates a schematic of UPR signaling events mediated by PERK.

FIG. 3 shows a schematic of phosphorylation of eIF2 and control of translation. The active eIF2 ternary complex is required to promote recognition of AUG codon on all mRNA. Phosphorylation at Ser51 prevents the recycling of eIF2 into an active GTP bound form. Knock-in gene targeting to block all eIF2 phosphorylation is exemplified in Example 1. In the exemplified system, AUG codon recognition cannot be downregulated.

FIG. 4 illustrates that heterozygous eIF2α^s/A mice gain more weight upon a high-fat (HF) diet. The S/A HF animals gain significantly more weight and become quite fat.

FIG. 5 illustrates blood glucose or insulin vs. time for heterozygous eIF2α^s/A mice. Heterozygous eIF2α^{s A} mice are glucose intolerant with impaired insulin secretion in vivo. The intolerance of eIF2α ^A mice is associated with reduced first phase insulin secretion.

FIG. 6 shows electron micrographs of beta cells from eIF2α^s/A mice fed a high-fat diet (HF) and a low-fat diet (LF), and of a control mouse fed a high-fat diet. Electron microscopy revealed ER dilation and stress in about 2/3 of the beta cells for eIF2α^s/A mice after 20 wks of a high-fat diet. In the cells with the most severe ER distention for the eIF2α^s/A mice after 20 wks of a high-fat diet, fewer insulin granules are apparent.

FIG. 7 illustrates that proinsulin is bound to BiP in the ER of islets isolated from eIF2α^s/A mice fed a high-fat diet.

FIG. 8 shows that CHOP deletion increases HF-induced obesity in eIF2α^s/A mice. A variety of nutrient deprivations and stresses activate eIF2 kinases and the Ser51Ala mutation blocks all of the pathways in unison so that there is no inhibition of translation, no preferential translation and no eIF2 UPR transcription.

FIG. 9 illustrates that CHOP deletion prevents HF diet-induced glucose intolerance in eIF2α^s/A mice. Blood glucose levels vs. time post glucose injection are shown. KlCi. lϋ shows that CHOP deletion enhances the obesity of db/db mice.

FIG. 11 shows that CHOP deletion normalizes glucose tolerance in diabetic db/db mice.

FIG. 12 shows how CHOP deletion may prevent glucose intolerance due to reduced ER stress signaling. The phenotype of the S/ A high fat diet mouse is one of obesity and diabetes, hi obesity, a reduced metabolic rate is observed while food intake is not increased. The animals are glucose intolerant with impaired insulin secretion, mild hyperglycemia, and hyperinsulinemia that increases over time.

FIG. 13 shows the nucleotide and amino acid sequences of human CHOP (SEQ ID NOs: 1 and 2, respectively.)

Detailed Description of the Invention

Definitions

As used herein, the following terms and phrases shall have the meanings set forth below. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art.

The singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise.

The term "agent" is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule (such as a nucleic acid, an antibody, a protein or portion thereof, e.g., a peptide), or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues. The activity of such agents may render it suitable as a "therapeutic agent" which is a biologically, physiologically, or pharmacologically active substance (or substances) that acts locally or systemically in a subject.

The term "C/EBP homologous protein" or "CHOP" is synonymous with the terms C/EBP zeta, CEBPZ, CHOPlO, DNA-damage inducible transcript 3, GADD153, Growth arrest and DNA-damage-inducible protein GADD 153, MGC4154, DNA-damage-inducible transcript 3, DDIT3. An example of an amino acid sequence of human CHOP protein may be found at the NCBI website, under NCBI accession No. NP_004074 (SEQ ID NO: 2). An example of a cDNA sequence for human CHOP protein may be found at the NCBI website, under NCBI accession No. NM_004083 (SEQ ID NO: 1). A "biological activity ot a CHUP protein" may be any of the following activities: transcriptional activity, inhibition of the DNA binding activity of C/EBP and LAP, forming heterodimers and to promote death, e.g., by apoptosis, of ER stressed. Cell death may be mediated by down-regulation of the anti-apoptotic gene Bcl2 (McCullough et al. MoI Cell Biol. 21 : 1249 ), and/or promoting protein synthesis and inducing oxidative stress

(Marciniak et al, Genes and Dev. 18: 3066). Proteins with which CHOP interacts include ATF3, C/EBPB (NF-IL6), C/EBP epsilon, CSNK2A1, c-FOS, c-JUN, JUN-D, MAPK14, PICALM, RPS3A, and TRIB3. DNA sequence with which CHOP heterodimers interact are set forth, e.g., in Ubeda et al. (1996) MoI. Cell. Biol. 16:1479. To "inhibit the activity of a CHOP protein" refers to any action that results in reducing or decreasing at least one biological activity of a CHOP protein, e.g., the transcriptional activity of the CHOP protein and/or its ability to bind to its target DNA sequence. Inhibition may be by a factor of at least about 50%, 75%, 100% (i.e., 2 fold), 3 fold, 5 fold, 10 fold, 20 fold, 50 fold or more. "Inhibit CHOP gene expression" or "inhibit CHOP expression" refers to any action that results in decreased production of a polypeptide encoded by the gene or decreased levels of an RNA encoded by the gene. Inhibiting gene expression includes, e. g., inhibiting transcription, translation or degrading the DNA template or RNA encoded thereby. Inhibition may be by a factor of at least about 50%, 75%, 100% (i.e., 2 fold), 3 fold, 5 fold, 10 fold, 20 fold, 50 fold or more.

The term "mammals" is inclusive of animals such as humans, rodents, mice, non- human primates, sheep, dog, cow, chickens, amphibians, reptiles, ovines, bovines, equines, canines, felines etc.

As used herein, the term "nucleic acid" refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). The term should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single (sense or antisense) and double-stranded polynucleotides.

A "protein" refers herein to any polymer consisting essentially of any of the 20 amino acids. Although "polypeptide" is often used in reference to relatively large polypeptides, and "peptide" is often used in reference to small polypeptides, these terms are used interchangeably herein. The term "protein" as used herein refers to peptides, proteins and polypeptides, unless otherwise noted. A "therapeutic composition" or "therapeutic" as used herein is defined as comprising a therapeutic, e.g., an inhibitor of the invention, and other physiologically compatible ingredients. The therapeutic composition may contain excipients such as water, minerals and carriers such as protein. An "effective amount" of an inhibitor of the invention in the context of treatment or prevention is that amount which produces a result or exerts an influence on the particular condition being treated such as glucose intolerance.

A "therapeutically effective amount" in the context of treatment or prevention is therefore that amount of a therapeutic composition that produces a result or exerts an influence on the particular condition being treated such as glucose intolerance.

An "expression vector" is a polynucleotide, such as a DNA plasmid, virus, or phage (among other common examples) which allows expression of at least one gene when the expression vector is introduced into a host cell. The vector may, or may not, be able to replicate in a cell. An "isolated nucleic acid" means an RNA or DNA polynucleotide, portion of genomic polynucleotide, cDNA or synthetic polynucleotide which, by virtue of its origin or manipulation: (i) is not associated with all of a polynucleotide with which it is associated in nature (e.g., is present in a host cell as an expression vector, or a portion thereof); or (ii) is linked to a nucleic acid or other chemical moiety other than that to which it is linked in nature; or (iii) does not occur in nature. By "isolated" it is further meant a polynucleotide sequence that is: (i) amplified in vitro by, for example, polymerase chain reaction (PCR); (ii) chemically synthesized; (iii) recombinantly produced by cloning; or (iv) purified, as by cleavage and gel separation.

When applied to polypeptides, the term "isolated" means a polypeptide or a portion thereof which, by virtue of its origin or manipulation: (i) is present in a host cell as the expression product of a portion of an expression vector; or (ii) is linked to a protein or other chemical moiety other than that to which it is linked in nature; or (iii) does not occur in nature. By "isolated" it is further meant a protein that is: (i) chemically synthesized; or (ii) expressed in a host cell and purified away from associated proteins. The teπn generally means a polypeptide that has been separated from other proteins and nucleic acids with which it naturally occurs. Preferably, the polypeptide is also separated from substances such as antibodies or gel matrices (polyacrylamide) which are used to purify it. The term "homologous" as used herein is synonymous with the term "identity" and refers to the sequence similarity between two polypeptides, molecules or between two nucleic acids. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit (for instance, if a position in each of the two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by a lysine), then the respective molecules are homologous at that position. The percentage homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared* 100. For instance, if 6 of 10 of the positions in two sequences are matched or are homologous, then the two sequences are 60% homologous. By way of example, the DNA sequences CTGACT and CAGGTT share 50% homology (3 of the 6 total positions are matched). Generally, a comparison is made when two sequences are aligned to give maximum homology. Such alignment can be provided using, for instance, the method of Needleman et al., J MoI Biol. 48: 443-453 (1970), implemented conveniently by computer programs such as the Align program (DNAstar, Inc.). Homologous sequences share identical or similar amino acid residues, where similar residues are conservative substitutions for, or "allowed point mutations" of, corresponding amino acid residues in an aligned reference sequence. In this regard, a "conservative substitution" of a residue in a reference sequence are those substitutions that are physically or functionally similar to the corresponding reference residues, e.g., that have a similar size, shape, electric charge, chemical properties, including the ability to form covalent or hydrogen bonds, or the like. Particularly preferred conservative substitutions are those fulfilling the criteria defined for an "accepted point mutation" in Dayhoff et al., 5: Atlas of Protein Sequence and Structure, 5: Suppl. 3, chapter 22: 354-352, Nat. Biomed. Res. Foundation, Washington, D.C. (1978). "Small molecule" as used herein, is meant to refer to a composition, which has a molecular weight of less than about 5 kD and most preferably less than about 4 kD. Small molecules can be nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids or other organic (carbon-containing) or inorganic molecules. Many pharmaceutical companies have extensive libraries of chemical and/or biological mixtures, often fungal, bacterial, or algal extracts, which can be screened with any of the assays described herein.

The term "inhibit" is intended to mean "prevent" or "reduce." A "CHOP protein inhibitor" is an agent that prevents or reduces levels of a CHOP protein and/or function/activity. The inhibition may occur at the gene, RNA, or protein level. The term "oligonucleotide" herein refers to polynucleotides comprising nucleotide units formed with naturally occurring bases and pentofuranosyl sugars joined by phosphodiester linkages. The term "copolymer" includes oligonucleotides and also structurally related molecules formed from non-naturally occurring or modified subunits of oligonucleotides. These modifications occur either on the base portion of a nucleotide, on the sugar portion of a nucleotide, or on the internucleotide linkage groups. Additional linkage groups are often also substituted for sugar and phosphate backbone of a natural oligonucleotide to generate a copolymer.

"Glucose intolerance" or "impaired glucose tolerance" (IGT) refers to a condition in which an individual has higher than nonnal levels of glucose in the blood upon fasting or following a carbohydrate-rich meal or ingestion of a glucose test solution but not high enough to be diagnostic of diabetes mellitus (Merriam- Webster's Medical Dictionary, © 2002 Merriam- Webster, Inc.). Impaired glucose tolerance is defined as two-hour glucose levels of 140 to 199 mg per dL (7.8 to 11.0 mmol) on the 75-g oral glucose tolerance test. In a "normal" or non-IGT individual, glucose levels rise during the first two hours to level less than 140 mg/dl and then drop rapidly. In an IGT individual, the blood glucose levels are higher and the drop-off level is at a slower rate.

"Impaired fasting glucose" (IFG) is a condition in which a blood glucose test, taken after an 8- to 12-hour fast, shows a level of glucose higher than normal but not high enough for a diagnosis of diabetes. IFG, also called pre-diabetes, is a level of 100 mg/dL to 125 mg/dL. A fasting blood glucose test is a check of a person's blood glucose level after the person has not eaten for 8 to 12 hours (usually overnight). This test is used to diagnose prediabetes and diabetes. It is also used to monitor people with diabetes.

An "insulin resistance related disorder" or "insulin-related disorder" is a disorder whereby an afflicted mammal has developed a resistance to insulin stimulation of glucose and lipid metabolism in the main insulin-sensitive tissues (muscle, liver and adipose tissues). A "glucose intolerance related disease" refers to a condition that is associated with glucose intolerance. Exemplary insulin resistance and glucose intolerance related diseases or disorders include: diabetes (such as type I and type II), obesity, metabolic syndrome, insulin-resistance syndromes, syndrome X, insulin resistance, high blood pressure, hypertension, high blood cholesterol, dyslipidemia, hyperlipidemia, dyslipidemia, atherosclerotic disease including stroke, coronary artery disease or myocardial infarction, hyperglycemia, hyperinsulinemia and/or hyperproinsulinemia, impaired glucose tolerance, impaired fasting glucose, delayed insulin release, diabetic complications, including coronary heart disease, angina pectoris, congestive heart failure, stroke, cognitive functions in dementia, retinopathy, peripheral neuropathy, nephropathy, glomerulonephritis, glomerulosclerosis, nephrotic syndrome, hypertensive nephrosclerosis some types of cancer (such as endometrial, breast, prostate, and colon), complications of pregnancy, poor female reproductive health (such as menstrual irregularities, infertility, irregular ovulation, polycystic ovarian syndrome (PCOS)), lipodystrophy, cholesterol related disorders, such as gallstones, cholescystitis and cholelithiasis, gout, obstructive sleep apnea and respiratory problems, osteoarthritis, and prevention and treatment of bone loss, e.g. osteoporosis. "Diabetes" refers to high blood sugar or ketoacidosis, as well as chronic, general metabolic abnormalities arising from a prolonged high blood sugar status or a decrease in glucose tolerance. "Diabetes" encompasses both the type I and type II (Non Insulin Dependent Diabetes Mellitus or NIDDM) forms of the disease. The risk factors for diabetes include the following factors: waistline of more than 40 inches for men or 35 inches for women, blood pressure of 130/85 mmHg or higher, triglycerides above 150 mg/dl, fasting blood glucose greater than 100 mg/dl or high-density lipoprotein of less than 40 mg/dl in men or 50 mg/dl in women.

The term "hyperinsulinemia" refers to a state in an individual in which the level of insulin in the blood is higher than normal. The term "insulin resistance" refers to a state in which a normal amount of insulin produces a subnormal biologic response relative to the biological response in a subject that does not have insulin resistance.

A "disease relating to an insulin dysfunction" includes diseases in which the insulin dysfunction is the result of an abnormality in a pancreatic islet cell (beta cell), such as an abnormality in an insulin gene. Abnormalities in beta cells typically result in type I diabetes, rather than type II diabetes. A disease relating to an insulin dysfunction also includes diseases in which the beta pancreatic islet cells are normal or functioning normally, e.g., by producing the normal amount of insulin, but there is a problem in the sensitivity of other cells to insulin or in the processing of insulin, such as due to an outside source, e.g., a high fat diet. These latter problems result, e.g., in insulin resistance and later may result in type II diabetes. Preferred diseases to be treated according to the methods described herein are those that relate to problems occurring due to exposure of the cells of an individual to an exogenous or outside stress, e.g., a high fat diet, rather than endogenous defects of beta pancreatic islet cells.

"Obese" individuals or individuals suffering from obesity are generally individuals having a body mass index (BMI) of at least 25 or greater. Obesity may or may not be associated with insulin resistance.

The terms "systemic administration," "administered systemically," "peripheral administration" and "administered peripherally" are art-recognized and refer to the administration of a subject composition, therapeutic or other material other than directly into the central nervous system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes.

The terms "parenteral administration" and "administered parenterally" are art- recognized and refer to modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articulare, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion.

Exemplary methods of treatment and prevention

The present invention is based at least in part on the finding that a knockout of the CHOP gene prevents the development of high-fat induced diabetes in eIF2α^{s A} mice. More specifically, the present invention is based on the finding that CHOP deletion prevents beta cell (β cell) failure in high-fat fed eIF2α^s/A mice. The data presented herein provide evidence that CHOP deletion may alleviate stress of environmental factors such as a high- fat diet on beta cells and thereby prevent a proapoptotic response in these cells. The results support that targeted intervention to prevent or reduce CHOP function might prevent or alleviate beta cell failure associated with glucose intolerance and/or insulin-related disorders such as Type 2 diabetes. Taken together, these discoveries suggest new methods for preventing or treating glucose intolerance, and in particular diabetes, and allow for the discovery of new drugs useful in the treatment of diseases relating to the same. In one aspect, the present invention relates to a method for preventing or reducing glucose intolerance or a disease relating thereto in a mammalian subject or in cells thereof. This method comprises contacting a mammalian cell with an inhibitor of a CHOP protein. The inhibitor contacted with the cell may be effective to prevent or reduce glucose intolerance in the mammalian cell. In a preferred embodiment, the mammalian cell is a beta cell. The inhibitor may be characterized as an inhibitor of a CHOP protein that reduces ER stress in the beta cell. The inhibitor is to be administered to cells using an appropriate delivery system such that the inhibitor is effective to reduce levels of a CHOP protein and/or activity in the contacted cell. The cell may be contacted directly with the inhibitor under conditions for cellular uptake. Alternatively, the cell may express an exogenous inhibitor composition provided herein from an introduced exogenous construct harboring an expressible nucleic acid construct or constructs. DNA or RNA compositions effective to inhibit CHOP activity in the cell may further be delivered to cells by injection. The inhibitor may comprise a recombinant agent obtained from cultured cell systems expressing the inhibitor.

Accordingly, the present invention relates to a method comprising contacting a cell, such as a beta cell, with an agent that inhibits the level or activity of a CHOP gene. In one embodiment, the beta cell is in a donor pancreatic islet, and the method comprises contacting the donor pancreatic islet with the agent. Contact with the inhibitor or agent may result in islet cell hyperplasia.

A method for preventing or reducing glucose intolerance in a mammalian cell may be carried out in vitro. Reduction of CHOP activity may be achieved in a cell in tissue culture. In vitro, CHOP may be exogenous or endogenous to the mammalian cell. In vitro, the methods provided herein may be used to study CHOP-related signaling events or for the development of therapeutics for modulating CHOP activity such as identifying an inhibitor of a CHOP protein. An identified inhibitor may be used in the methods provided herein for preventing or reducing glucose intolerance in a mammalian cell.

A method for preventing or reducing glucose intolerance may also comprise contacting the mammalian cell with an inhibitor of a CHOP protein in vivo or ex vivo. Accordingly, an array of therapeutic modalities fall within the scope of the present invention, and as such, any composition associated with such modalities also fall within the scope of the present invention. Cells that are targeted for CHOP inhibition include beta pancreatic cells. The present invention relates to a method for preventing glucose intolerance in a mammalian beta cell, either in vivo or ex vivo.

With respect to ex vivo applications, beta cells may be isolated from a mammal and an ex vivo culture may be established. Such cultures can be established from a population of beta cells obtained from a mammal, and the population may comprise abnormal beta cells, with or without separation from accompanying normal cells to be treated. Alternatively, the beta cells may be obtained from cell lines or clones from such cell lines. Alternatively, such beta cells may be obtained from established beta cell lines from unrelated patients or as explants of fresh islet cell tissue. Another aspect of the present invention relates to a method for transplanting a beta cell from a donor to a recipient, such as a recipient suffering from glucose intolerance or a disease relating thereto. This method comprises obtaining a beta cell from a donor. The donor may be glucose tolerant or intolerant. The donor may further be the recipient, although this is not a requirement. The method further comprises contacting the beta cell with an agent that reduces the level or activity of a CHOP protein in the beta cell. The beta cell is contacted with the agent in an amount and for a sufficient time, effective to reduce the level or activity of a CHOP protein in the beta cell. An agent may be any inhibitor as described herein and may be used in conjunction with an additional agent or agents as described herein. A method may further comprise administering the beta cell to a recipient, e.g., a subject having a glucose intolerance related disorder or an insulin resistance related disorder. The insulin resistance related disorder may be type 2 diabetes. The beta cell may be contacted with the agent ex vivo and the resulting beta cell administered to a recipient. Alternatively, the beta cell may be contacted with the agent in vivo, hi this case, the beta cell may be administered to the recipient prior to treatment of the beta cell with the agent. In the case that the beta cell contacted is in a donor pancreatic islet, the method may comprise contacting the pancreatic islet with the agent and administering the pancreatic islet to the recipient.

An inhibitor of the present invention may be modified or contacted with a mammalian cell together with an additional agent or agents to enhance some property of the inhibitor to enable prevention or reduction of glucose intolerance in a mammalian cell. Such properties include the molecular size of the inhibitor, permeation properties, hydrophobicity, hydrophilicity, and/or charge that may facilitate entry of the inhibitor into a beta cell. In vivo, the inhibitor may be modified and/or delievered in conjunction with another agent to enable delivery of the inhibitor to beta cells within a mammal. For example, the inhibitor may be modified with the addition of specific ligands that allow the inhibitor to be directed to a specific target via molecular recognition, or to facilitate entry of the inhibitor into a cell. The ligands may recognize and bind to beta cell receptors to which delivery is desired. As an example, the inhibitor may be fused to an antibody that specifically recognizes an antigen on the surface of beta cells. Where the inhibitor is to be used as a therapeutic, cell or tissue targeting provides the distinct advantage of lowering the required dosage for effective treatment, thereby reducing cellular toxicity. Non-limiting examples of ligands suitable for targeting molecules to specific cell types which maybe used in conjunction with the invention include proteins, peptides, and peptoids.

An inhibitor of the present invention may further be contacted with a mammalian cell in conjunction with an inhibitor of eIF2α, e.g., an agent that decreases the protein level or activity of an eIF2α protein.

An inhibitor of a CHOP protein may act directly or indirectly on the activity of the CHOP gene product. When the activity of the CHOP gene product is to be decreased directly, the inhibitor contacted with the cell is effective to decrease transcription, translation, and/or stability of an endogenous CHOP protein. The inhibitor may also modify the binding activity of the endogenous CHOP protein to any of its biologically active ligands, wherein the modification of binding activity is effective to decrease CHOP protein activity in the cell. The inhibitor may increase or decrease levels and/or activity of a molecule or molecules upstream of a CHOP protein, wherein the increase or decrease in levels and/or activity result in inhibiting the activity of a CHOP protein. The inhibitor may decrease the affinity of endogenous CHOP protein to one of it is biologically active ligands, or alternatively increase the affinity of endogenous CHOP protein to a negative regulator. The inhibitor may target CHOP RNA, DNA, or protein sequence directly, or indirectly by targeting another signaling molecule or molecules upstream of an endogenous CHOP protein.

Another aspect of the present invention relates to a method for preventing or treating glucose intolerance or a related disorder in a mammal. A method may comprise administering to a mammal in need thereof a therapeutically effective amount of an inhibitor of A CHOP protein. As used herein, "treatment" of a subject includes the application or administration of a therapeutic agent to a subject, or application or administration of a therapeutic agent to a cell or tissue from a subject, who has a diseases or disorder, has a symptom of a disease or disorder, or is at risk of (or susceptible to) a disease or disorder, with the purpose of curing, inhibiting, healing, alleviating, relieving, altering, remedying, ameliorating, improving, or affecting the disease or disorder, the symptom of the disease or disorder, or the risk of (or susceptibility to) the disease or disorder. As used herein, a "therapeutic agent" or "compound" includes, but is not limited to, small molecules, peptides, peptidomimetics, polypeptides, RNA interfering agents, e.g., siRNA molecules, antibodies, ribozymes, and antisense oligonucleotides. The method may be used such as for example treating a mammal having a condition that would benefit from the prevention or treatment of glucose intolerance such as an animal predisposed to or diagnosed with glucose intolerance or an insulin resistance related disorder.

In one embodiment, a method is for treating a subject having a glucose intolerance related disease, such as glucose intolerance, impaired fasting glucose or insulin-resistance. A subject to be treated may also be a subject who has or is likely to develop a disease that results from a stress, e.g., an exogenous stress, that is applied to pancreatic beta cells, such as a high fat diet. Accordingly, a glucose intolerance disease that may be treated may be a disease that is caused or contributed to by a high fat diet and/or obesity. A high fat diet may be a diet that contains more fat that is recommended for maintaining the body weight of a subject having a normal body weight. A high fat diet may result in the subject becoming over- weight, potentially obese, and developing any of the conditions relating thereto, e.g., insulin resistance and diabetes.

In certain embodiments, a subject that is to be treated as described herein, is a subject who does not have a disease that is associated with an abnormal insulin or pro- insulin gene that results in the production of insulin in lesser amounts or in a less effective form. For example, the animal model called the Akita mouse, which spontaneously develops hyperglycemia has a mutation in the insulin 2 gene (Ins2 (Cys96Tyr)) that is responsible for the diabetic phenotype of this mouse. A subject may also be a subject who does not have type I diabetes. In certain embodiments, a subject is a subject that does not have one of the diseases set forth herein. Accordingly, in certain embodiments, a subject to be treated (ex vivo or in vivo) is a subject who has a glucose intolerance related disease, provided that the disease is not a disease that is associated with a defect in an insulin or pro- insulin gene, e.g., a mutation that inactivates the insulin produced.

Other methods provided herein are diagnostic methods. A method may comprise determining the level or activity of a CHOP protein in a tissue sample of a subject. A higher level of protein or activity of a CHOP protein may indicate that a subject has or is likely to develop a disease relating to glucose intolerance. A tissue sample may be a pancreatic islet sample, e.g., one or more beta cells from a pancreatic islet. A higher level of protein or activity of a CHOP protein or rnRNA may be a level that is at least about 25%, 50%, 75%, 100% (i.e., 2 fold), 3 fold, 5 fold or more higher than that in a normal cell oi the same type. An exemplary method may comprise (i) obtaining a tissue sample from a subject; and (ii) determining the level of^" protein, gene expression (e.g., mRNA) or activity of a CHOP protein, wherein the presence of a higher level of a CHOP protein or gene expression indicates that the subject has or is likely to develop a glucose intolerance related disease.

Exemplary CHOP inhibitors

Inhibitors of the invention include inhibitory nucleic acid molecules which are introduced into a cell, e.g., a beta cell, and directly inhibit CHOP protein synthesis by binding to the CHOP mRNA, or inhibit CHOP transcription by binding to the CHOP gene. An inhibitor of the invention may include any biologically active agent such as a nucleic acid, protein, polypeptide, peptide, small molecule, and/or other compound. A nucleic acid may comprise a single-stranded or double-stranded DNA or RNA molecule and may further include an oligonucleotide, plasmid, or vector. Examples of nucleic acid compositions effective to inhibit CHOP activity include ribozymes, antisense oligonucleotides, antisense RNAs, and short or small interfering RNAs (siRNAs). Such ribozymes, antisense oligonucleotides, antisense RNAs, and short or small interfering RNAs (siRNAs) may target CHOP RNA directly or indirectly such as by targeting upstream regulators of CHOP, as described above. Antisense oligonucleotides, ribozymes, antisense RNAs, and siRNAs may be designed to form hybrids with target mRNA for effecting suppression of activity of the targeted molecule.

The principles for design of such compositions are well known in the art and may be used for providing a composition effective for inhibiting CHOP activity. These inhibitor compositions contain nucleotide base sequences which are complementary to a targeted portion of the RNA molecule. A complementary oligonucleotide or RNA may be designed to specifically inhibit translation of a target such as CHOP as stated above. Nucleic acids for targeting CHOP activity may be synthesized and administered to beta cells using standard techniques known in the art.

Inhibitory nucleic acids hybridize to target RNA to form hybrids, such as by Watson-Crick base pairing. The sequence of a copolymer is thus defined by the complementary sequence of the target RNA. The copolymers may be synthesized chemically with nucleotide sequence lengths which span at least 6 complementary nucleotides of the target RNA, with 12-25 being most common. Statistically, a sequence of about 15 nucleotides is unique within the population of all RNAs within a cell, enabling any particular RNA to be targeted with a high degree of specificity. Binding to RNA is also very stable with Kd values around 10^'17 M, for a copolymer encompassing 20 base pairs.

In one embodiment a CHOP inhibitor is an interfering RNA. An "RNA interfering agent" or "interfering RNA", is defined as any agent which interferes with or inhibits expression of a target gene, e.g., a CHOP gene, by RNA interference (RNAi). Such RNA interfering agents include, but are not limited to, nucleic acid molecules including RNA molecules which are homologous to the target gene, e.g., a CHOP gene, or a fragment thereof, short interfering RNA (siRNA), and small molecules which interfere with or inhibit expression of a target gene by RNA interference (RNAi).

"RNA interference (RNAi)" is an evolutionally conserved process whereby the expression or introduction of RNA of a sequence that is identical or highly similar to a target gene results in the sequence specific degradation or specific post-transcriptional gene silencing (PTGS) of messenger RNA (mRNA) transcribed from that targeted gene (see Coburn, G. and Cullen, B. (2002) J. of Virology 76(18):9225), thereby inhibiting expression of the target gene, hi one embodiment, the RNA is double stranded RNA (dsRNA). This process has been described in plants, invertebrates, and mammalian cells. In nature, RNAi is initiated by the dsRNA-specific endonuclease Dicer, which promotes processive cleavage of long dsRNA into double-stranded fragments termed siRNAs. siRNAs are incorporated into a protein complex that recognizes and cleaves target mRNAs. RNAi can also be initiated by introducing nucleic acid molecules, e.g., synthetic siRNAs or RNA interfering agents, to inhibit or silence the expression of target genes. As used herein, "inhibition of target gene expression" or "inhibition of a CHOP gene expression" includes any decrease in expression or protein activity or level of the target gene or protein encoded by the target gene, e.g., a CHOP protein. The decrease may be of at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more as compared to the expression of a target gene or the activity or level of the protein encoded by a target gene which has not been targeted by an RNA interfering agent.

"Short interfering RNA" (siRNA), also referred to herein as "small interfering RNA" is defined as an agent which functions to inhibit expression of a target gene, e.g., by RNAi. An siRNA may be chemically synthesized, may be produced by in vitro transcription, or may be produced within a host cell, hi one embodiment, siRNA is a double stranded RNA (dsRNA) molecule of about 15 to about 40 nucleotides in length, preferably about 15 to about 28 nucleotides, more preferably about 19 to about 25 nucleotides in length, and more preferably about 19, 20, 21, or 22 nucleotides in length, and may contain a 3' and/or 5' overhang on each strand having a length of about 0, 1, 2, 3, 4, or 5 nucleotides. The length of the overhang is independent between the two strands, i.e., the length of the over hang on one strand is not dependent on the length of the overhang on the second strand. Preferably the siKNA is capable of promoting RNA interference through degradation or specific post-transcriptional gene silencing (PTGS) of the target messenger RNA (mRNA).

In another embodiment, an siRNA is a small hairpin (also called stem loop) RNA (shRNA). In one embodiment, these shRNAs are composed of a short (e.g., 19-25 nucleotide) antisense strand, followed by a 5-9 nucleotide loop, and the analogous sense strand. Alternatively, the sense strand may precede the nucleotide loop structure and the antisense strand may follow. These shRNAs may be contained in plasmids, retroviruses, and lentiviruses and expressed from, for example, the pol III U6 promoter, or another promoter (see, e.g., Stewart, et al. (2003) RNA Apr;9(4):493-501).

CHOP RNA interfering agents, e.g., siRNA molecules, may be administered to a patient having or at risk of having glucose intolerance or a disease relating thereto, to inhibit expression of a CHOP gene and thereby treat, prevent, or inhibit the disease in the subject. A CHOP inhibitor may also be an antisense nucleic acid molecule, e.g., a CHOP antisense nucleic acid. An antisense nucleic acid is a molecule that is complementary to a sense nucleic acid, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA. Accordingly, an antisense nucleic acid molecule of the invention can hydrogen bond to (i.e. anneal with) a sense nucleic acid of the invention. The antisense nucleic acid can be complementary to an entire coding strand, or to only a portion thereof, e.g., all or part of the protein coding region (or open reading frame). An antisense nucleic acid molecule can also be antisense to all or part of a non- coding region of the coding strand of a nucleotide sequence encoding a CHOP polypeptide. The non-coding regions ("5¹ and 3' untranslated regions") are the 5' and 3' sequences which flank the coding region and are not translated into amino acids.

An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides in length. An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. Examples of modified nucleotides which can be used to generate the antisense nucleic acid include 5-fiuorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5- carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1- methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5- methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5- methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2- carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been sub-cloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further herein).

The antisense nucleic acid molecules of the invention, e.g., CHOP antisense nucleic acids, are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a polypeptide, such as a CHOP polypeptide, to thereby inhibit expression of the polypeptide, e.g., by inhibiting transcription and/or translation. The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix. Examples of a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site or infusion of the antisense nucleic acid into a lung-associated body fluid. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens. The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.

An antisense nucleic acid molecule of the invention can be an α-anomeric nucleic acid molecule. An α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual α-units, the strands run parallel to each other (Gaultier et al, 1987, Nucleic Acids Res. 15:6625-6641). The antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (Inoue et al, 1987, Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al, 1987, FEBS Lett. 215:327-330).

The invention also encompasses ribozymes, e.g., CHOP ribozymes. Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single- stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes {e.g. , hammerhead ribozymes as described in Haselhoff and Gerlach, 1988, Nature 334:585-591) can be used to catalytically cleave mRNA transcripts to thereby inhibit translation of the protein encoded by the mRNA. A ribozyme having specificity for a nucleic acid molecule encoding a polypeptide corresponding to a protein of the invention, e.g., a CHOP protein, can be designed based upon the nucleotide sequence of a cDNA corresponding to the CHOP protein. For example, a derivative of a Tetrahymena L- 19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved (see Cech et al. U.S. Patent No. 4,987,071; and Cech et al. U.S. Patent No. 5,116,742). Alternatively, an mRNA encoding a polypeptide of the invention can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules (see, e.g., Bartel and Szostak, 1993, Science 261:1411-1418).

The invention also encompasses nucleic acid molecules which form triple helical structures. For example, expression of a polypeptide of the invention can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the gene encoding the polypeptide (e.g., the promoter and/or enhancer) to form triple helical structures that prevent transcription of the gene in target cells. See generally Helene (1991) Anticancer Drug Des. 6(6):569-84; Helene (1992) Ann. NY. Acad. ScL 660:27-36; and Maher (1992) Bioassays 14(12):807-15. In various embodiments, the nucleic acid molecules of the invention can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule. For example, the deoxyribose phosphate backbone of the nucleic acid molecules can be modified to generate peptide nucleic acid molecules (see Hyrup et ah, 1996, Bioorganic & Medicinal Chemistry 4(1): 5- 23). As used herein, the terms "peptide nucleic acids" or "PNAs" refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup et a (1996), supra; Perry-O'Keefe et a (1996) Proc. Natl. Acad. ScL USA 93:14670-675.

PNAs can be used in therapeutic and diagnostic applications. For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication. PNAs can also be used, e.g., in the analysis of single base pair mutations in a gene by, e.g., PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., Sl nucleases (Hyrup (1996), supra; or as probes or primers for DNA sequence and hybridization (Hyrup, 1996, supra; Perry-O'Keefe et ah, 1996, Proc. Natl. Acad. ScL USA 93:14670-675). In another embodiment, PNAs can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art. For example, PNA-DNA chimeras can be generated which can combine the advantageous properties of PNA and DNA. Such chimeras allow DNA recognition enzymes, e.g., RNASE H and DNA polymerases, to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity. PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup, 1996, supra). The synthesis of PNA-DNA chimeras can be performed as described in Hyrup (1996), supra, and Finn et al (1996) Nucleic Acids Res. 24(17):3357-63. For example, a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry and modified nucleoside analogs. Compounds such as 5'-(4-methoxytrityl)amino-5'-deoxy- thymidine phosphoramidite can be used as a link between the PNA and the 5' end of DNA (Mag et al, 1989, Nucleic Acids Res. 17:5973-88). PNA monomers are then coupled in a step-wise manner to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment (Finn et al, 1996, Nucleic Acids Res. 24(17):3357-63). Alternatively, chimeric molecules can be synthesized with a 5' DNA segment and a 3' PNA segment (Peterser et al, 1975, Bioorganic Med. Chem. Lett. 5:1119-11124).

In other embodiments, the oligonucleotide can include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al, 1989, Proc. Natl. Acad. ScL USA 86:6553-6556; Lemaitre et al, 1987, Proc. Natl. Acad. Sd. USA 84:648-652; PCT Publication No. WO 88/09810) or the blood-brain barrier (see, e.g. , PCT Publication No. WO 89/10134). In addition, oligonucleotides can be modified with hybridization-triggered cleavage agents (see, e.g., Krol et al, 1988, Bio/Techniques 6:958-976) or intercalating agents (see, e.g., Zon, 1988, Pharm. Res. 5:539-549). To this end, the oligonucleotide can be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.

Other agents that reduce the level of protein or activity of a CHOP protein include dominant negative mutants, as well as agents that inhibit the transcription of a CHOP gene and agents that decrease the activity of a CHOP protein. Agents may be identified as further described herein. Cells in culture may spontaneously take up agents in a sufficient amount to achieve a useful effect. Such uptake appears to be an active process requiring biochemical energy and participation of certain cell surface proteins. Uptake can also occur by pinocytosis. This route can be enhanced by incubating cells in a hypertonic medium containing a copolymer followed by resuspension of the cells in a slightly hypotonic medium to induce bursting of intracellular pinocytotic vesicles. In other cases, uptake can be assisted by use of lipids, liposomes, or polyalkyloxy copolymers, by electroporation, or by streptolysin O treatment to permeabilize the cell membrane. Cells in vivo often take up copolymers more readily than do cultured cells. Potential sites of the target RNA are those open for binding of functional complexes of proteins, and additional sites which are otherwise open for copolymer binding. Such sites can be identified using ribonuclease H (RNase H), an enzyme which cleaves RNA that is hybridized to DNA. By adding DNA oligonucleotides, singly or in mixtures, to 5'- radiophosphorus-labeled RNA in the presence of ribonuclease H, the sites on the RNA where oligonucleotides and other copolymers hybridize are identified after gel electrophoresis of the RNA and autoradiography.

Inhibitors of the invention also include reverse gene constructs which are introduced into a cell as a nucleic acid construct which is subsequently transcribed into an RNA molecule which inhibits CHOP expression after specific hybridization.

An additional strategy for inhibiting CHOP activity includes the use of an antibody or antibody fragment as an inhibitor of a CHOP protein. As is known in the art, binding of a blocking antibody or antibody fragment specifically to its target inhibits function of that target. Antibodies or antibody fragments may be used to bind directly to CHOP protein and thereby inhibit CHOP activity. Upstream positive regulators of CHOP may be targeted with an antibody or antibody fragment for indirectly reducing CHOP activity.

The terms "antibody" and "antibody substance" as used interchangeably herein refer to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds an antigen, such as a CHOP polypeptid. A molecule which specifically binds to a given polypeptide of the invention is a molecule which binds the polypeptide, but does not substantially bind other molecules in a sample, e.g., a biological sample, which naturally contains the polypeptide. Examples of immunologically active portions of immunoglobulin molecules include F(ab) and F(ab')₂ fragments which can be generated by treating the antibody with an enzyme such as pepsin. The invention provides polyclonal and monoclonal antibodies. The term "monoclonal antibody" or "monoclonal antibody composition", as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope. Polyclonal antibodies can be prepared as described above by immunizing a suitable subject with a polypeptide of the invention as an immunogen. The antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized polypeptide. If desired, the antibody molecules can be harvested or isolated from the subject {e.g., from the blood or serum of the subject) and further purified by well-known techniques, such as protein A chromatography to obtain the IgG fraction. At an appropriate time after immunization, e.g., when the specific antibody titers are highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein (1975) Nature 256:495- 497, the human B cell hybridoma technique (see Kozbor et al, 1983, Immunol. Today 4:72), the EBV-hybridoma technique (see Cole et al, pp. 77-96 hi Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., 1985) or trioma techniques. The technology for producing hybridomas is well known (see generally Current Protocols in Immunology, Coligan et al. ed., John Wiley & Sons, New York, 1994). Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supeniatants for antibodies that bind the polypeptide of interest, e.g., using a standard ELISA assay.

Alternative to preparing monoclonal antibody-secreting hybridomas, a monoclonal antibody directed against a polypeptide, e.g., a CHOP polypeptide, can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with the polypeptide of interest. Kits for generating and screening phage display libraries are commercially available {e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene SurfZAP Phage Display Kit, Catalog No. 240612). Additionally, examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, U.S. Patent No. 5,223,409; PCT Publication No. WO 92/18619; PCT Publication No. WO 91/17271; PCT Publication No. WO 92/20791; PCT Publication No. WO 92/15679; PCT Publication No. WO 93/01288; PCT Publication No. WO 92/01047; PCT Publication No. WO 92/09690; PCT Publication No. WO 90/02809; Fuchs et al.

(1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas 3:81- 85; Huse et al. (1989) Science 246:1275- 1281; Griffiths et al. (1993) EMBO J. 12:725-734. Additionally, recombinant antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in PCT Publication No. WO 87/02671; European Patent Application 184,187; European Patent Application 171,496; European Patent Application 173,494; PCT Publication No. WO 86/01533; U.S. Patent No. 4,816,567; European Patent Application 125,023; Better et al. (1988) Science 240:1041-1043; Liu et al. (1987) Proc. Natl. Acad. ScL USA 84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521- 3526; Sun et al. (1987) Proc. Natl. Acad. Sd. USA 84:214- 218; Nishimura et al. (1987) Cancer Res. 47:999-1005; Wood et al. (1985) Nature

314:446-449; and Shaw et al. (1988) J. Natl. Cancer Inst. 80:1553-1559); Morrison (1985) Science 229:1202-1207; Oi et al. (1986) Bio/Techniques 4:214; U.S. Patent 5,225,539; Jones et al. (1986) Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; and Beidler et al. (1988) J. Immunol. 141:4053-4060. Completely human antibodies are particularly desirable for therapeutic treatment of human subjects. Such antibodies can be produced using transgenic mice which are incapable of expressing endogenous immunoglobulin heavy and light chains genes, but which can express human heavy and light chain genes. The transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a CHOP polypeptide. Monoclonal antibodies directed against the antigen can be obtained using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar (1995) Int. Rev. Immunol. 13:65-93). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., U.S. Patent 5,625,126; U.S. Patent 5,633,425; U.S. Patent 5,569,825; U.S. Patent 5,661,016; and U.S. Patent 5,545,806. hi addition, companies such as Abgenix, Inc. (Freemont, CA), can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above.

Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as "guided selection." In this approach a selected non-human monoclonal antibody, e.g., a murine antibody, is used to guide the selection of a completely human antibody recognizing the same epitope (Jespers et al., 1994, Bio/technology 12:899- 903).

An inhibitor of CHOP may be packaged alone or in combination with other agents and may further be packaged in dosage forms. Such agents include both active and inert agents such as a carrier that allows administration of the inhibitor as a tablet, capsule, or implant. Therapeutically, delivery of an inhibitor of CHOP is not to be limited to any particular route. Delivery of the inhibitor may be achieved orally, intranasally, peritoneally, subcutaneously, or parenterally. In a method of the present invention, an inhibitor composition effective to prevent or reduce CHOP levels and/or activity may be administered to a mammal in a therapeutically effective amount . The inhibitor composition may be administered alone or in combination with other therapies and may be delivered systemically or locally to the mammal in need of such treatment.

In mammals, the inhibitor or agent that reduces the level of protein or activity of a CHOP protein may be introduced into the cell by methods of gene therapy, which are known in the art. In such methods, a nucleic acid encoding a biologically active agent such as a peptide, polypeptide, protein, or RNA is delivered to a cell in a form which allows its entry into the cell and allows it to encode for the biologically active inhibitor such as the peptide, polypeptide, protein, or RNA, sufficient to prevent or reduce CHOP levels or activity in the cell. Retroviral, adenoviral, or other viral vector may be used as a gene transfer delivery system using standard delivery methods known in the art. Non- viral carriers such as liposomes may also be employed for gene transfer delivery of an inhibitor composition effective to prevent or reduce CHOP levels or activity in the cell.

For in vitro, ex vivo, and in vivo applications, a nucleic acid encoding a biologically active inhibitor such as a peptide, polypeptide, protein, or RNA may be expressed from a suitable promoter. Expression of the encoded inhibitor may be constitutive. Alternatively, the expression of the encoded inhibitor may be regulated by a condition-, tissue-, or cell- specific promoter or enhancer such that expression of the biologically active inhibitor may be preferentially directed to desired cells and/or for a specified time. For example, wherein the desired cells to be contacted with the inhibitor are beta cells, expression of a biologically active inhibitor may be controlled with the use of a beta-cell specfϊc promoter. An inhibitor composition delivered systemically can thus be specifically activated only in beta cells within the mammal. An inhibitor composition that is constitutively expressed may be delivered locally, if desired.

Exemplary screening assays

The participation of CHOP in mediating glucose tolerance may be exploited for identifying pharmaceuticals useful in the treatment of diseases or conditions relating to glucose intolerance and/or insulin resistance and more specifically relating to type 2 diabetes. As such, provided herein are methods for identifying an agent, e. g., a compound that reduces glucose intolerance in a mammalian beta cell. In one aspect this method comprises contacting a mammalian cell that expresses CHOP with a candidate compound to be screened for an activity of reducing glucose intolerance; determining the activity of CHOP in the cell relative to a similar cell that has not been contacted with the candidate compound; and identifying the compound as a compound that reduces glucose intolerance in a mammalian β cell, if the activity of CHOP in step (b) is reduced in the cell that has been contacted with the candidate compound relative the identical cell that has not been contacted with the candidate compound. The method may further comprise subjecting the test agent to a glucose intolerance test.

In another aspect, a method for identifying a compound that reduces glucose intolerance in a mammalian β cell comprises subjecting a test agent that interacts with a CHOP protein and/or reduces the level of protein or activity of a CHOP protein to an in vitro or in vivo model of glucose intolerance. The test agent that inhibits glucose intolerance in the model of glucose intolerance is a compound that reduces glucose intolerance in a mammalian β cell.

In yet another aspect this method comprises contacting a CHOP protein or a functional homolog thereof with a test agent; determining whether the test agent interacts with a CHOP protein and/or reduces the level of protein or activity of a CHOP protein; and subjecting the test agent to an in vitro or in vivo glucose intolerance test, wherein a test agent that interacts with a CHOP protein or functional homolog thereof and reduces glucose intolerance in the glucose intolerance test is a compound that reduces glucose intolerance in a mammalian β cell. Determining whether the test agent interacts with a CHOP protein may comprise labeling CHOP or the test agent with a detectable substance (e.g., a radiolabel), isolating the non-labeled CHOP protein or test agent, and quantitating the amount of detectable substance that has become associated with the non-labeled CHOP protein or test agent. Alternatively in vitro cell culture assays may be used such as a two-hybrid assay that allow identification of an interaction between an expressed CHOP protein and another expressed candidate test agent.

A person of skill in the art will understand that instead of using a CHOP protein itself in a screening assay, a functional homolog of a CHOP protein may be used. A "functional homolog of a CHOP protein" of a "functional analog of a CHOP protein" refers to a protein that shares a certain similarity with a CHOP protein and which has at least one activity of a CHOP protein. A functional homolog may be a homolog that is at least about 70%, 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of a CHOP protein or a portion thereof, such as the active domain. A functional homolog may also be a homolog that is encoded by a nucleic acid comprising a nucleotide sequence that is at least about 70%, 80%, 90%, 95%, 98%, or 99% identical to the nucleotide sequence of a nucleic acid encoding a CHOP protein or a portion thereof. Yet other functional homo logs are those that are encoded by nucleic acids that hybridize to a nucleic acid that encodes a CHOP protein or a portion thereof under stringent, medium or mild hybridization conditions (e.g., comprising a hybridization and/or wash in 2, 3, 5 or 6 x SSC).

To determine the percent identity of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e. , % identity = # of identical positions/total # of positions (e.g., overlapping positions) xlOO). In one embodiment the two sequences are the same length.

The determination of percent identity between two sequences can be accomplished using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. ScL USA 87:2264-2268, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. ScL USA 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul, et al. (1990) J. MoI. Biol. 215:403-410. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to a nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to a protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-Blast can be used to perform an iterated search which detects distant relationships between molecules. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g. , XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov. Another preferred, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, (1988) Comput Appl Biosci, 4:11-7. Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAMl 20 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Yet another useful algorithm for identifying regions of local sequence similarity and alignment is the FASTA algorithm as described in Pearson and Lipman (1988) Proc. Natl. Acad. Sd. USA 85:2444-2448. When using the FASTA algorithm for comparing nucleotide or amino acid sequences, a PAM120 weight residue table can, for example, be used with a &-tuple value of 2.

The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, only exact matches are counted.

A functional homolog may be a portion of a CHOP protein comprising essentially a biologically active domain, e.g., that comprising the DNA binding activity of the protein, the transcriptional activity, or a dimerization domain, or a homolog thereof. A functional homolog may also be a protein comprising a conserved domain that is found in CHOP proteins of various species. For example, the amino acid sequence of CHOP proteins of various species may be aligned and conserved domains identified. These conserved domains are likely to be biologically active domains. An exemplary functional analog of a human CHOP protein comprises, consists essentially of, or consists of amino acids 101-156 of SEQ ID NO: 2 (which correspond to the basic region leucine zipper, i.e., the basic domain that binds DNA and the leucine zipper that mediates interaction with other proteins). A functional homolog may also differ from a naturally-occurring CHOP protein or fragment thereof by one or more conservative amino acid substitutions. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains {e.g., lysine, arginine, histidine), acidic side chains {e.g., aspartic acid, glutamic acid), uncharged polar side chains {e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains {e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains {e.g., threonine, valine, isoleucine) and aromatic side chains {e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity. Following mutagenesis, the encoded protein can be expressed recombinantly and the activity of the protein can be determined.

Variants of a protein, e.g., a CHOP protein that function as either agonists (e.g., mimetics), for use, e.g., in screening assays, or as antagonists, for use, e.g., as inhibitors of CHOP, can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of the protein of the invention for agonist or antagonist activity. In one embodiment, a variegated library of variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A variegated library of variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential protein sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins {e.g. , for phage display). There are a variety of methods which can be used to produce libraries of potential variants of the polypeptides of the invention from a degenerate oligonucleotide sequence. Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang, 1983, Tetrahedron 39:3; Itakura et al, 1984, Anna. Rev. Biochem. 53:323; Itakura et al, 1984, Science 198:1056; Ike et al, 1983 Nucleic Acid Res. 11:477).

In addition, libraries of fragments of the coding sequence of a CHOP polypeptide can be used to generate a variegated population of polypeptides for screening and subsequent selection of variants. For example, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of the coding sequence of interest with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with Sl nuclease, and ligating the resulting fragment library into an expression vector. By this method, an expression library can be derived which encodes amino terminal and internal fragments of various sizes of the CHOP protein. Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property. The most widely used techniques, which are amenable to high throughput analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected. Recursive ensemble mutagenesis (REM), a technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify variants of a protein of the invention (Arkin and Yourvan, 1992, Proc. Natl. Acad. ScL USA 59:7811-7815; Delgrave et al, 1993, Protein Engineering 6(3):327- 331).

A variety of cell types are suitable for use in determining whether the test agent interacts with a CHOP protein or whether the test agent reduces the level of protein or activity of CHOP. In one embodiment, CHOP is endogenously expressed by the cell. In another embodiment, CHOP is expressed exogenously from an exogenous construct.

Standard methods for measuring the levels and/or activity of CHOP are well known in the art and may be used in conjunction with the present invention for such purposes.

Reporter genes are known in the art and may be used for determining whether the test agent reduces the levels of CHOP protein and/or activity such as by reducing CHOP gene expression. In this embodiment, a CHOP regulatory sequence or sequences (e.g. promoter, enhancer sequences) are operatively linked to a reporter gene. A variety of reporter genes are known in the art and are suitable for such uses. Examples of such reporter genes include those which encode chloramphenicol acetyltransferase, beta- galactosidase, alkaline phosphatase or luciferase. The reporter genes may be used in cell culture or in cell-free assays.

Cell-free assays may be used to determine whether the agent reduces the levels of CHOP protein and/or activity. As an example, an agent's ability to inhibit CHOP expression may be monitored in an in vitro transcription assay. An agent is identified as an inhibitor if CHOP expression is reduced in the presence of the agent relative to control reaction wherein the agent is not present. Similarly, CHOP protein activity may be assayed in the presence and absence of a test agent, such as in a binding reaction. In this assay, CHOP protein binding to a binding partner is assayed in the presence and absence of a test agent. An agent is identified as an inhibitor if CHOP binding is modulated in the presence of the agent relative to control reaction wherein the agent is not present. As an example, an agent which decreases the affinity of endogenous CHOP protein to one of it is biologically active ligands, or alternatively increase the affinity of endogenous CHOP protein to a negative regulator, relative to control binding reactions wherein the agent is not present, may be considered an agent that reduces the activity of CHOP.

In such cell-free assays as described herein that utilize CHOP protein or nucleic acid, the protein or nucleic acid is preferably isolated or substantially pure, although this is not a requirement, so long as any impurities do not effect assay detection. Test agents, e.g., compounds, may be obtained from any available source, including systematic libraries of natural and/or synthetic agents. Test agents may also be obtained by any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone which are resistant to enzymatic degradation but which nevertheless remain bioactive; see, e.g., Zuckermann et al., 1994, J. Med. Chern. 37:2678-85); spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the 'one-bead one-agent' library method; and synthetic library methods using affinity chromatography selection. The biological library and peptoid library approaches are limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of agents (Lam, 1997, Anticancer Drug D ^]es. 12:145).

Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al. (1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; and in Gallop et al. (1994) J. Med. Chem. 37:1233. Libraries of agents may be presented in solution {e.g., Houghten, 1992, Biotechniques 13:412-421), or on beads (Lam, 1991, Nature 354:82-84), chips (Fodor, 1993, Nature 364:555-556), bacteria and/or spores, (Ladner, USP 5,223,409), plasmids (Cull et al, 1992, Proc Natl Acad Sd USA 89:1865-1869) or on phage (Scott and Smith, 1990, Science 249:386-390; Devlin, 1990, Science 249:404-406; Cwirla et al, 1990, Proc. Natl. Acad. ScL 87:6378-6382; Felici, 1991, J. MoI. Biol. 222:301-310; Ladner, supra.). The invention provides assays for screening candidate or test agents which bind to a

CHOP protein or biologically active portion thereof. Determining the ability of the test agent to directly bind to a CHOP protein can be accomplished, for example, by coupling the agent with a radioisotope or enzymatic label such that binding of the agent to the CHOP protein can be determined by detecting the labeled agent in a complex. For example, agents can be labeled with ¹²⁵1, ³⁵S, ¹⁴C, or ³H, either directly or indirectly, and the radioisotope detected by direct counting of radioemission or by scintillation counting. Alternatively, assay components can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product. In another embodiment, the invention provides assays for screening candidate or test agents which modulate the activity of a CHOP protein or a biologically active portion thereof. A CHOP protein can, in vivo, interact with one or more molecules, such as, but not limited to, peptides, proteins, cofactors and nucleic acids. For the purposes of this discussion, such molecules are referred to herein as "binding partners." Assays may be devised for the purpose of identifying agents which modulate {e.g., affect either positively or negatively) interactions between a CHOP protein and one or more of its binding partners. Such agents can include, but are not limited to, molecules such as antibodies, peptides, hormones, oligonucleotides, nucleic acids, and analogs thereof. Such agents may also be obtained from any available source, including systematic libraries of natural and/or synthetic agents.

The basic principle of the assay systems used to identify agents that interfere with the interaction between the CHOP protein and its binding partner involves preparing a reaction mixture containing the CHOP protein and its binding partner under conditions and for a time sufficient to allow the two products to interact and bind, thus forming a complex. In order to test an agent for inhibitory activity, the reaction mixture is prepared in the presence and absence of the test agent. The test agent can be initially included in the reaction mixture, or can be added at a time subsequent to the addition of the CHOP protein and its binding partner. Control reaction mixtures are incubated without the test agent or with a placebo. The formation of any complexes between the CHOP protein and its binding partner is then detected. The formation of a complex in the control reaction, but less or no such formation in the reaction mixture containing the test agent, indicates that the agent interferes with the interaction of the CHOP protein and its binding partner. Conversely, the formation of more complex in the presence of agent than in the control reaction indicates that the agent may enhance interaction of the CHOP protein and its binding partner.

The assay for agents that interfere with the interaction of the CHOP protein with its binding partner may be conducted in a heterogeneous or homogeneous format. Heterogeneous assays involve anchoring either the CHOP protein or its binding partner onto a solid phase and detecting complexes anchored to the solid phase at the end of the reaction. In homogeneous assays, the entire reaction is carried out in a liquid phase. In either approach, the order of addition of reactants can be varied to obtain different information about the agents being tested. For example, test agents that interfere with the interaction between the CHOP proteins and the binding partners (e.g., by competition) can be identified by conducting the reaction in the presence of the test substance, i.e., by adding the test substance to the reaction mixture prior to or simultaneously with the CHOP protein and its interactive binding partner. Alternatively, test agents that disrupt preformed complexes, e.g., agents with higher binding constants that displace one of the components from the complex, can be tested by adding the test agent to the reaction mixture after complexes have been formed. The various formats are briefly described below.

In a heterogeneous assay system, either the CHOP protein or its binding partner is anchored onto a solid surface or matrix, while the other corresponding non-anchored component may be labeled, either directly or indirectly. In practice, microtitre plates are often utilized for this approach. The anchored species can be immobilized by a number of methods, either non-covalent or covalent, that are typically well known to one who practices the art. Non-covalent attachment can often be accomplished simply by coating the solid surface with a solution of the CHOP protein or its binding partner and drying. Alternatively, an immobilized antibody specific for the assay component to be anchored can be used for this purpose. Such surfaces can often be prepared in advance and stored. In related embodiments, a fusion protein can be provided which adds a domain that allows one or both of the assay components to be anchored to a matrix. For example, glutathione- S-transferase/CHOP protein fusion proteins or glutathione-S- transferase/binding partner can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtiter plates, which are then combined with the test agent or the test agent and either the non-adsorbed CHOP protein or its binding partner, and the mixture incubated under conditions conducive to complex formation (e.g., physiological conditions). Following incubation, the beads or microtiter plate wells are washed to remove any unbound assay components, the immobilized complex assessed either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of CHOP protein binding or activity determined using standard techniques.

Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention. For example, either a CHOP protein or a CHOP protein binding partner can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated CHOP protein or target molecules can be prepared from biotin-NHS (N- hydroxy-succinimide) using techniques known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, IL), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). In certain embodiments, the protein-immobilized surfaces can be prepared in advance and stored.

In order to conduct the assay, the corresponding partner of the immobilized assay component is exposed to the coated surface with or without the test agent. After the reaction is complete, unreacted assay components are removed (e.g., by washing) and any complexes formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the non-immobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the non-immobilized component is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the initially non-immobilized species (the antibody, in turn, can be directly labeled or indirectly labeled with, e.g., a labeled anti-Ig antibody). Depending upon the order of addition of reaction components, test agents which modulate (inhibit or enhance) complex formation or which disrupt preformed complexes can be detected. In an alternate embodiment of the invention, a homogeneous assay may be used.

This is typically a reaction, analogous to those mentioned above, which is conducted in a liquid phase in the presence or absence of the test agent. The formed complexes are then separated from unreacted components, and the amount of complex formed is determined. As mentioned for heterogeneous assay systems, the order of addition of reactants to the liquid phase can yield information about which test agents modulate (inhibit or enhance) complex formation and which disrupt preformed complexes.

In such a homogeneous assay, the reaction products may be separated from unreacted assay components by any of a number of standard techniques, including but not limited to: differential centrifugation, chromatography, electrophoresis and immunoprecipitation. In differential centrifugation, complexes of molecules may be separated from uncomplexed molecules through a series of centrifugal steps, due to the different sedimentation equilibria of complexes based on their different sizes and densities (see, for example, Rivas, G., and Minton, A.P., Trends Biochem Sd 1993 Aug;18(8):284- 7). Standard chromatographic techniques may also be utilized to separate complexed molecules from uncomplexed ones. For example, gel filtration chromatography separates molecules based on size, and through the utilization of an appropriate gel filtration resin in a column format, for example, the relatively larger complex may be separated from the relatively smaller uncomplexed components. Similarly, the relatively different charge properties of the complex as compared to the uncomplexed molecules may be exploited to differentially separate the complex from the remaining individual reactants, for example through the use of ion-exchange chromatography resins. Such resins and chromatographic techniques are well known to one skilled in the art (see, e.g., Heegaard, 1998, JMoI. Recognit. 11 : 141-148; Hage and Tweed, 1997, J. Chromatogr. B. Biomed. ScL Appl,

699:499-525). Gel electrophoresis may also be employed to separate complexed molecules from unbound species (see, e.g., Ausubel et al (eds.), In: Current Protocols in Molecular Biology, J. Wiley & Sons, New York. 1999). In this technique, protein or nucleic acid complexes are separated based on size or charge, for example. In order to maintain the binding interaction during the electrophoretic process, nondenaturing gels in the absence of reducing agent are typically preferred, but conditions appropriate to the particular interactants will be well known to one skilled in the art. Immunoprecipitation is another common technique utilized for the isolation of a protein-protein complex from solution (see, e.g., Ausubel et al (eds.), In: Current Protocols in Molecular Biology, J. Wiley & Sons, New York. 1999). In this technique, all proteins binding to an antibody specific to one of the binding molecules are precipitated from solution by conjugating the antibody to a polymer bead that may be readily collected by centrifugation. The bound assay components are released from the beads (through a specific proteolysis event or other technique well known in the art which will not disturb the protein-protein interaction in the complex), and a second immunoprecipitation step is performed, this time utilizing antibodies specific for the correspondingly different interacting assay component. In this manner, only formed complexes should remain attached to the beads. Variations in complex formation in both the presence and the absence of a test agent can be compared, thus offering information about the ability of the agent to modulate interactions between the CHOP protein and its binding partner.

Also within the scope of the present invention are methods for direct detection of interactions between the CHOP protein and its natural binding partner and/or a test agent in a homogeneous or heterogeneous assay system without further sample manipulation. For example, the technique of fluorescence energy transfer may be utilized (see, e.g., Lakowicz et al, U.S. Patent No. 5,631,169; Stavrianopoulos et al, U.S. Patent No. 4,868,103). Generally, this technique involves the addition of a fluorophore label on a first 'donor' molecule (e.g., CHOP protein or test agent) such that its emitted fluorescent energy will be absorbed by a fluorescent label on a second, 'acceptor' molecule (e.g., CHOP protein or test agent), which in turn is able to fluoresce due to the absorbed energy. Alternately, the 'donor' protein molecule may simply utilize the natural fluorescent energy of tryptophan residues. Labels are chosen that emit different wavelengths of light, such that the 'acceptor' molecule label may be differentiated from that of the 'donor'. Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, spatial relationships between the molecules can be assessed. In a situation in which binding occurs between the molecules, the fluorescent emission of the 'acceptor' molecule label in the assay should be maximal. An FET binding event can be conveniently measured through standard fiuorometric detection means well known in the art (e.g., using a fluorimeter). A test substance which either enhances or hinders participation of one of the species in the preformed complex will result in the generation of a signal variant to that of background. In this way, test substances that modulate interactions between a CHOP protein and its binding partner can be identified in controlled assays.

In another embodiment, modulators of CHOP protein expression are identified in a method wherein a cell is contacted with a candidate agent and the expression of mRNA or protein, corresponding to a CHOP protein in the cell, is determined. The level of expression of mRNA or protein in the presence of the candidate agent is compared to the level of expression of mRNA or protein in the absence of the candidate agent. The candidate agent can then be identified as a modulator of CHOP protein expression based on this comparison. For example, when expression of CHOP mRNA or protein is greater (statistically significantly greater) in the presence of the candidate agent than in its absence, the candidate agent is identified as a stimulator of CHOP mRNA or protein expression. Conversely, when expression of CHOP mRNA or protein is less (statistically significantly less) in the presence of the candidate agent than in its absence, the candidate agent is identified as an inhibitor of CHOP mRNA or protein expression. The level of CHOP mRNA or protein expression in the cells can be determined by methods described herein for detecting CHOP mRNA or protein. In another aspect, the invention pertains to a combination of two or more of the assays described herein. For example, a modulating agent can be identified using a cell-based or a cell free assay, and the ability of the agent to modulate the activity of a CHOP protein can be further confirmed in vivo, e.g., in a whole animal model for glucose intolerance or diseases relating thereto, e.g., diabetes. In vivo glucose intolerance tests are known in the art and may comprise providing an animal in a fasting state, wherein the animal is at least somewhat glucose intolerant relative to a normal animal in a fasting state, and administering a known amount of glucose to the glucose intolerant animal. A glucose intolerant animal is an animal that has elevated levels of glucose in a fasting state, relative to a normal animal in the same state. Blood is obtained from the animal in a fasting state and the level of glucose measured to provide a reference point. Glucose is then administered to the animal, for example, orally or intravenously. Blood is obtained subsequent to glucose administration, and may be obtained at multiple, measured time points from glucose administration, such as every 30 to 60 minutes after the glucose is administered for up to 3 hours. The test agent may be administered to the animal prior to, subsequent to, or simultaneous with the glucose administration. The test agent may be administered intermittently or continuously throughout the glucose intolerance test. Blood glucose levels above normal at the times measured is indicative of glucose intolerance. The results obtained from an animal administered the test agent may be compared to an animal to which the agent was not administered to determine whether or not the agent increases glucose tolerance (or reduces glucose intolerance) in the animal. An agent that tests positive in the glucose intolerance test is an agent that provides the animal with an increased level of glucose tolerance (or reduced level of glucose intolerance), relative to a similar animal that has not been treated with the agent. Examples of glucose intolerance test results are shown in FIGS. 5, 9, and 11.

Exemplary pharmaceutical compositions The small molecules, peptides, peptoids, peptidomimetics, polypeptides, RNA interfering agents, e.g., siRNA molecules, antibodies, ribozymes, and antisense oligonucleotides, e.g., those that reduce protein level or activity of a CHOP protein (also referred to herein as "active compounds" or "compounds") of the invention can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the small molecules, peptides, peptoids, peptidomimetics, polypeptides, RNA interfering agents, e.g., siRNA molecules, antibodies, ribozymes, or antisense oligonucleotides and a pharmaceutically acceptable carrier. As used herein the language "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions. The invention includes methods for preparing pharmaceutical compositions for treating or preventing diseases and conditions described herein. Such methods comprise formulating a pharmaceutically acceptable carrier with an agent which modulates expression or activity of a polypeptide or nucleic acid described herein. Such compositions can further include additional active agents. It is understood that appropriate doses of small molecule agents and protein or polypeptide agents depends upon a number of factors within the knowledge of the ordinarily skilled physician, veterinarian, or researcher. The dose(s) of these agents will vary, for example, depending upon the identity, size, and condition of the subject or sample being treated, further depending upon the route by which the composition is to be administered, if applicable, and the effect which the practitioner desires the agent to have. Small molecules include, but are not limited to, peptides, peptidomimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic compounds (i.e., including heteroorganic and organometallic compounds) having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds.

Exemplary doses of a small molecule include milligram or microgram amounts per kilogram of subject or sample weight (e.g. about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram).

As defined herein, a therapeutically effective amount of an RNA interfering agent, e.g., siRNA, (i.e., an effective dosage) ranges from about 0.001 to 3,000 mg/kg body weight, preferably about 0.01 to 2500 mg/kg body weight, more preferably about 0.1 to 2000, about 0.1 to 1000 mg/kg body weight, 0.1 to 500 mg/kg body weight, 0.1 to 100 mg/kg body weight, 0.1 to 50 mg/kg body weight, 0.1 to 25 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. Treatment of a subject with a therapeutically effective amount of an RNA interfering agent can include a single treatment or, preferably, can include a series of treatments. In a preferred example, a subject is treated with an RNA interfering agent in the range of between about 0.1 to 20 mg/kg body weight, one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks.

Exemplary doses of a protein or polypeptide include gram, milligram or microgram amounts per kilogram of subject or sample weight (e.g. about 1 microgram per kilogram to about 5 grains per kilogram, about 100 micrograms per kilogram to about 500 milligrams per kilogram, or about 1 milligram per kilogram to about 50 milligrams per kilogram). It is furthermore understood that appropriate doses of one of these agents depend upon the potency of the agent with respect to the expression or activity to be modulated. Such appropriate doses can be determined using the assays described herein. When one or more of these agents is to be administered to an animal (e.g. a human) in order to modulate expression or activity of a polypeptide or nucleic acid of the invention, a physician, veterinarian, or researcher can, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular animal subject will depend upon a variety of factors including the activity of the specific agent employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.

A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediamine-tetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL (BASF; Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin. Sterile injectable solutions can be prepared by incorporating an active compound

(e.g., a polypeptide or interfering RNA) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium, and then incorporating the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed.

Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches, and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as micro crystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a pressurized container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known m the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes having monoclonal antibodies incorporated therein or thereon) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811. It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

For antibodies, the preferred dosage is 0.1 mg/kg to 100 mg/kg of body weight (generally 10 mg/kg to 20 mg/kg). If the antibody is to act in the brain, a dosage of 50 mg/kg to 100 mg/kg is usually appropriate. Generally, partially human antibodies and fully human antibodies have a longer half-life within the human body than other antibodies. Accordingly, lower dosages and less frequent administration is often possible. Modifications such as lipidation can be used to stabilize antibodies and to enhance uptake and tissue penetration (e.g., into the epithelium). A method for lipidation of antibodies is described by Cruikshanlc et al. (1997) J. Acquired Immune Deficiency Syndromes and Human Retrovirology 14:193. Nucleic acid molecules can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (U.S. Patent 5,328,470), or by stereotactic injection (see, e.g., Chen et al, 1994, Proc. Natl. Acad. ScL USA 91:3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector 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 vector can be produced intact from recombinant cells, e.g. retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

The RNA interfering agents, e.g., siRNAs used in the methods of the invention can be inserted into vectors. These constructs can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Patent 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. ScL USA 91:3054-3057). The pharmaceutical preparation of the vector can include the RNA interfering agent, e.g., the siRNA vector 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 vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration. Also provided herein are kits, e.g., kits comprising one or more pharmaceutical agents for treating or preventing diseases and conditions described herein. A kit may comprise a CHOP inhibitor and optionally a device for administering the CHOP inhibitor. Other kits provided herein are kits for screening assays. Exemplary kits may comprise a CHOP protein or functional homo log thereof and optionally a binding partner. Kits may also comprise buffers.

The present description is further illustrated by the following examples, which should not be construed as limiting in any way. The contents of all cited references (including literature references, issued patents, published patent applications and GenBank Accession numbers as cited throughout this application) are hereby expressly incorporated by reference.

The practice of the present methods will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2^nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Patent No: 4,683,195; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984);

Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N. Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, VoIs. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1986).

Exemplification

Example 1. CHOP deletion protects from high-fat diet-induced diabetes in mice with defective translational control Signaling of the unfolded protein response through regulation of mRNA translation by PERK-mediated phosphorylation of eukaryotic initiation factor 2 (eIF2α) is essential to preserve the integrity of the endoplasmic reticulum (ER) and to increase insulin production to meet the demand imposed by a high-fat diet (Scheuner et al. 2005 Nature Med. 11 : 757- 764). Although mice with a heterozygous SerSIAla knock-in mutation at the regulatory eIF2q phosphorylation site (eIF2α^SA) do not have a significant phenotype, they became obese and diabetic on a high- fat diet with abnormal pancreatic beta cell function accompanied by a loss of glucose-regulated insulin secretion, accumulation of proinsulin in a distended ER lumen, and a reduced number of insulin granules. They are characterized by 40% body fat, hyperleptinemia, and a reduced metabolic rate. They are characterized by glucose intolerance, impaired glucose-stimulated insulin secretion, mild fasting hyperglycemia, beta cell ER stress, elevated fasting blood glucose levels, and proinsulin retention with the ER chaperone BiP. CHOP is one gene induced through the PERK/eIF2α phosphorylation signaling sub- pathway of the unfolded protein response. CHOP is a transcription factor homologous to C/EBP that induces a pro-apoptotic response. As shown in the attached figures, knockout of the CHOP gene prevents the development of high- fat diet-induced glucose intolerance in eIF2α^SA mice. CHOP deletion in this diabetes model is associated with significant islet hyperplasia. Interestingly, CHOP deletion actually increases obesity, but prevents beta cell failure in high- fat-fed eIF2α^SA mice.

The phenotype of the S/A high fat diet mouse is one of obesity and diabetes. In the obesity, a reduced metabolic rate is observed while food intake is not increased. The animals are glucose intolerant with impaired insulin secretion, mild hyperglycemia, and hyperinsulinemia that increases over time. Equivalents

While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

Claims

1. A method for treating or preventing a glucose intolerance related disease in a mammal, comprising administering to a mammal in need thereof a therapeutically effective amount of an inhibitor of C/EBP homologous protein (CHOP), wherein the glucose intolerance disease is not a disease resulting from a defect in a pro-insulin gene.

2. The method of claim 1, wherein the mammal has or is predisposed to a glucose intolerance related disease.

3. The method of claim 2, wherein the glucose intolerance related disease is caused or contributed to by obesity.

4. The method of claim 2, wherein the glucose intolerance related disease is caused or contributed to by a high fat diet.

5. The method of claim 3, wherein the glucose intolerance related disease is glucose intolerance or insulin-resistance.

6. The method of claim 1 , wherein the mammal is a human.

7. The method of claim 1, further comprising administering to the mammal an inhibitor of eIF2α.

8. The method of claim 1 , wherein the treatment or prevention results in islet cell hyperplasia.

9. The method of claim 1 , wherein the inhibitor of CHOP is a CHOP siRNA.

10. The method of claim 1 , wherein the inhibitor of CHOP is a nucleic acid encoding a CHOP siRNA.

11. A method for preventing or reducing glucose intolerance in a mammalian cell, comprising contacting a mammalian cell with an inhibitor of CHOP, wherein the cell is not a pancreatic β cell having a defect in a pro-insulin gene.

12. The method of claim 11 , wherein the step of contacting occurs in vitro or ex vivo.

13. The method of claim 11, wherein the step of contacting occurs in vivo.

14. The method of claim 11 , wherein the mammalian cell is a human cell.

15. The method of claim 11 , wherein the mammalian cell is a β cell.

16. The method of claim 11 , further comprising contacting the mammalian cell with an inhibitor of eIF2α.

17. The method of claim 15 , wherein the inhibitor of CHOP reduces ER stress in the β cell.

18. A method comprising contacting a β cell with a CHOP inhibitor in the cell.

19. The method of claim 18, wherein the β cell is in a donor pancreatic islet, and the method comprises contacting the donor pancreatic islet with the CHOP inhibitor.

20. A method for treating a glucose intolerance related disease in a subject, comprising a. providing a β cell; b. contacting the β cell with a CHOP inhibitor; and c. administering the β cell to a subject having a glucose intolerance related disease.

21. The method of claim 20, for transplanting a β cell from a donor to a recipient having a glucose intolerance related disease, comprising: a. obtaining a β cell from a donor; b. contacting the β cell with a CHOP inhibitor; and c. administering the β cell to a recipient having a glucose intolerance related disease.

22. The method of claim 21 , wherein the β cell is in a donor pancreatic islet and the method comprises contacting the donor pancreatic islet with the CHOP inhibitor ex vivo; and administering the pancreatic islet to the recipient.

23. The method of claim 21, wherein the glucose intolerance related disease is not a disease resulting from a defect in a pro-insulin gene.

24. The method of claim 23, wherein the glucose intolerance related disease is glucose intolerance or insulin resistance.

25. A method for identifying an agent that reduces glucose intolerance in a mammalian β cell, comprising: a. contacting a CHOP protein or a functional homolog thereof with a test agent; b. determining whether the test agent interacts with the CHOP protein and/or reduces the level of protein or activity of the CHOP protein; and c. subjecting the test agent to an in vitro or in vivo glucose intolerance test, wherein a test agent that interacts with a CHOP protein or functional homolog thereof and reduces or prevents glucose intolerance in the glucose intolerance test is an agent that reduces glucose intolerance in a mammalian β cell.

26. A method for identifying an agent that reduces glucose intolerance in a mammalian β cell, comprising subjecting a test agent to an in vitro or in vivo model of glucose intolerance, wherein the test agent interacts with a CHOP protein and/or reduces the level of protein or activity of a CHOP protein in a cell, and wherein a test agent that inhibits glucose intolerance in the model of glucose intolerance is an agent that reduces glucose intolerance in a mammalian β cell.

27. A method for identifying an agent that reduces glucose intolerance in a mammalian β cell, comprising: a. contacting a mammalian cell that expresses CHOP with a test agent; b. determining the activity or level of expression of CHOP in the cell relative to a similar cell that has not been contacted with the test agent; and c. identifying the agent as an agent that reduces glucose intolerance in a mammalian β cell, if the activity or level of expression of CHOP in step (b) is reduced in the cell that has been contacted with the test agent relative the similar cell that has not been contacted with the test agent.

28. The method of claim 27, further comprising subjecting the test agent to a glucose intolerance test.

29. The method of claim 27, wherein CHOP is a heterologous protein that is expressed from a heterologous nucleic acid that was introduced into the β cell.

30. A method for identifying an agent that reduces glucose intolerance in a mammalian β cell, comprising: a. contacting a β cell comprising a promoter of a CHOP gene operably linked to a reporter gene with a test agent; b. determining the level of expression of the reporter gene; and c. subjecting a test agent that reduces the level of expression of the reporter gene to a glucose tolerance test, wherein a lower level of expression of the reporter gene in a cell contacted with the test agent relative to a cell that was not contacted with a test agent indicates that the test agent is an agent that reduces glucose intolerance in a mammalian β cell.