WO2005124359A2 - Procede de diagnostic et de traitement du diabete et de l’insulinoresistance - Google Patents

Procede de diagnostic et de traitement du diabete et de l’insulinoresistance Download PDF

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WO2005124359A2
WO2005124359A2 PCT/US2005/021296 US2005021296W WO2005124359A2 WO 2005124359 A2 WO2005124359 A2 WO 2005124359A2 US 2005021296 W US2005021296 W US 2005021296W WO 2005124359 A2 WO2005124359 A2 WO 2005124359A2
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polypeptide
expression
protein
cells
polypeptides
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PCT/US2005/021296
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WO2005124359A3 (fr
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Shonna A. Moodie
Brian E. Lavan
Thomas A. Gustafson
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Metabolex, Inc.
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Priority to JP2007516736A priority Critical patent/JP2008502909A/ja
Publication of WO2005124359A2 publication Critical patent/WO2005124359A2/fr
Publication of WO2005124359A3 publication Critical patent/WO2005124359A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/66—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood sugars, e.g. galactose
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism

Definitions

  • Type 1 diabetes mellitus can be divided into two clinical syndromes, Type 1 and Type 2 diabetes mellitus.
  • Type 1 or insulin-dependent diabetes mellitus (IDDM)
  • IDDM insulin-dependent diabetes mellitus
  • Type 2 diabetes mellitus is a chronic autoimmune disease characterized by the extensive loss of beta cells in the pancreatic Islets of Langerhans, which produce insulin. As these cells are progressively destroyed, the amount of secreted insulin decreases, eventually leading to hyperglycemia (abnormally high level of glucose in the blood) when the amount of secreted insulin drops below the level required for euglycemia (normal blood glucose level).
  • hyperglycemia abnormally high level of glucose in the blood
  • euglycemia normal blood glucose level
  • Type 2 diabetes also referred to as non-insulin dependent diabetes mellitus (NIDDM)
  • NIDDM non-insulin dependent diabetes mellitus
  • Type 2 diabetes develops when muscle, fat and liver cells fail to respond normally to insulin. This failure to respond (called insulin resistance) may be due to reduced numbers of insulin receptors on these cells, or a dysfunction of signaling pathways within the cells, or both.
  • the beta cells initially compensate for this insulin resistance by increasing insulin output. Over time, these cells become unable to produce enough insulin to maintain normal glucose levels, indicating progression to Type 2 diabetes.
  • Type 2 diabetes is brought on by a combination of genetic and acquired risk factors - including a high-fat diet, lack of exercise, and aging.
  • Type 2 diabetes has become an epidemic, driven by increases in obesity and a sedentary lifestyle, widespread adoption of western dietary habits, and the general aging of the population in many countries.
  • an estimated 30 million people worldwide had diabetes ⁇ by 2000, this figure had increased 5-fold, to an estimated 154 million people.
  • the number of people with diabetes is expected to double between now and 2025, to about 300 million.
  • Type 2 diabetes is a complex disease characterized by defects in glucose and lipid metabolism. Typically there are perturbations in many metabolic parameters including increases in fasting plasma glucose levels, free fatty acid levels and triglyceride levels, as well as a decrease in the ratio of HDL/LDL.
  • the molecular target of TZDs is a member of the PPAR family of ligand-activated transcription factors called PPAR gamma. This transcription factor is highly expressed in adipose tissue with much lower levels being observed in muscle. Binding of TZDs to PPAR gamma in target cells and tissues such as fat and muscle brings about a change in gene expression. The link between TZD-altered gene expression in fat and muscle and increased insulin sensitivity is unknown. The present invention addresses this and other problems.
  • the present invention provides methods for identifying an agent for treating a diabetic or pre-diabetic individual.
  • the methods comprise the steps of: (i) contacting an agent to a polypeptide encoded by a polynucleotide that is substantially identical to or hybridizes to a nucleic acid encoding a polypeptide listed in Table 1 under hybridization conditions of 50% formamide, 5X SSC, and 1% SDS at 42°C followed by a wash in 0.2X SSC, and 0.1% SDS at 55°C, wherein the polypeptide optionally has the activity listed in Table 1; and (ii) selecting an agent that modulates the expression or activity of the polypeptide or that binds to the polypeptide, thereby identifying an agent for treating a diabetic or pre-diabetic individual.
  • the polypeptide comprises an amino acid sequence at least 95% identical to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 or a protein domain thereof.
  • the methods further comprise selecting an agent that modulates insulin sensitivity. Table 1: List of Polypeptides, SEQ ID numbers and Proposed Activity
  • step (ii) comprises selecting an agent that modulates expression of the polypeptide. In some embodiments, step (ii) comprises selecting an agent that modulates the activity of the polypeptide. In some embodiments, step (ii) comprises selecting an agent that specifically binds to the polypeptide. In some embodiments, the polypeptide is expressed in a cell and the cell is contacted with the agent. In some embodiments, the polypeptide comprises SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, or 38. [10] The present invention also provides methods of treating a diabetic or pre-diabetic animal.
  • the methods comprise administering to the animal a therapeutically effective amount of an agent that modulates the activity or expression of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, or 38.
  • the agent is identified by a method comprising (i) contacting an agent to a polypeptide encoded by a polynucleotide that is substantially identical to or hybridizes to a nucleic acid encoding a polypeptide listed in Table 1 under hybridization conditions of 50% formamide, 5X SSC, and 1% SDS at 42°C followed by a wash in 0.2X > ⁇ SSC, and 0.1% SDS at 55°C; and (ii) selecting an agent that modulates the expression or " >i ⁇ activity of the polypeptide or that binds to the polypeptide.
  • the polypeptide comprises an amino acid sequence at least 95% identical to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 or a protein domain thereof.
  • the agent is an antibody.
  • the antibody is a monoclonal antibody.
  • the animal is a human.
  • the present invention also provides methods of introducing an expression cassette into a cell.
  • the methods comprise introducing into the cell an expression cassette comprising a promoter operably linked to a polynucleotide encoding a polypeptide, wherein the polynucleotide is substantially identical to or hybridizes to a nucleic acid encoding a polypeptide listed in Table 1 under hybridization conditions of 50% formamide, 5X SSC, and 1% SDS at 42°C followed by a wash in 0.2X SSC, and 0.1% SDS at 55°C, and the polypeptide optionally has the activity listed in Table 1.
  • the polypeptide comprises an amino acid sequence at least 95% identical to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 or a protein domain thereof.
  • the polypeptide comprises SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, or 38.
  • the cell is selected from the group consisting of an adipocyte and a skeletal muscle cell.
  • the methods further comprising introducing the cell into a human.
  • the human is diabetic.
  • the human is prediabetic.
  • the cell is from the human.
  • the present invention also provides methods of diagnosing an individual who has Type 2 diabetes or is prediabetic.
  • the method comprises, detecting in a sample from the individual the level of a polypeptide or the level of a polynucleotide encoding the polypeptide, wherein the polynucleotide is substantially identical to or hybridizes to a nucleic acid encoding a polypeptide listed in Table 1 under hybridization conditions of 50% formamide, 5X SSC, and 1% SDS at 42°C followed by a wash in 0.2X SSC, and 0.1% SDS at 55°C, wherein a modulated level of the polypeptide or polynucleotide in the sample compared to a level of the polypeptide or polynucleotide in either a lean individual or a previous sample from the individual indicates that the individual is diabetic or prediabetic, and the polypeptide optionally has the activity listed in Table 1.
  • the detecting step comprises contacting the sample with an antibody that specifically binds to the polypeptide.
  • the amino acid sequence comprises SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, or 38.
  • the detecting step comprises quantifying mRNA encoding the polypeptide.
  • the mRNA is reverse transcribed and amplified in a polymerase chain reaction.
  • the sample is a blood, urine or tissue sample.
  • the present invention also provides isolated nucleic acids that is substantially identical to or hybridizes to a nucleic acid encoding a polypeptide listed in Table 1 under hybridization conditions of 50% formamide, 5X SSC, and 1% SDS at 42°C followed by a wash in 0.2X SSC, and 0.1% SDS at 55°C.
  • the nucleic acid comprises SEQ ID NOs: 1, 3,
  • the nucleic acid encodes SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, or 38.
  • the present invention also provides an expression cassette comprising a heterologous promoter operably linked to a polynucleotide that is substantially identical to or hybridizes to a nucleic acid encoding a polypeptide listed in Table 1 under hybridization conditions of 50% formamide, 5X SSC, and 1% SDS at 42°C followed by a wash in 0.2X SSC, and 0.1% SDS at 55°C.
  • the present invention also provides host cells transfected with a polynucleotide that is substantially identical to or hybridizes to a nucleic acid encoding a polypeptide listed in Table 1 under hybridization conditions of 50% formamide, 5X SSC, and 1% SDS at 42°C followed by a wash in 0.2X SSC, and 0.1% SDS at 55°C.
  • the host cell is a human cell.
  • the host cell is a bacterium.
  • the cell is selected from the group consisting of an adipocyte and a skeletal muscle cell.
  • the present invention also provides isolated polypeptides comprising an amino acid sequence at least 70% identical to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, or 38.
  • the polypeptide comprises SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38.
  • the present invention also provides pharmaceutical compositions comprising polypeptides comprising an amino acid sequence at least 70% identical to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, or 38 or fragments thereof, and a pharmaceutically-acceptable excipient.
  • Insulin sensitivity refers to the ability of a cell or tissue to respond to insulin. Responses include, e.g., glucose uptake of a cell or tissue in response to insulin stimulation. Sensitivity can be determined at an organismal, tissue or cellular level. For example, blood or urine glucose levels following a glucose tolerance test are indicative of insulin sensitivity. Other methods of measuring insulin sensitivity include, e.g., measuring glucose uptake (see, e.g., Garcia de Herreros, A., and Bi nbaum, M. J. J. Biol. Chem. 264, 19994-19999 (1989); Klip, A., Li, G., and Logan, WJ. Am. J. Physiol.
  • lipid metabolism refers to the in vivo process of catabolism (decomposition) and anabolism (accumulation) of lipids (e.g., triglycerides derived from food) and is intended to include, in the broad sense, reactions for transforming lipids into energy, biosynthesis of fatty acids, acylglycerol, phospholipid metabolism and cholesterol metabolism.
  • Activity of a polypeptide of the invention refers to structural, regulatory, or biochemical functions of a polypeptide in its native cell or tissue. Examples of activity of a polypeptide include both direct activities and indirect activities.
  • Exemplary direct activities are the result of firect interaction with the polypeptide, , e.g., enzymatic activity, ligand binding, production or depletion of second messengers (e.g., cAMP, cGMP, IP 3 , DAG, or Ca 2+ ), ion flux, phosphorylation levels, transcription levels, and the like.
  • Exemplary indirect activities are observed as a change in phenotype or response in a cell or tissue to a polypeptide's directed activity, e.g., modulating insulin sensitivity of a cell as a result of the interaction of the polypeptide with other cellular or tissue components.
  • Predisposition for diabetes occurs in a person when the person is at high risk for developing diabetes.
  • a number of risk factors are known to those of skill in the art and include: genetic factors (e.g., carrying alleles that result in a higher occurrence of diabetes than in the average population or having parents or siblings with diabetes); overweight (e.g., body mass index (BMI) greater or equal to 25 kg/m 2 ); habitual physical inactivity, race/ethnicity (e.g., African- American, Hispanic-American, Native Americans, Asian- Americans, Pacific Islanders); previously identified impaired fasting glucose or impaired glucose tolerance, hypertension (e.g., greater or equal to 140/90 mmHg in adults); HDL cholesterol less than or equal to 35 mg/dl; triglyceride levels greater or equal to 250 mg/dl; a history of gestational diabetes or delivery of a baby over nine pounds; and/or polycystic ovary syndrome.
  • genetic factors e.g., carrying alleles that result in a higher occurrence of diabetes than in the average population or having parents or siblings with diabetes
  • overweight e.g
  • a “2 hour PG” refers to the level of blood glucose after challenging a patient to a glucose load containing the equivalent of 75g anhydrous glucose dissolved in water. The overall test is generally referred to as an oral glucose tolerance test (OGTT).
  • OGTT oral glucose tolerance test
  • the level of a polypeptide in a lean individual can be a reading from a single individual, but is typically a statistically relevant average from a group of lean individuals.
  • the level of a polypeptide in a lean individual can be represented by a value, for example in a computer program.
  • a "pre-diabetic individual,” when used to compare with a sample from a patient, refers to an adult with a fasting blood glucose level greater than 110 mg/dl but less than 126 mg/dl or a 2 hour PG reading of greater than 140 mg/dl but less than 200mg/dl.
  • a “diabetes-related nucleic acid” or “diabetes-related polynucleotide” (also referred to as a “nucleic acid of the invention” or a “polynucleotide of the invention”) of the invention is a subsequence or full-length polynucleotide sequence of a gene that encodes a polypeptide, whose activity modulates diabetes or insulin sensitivity, or whose presence or absence is indicative of diabetes or altered insulin sensitivity.
  • nucleic acids of the invention include those sequences substantially identical to SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, or 37 or encode polypeptides substantially identical to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, or 38.
  • An "agonist” refers to an agent that binds to, stimulates, increases, activates, facilitates, enhances activation, sensitizes or up regulates the activity or expression of a polypeptide of the invention.
  • an "antagonist” refers to an agent that binds to, partially or totally blocks stimulation, decreases, prevents, delays activation, inactivates, desensitizes, or down regulates the activity or expression of a polypeptide of the invention.
  • Antibody refers to a polypeptide substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof which specifically bind and recognize an analyte (antigen).
  • the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda.
  • An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the terms variable light chain (VL) and variable heavy chain (N ⁇ ) refer to these light and heavy chains respectively.
  • Antibodies exist, e.g., as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases.
  • pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)' 2; a dimer of Fab which itself is a light chain joined to V H -C H I by a disulfide bond.
  • the F(ab)' 2 may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)' 2 dimer into an Fab' monomer.
  • the Fab' monomer is essentially an Fab with part of the hinge region (see, Paul (Ed.) Fundamental Immunology, Third Edition, Raven Press, ⁇ Y (1993)). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by utilizing recombinant D ⁇ A methodology. Thus, the term antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant D ⁇ A methodologies (e.g., single chain Fv).
  • peptidomimetic and “mimetic” refer to a synthetic chemical compound that has substantially the same structural and functional characteristics of the antagonists or agonists of the invention.
  • Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of the template peptide. These types of non-peptide compound are termed “peptide mimetics” or “peptidomimetics” (Fauchere, J. Adv. Drug Res. 15:29 (1986); Neber and Freidinger TINS p. 392 (1985); and Evans et al. J. Med. Chem. 30:1229 (1987), which are incorporated herein by reference).
  • Peptide mimetics that are structurally similar to therapeutically useful peptides may be used to produce an equivalent or enhanced therapeutic or prophylactic effect.
  • a paradigm polypeptide i.e., a polypeptide that has a biological or pharmacological activity
  • a linkage selected from the group consisting of, e.g., -CH2NH-, -CH2S-, -CH2-CH2-, -
  • the mimetic can be either entirely composed of synthetic, non-natural analogues of amino acids, or, is a chimeric molecule of partly natural peptide amino acids and partly non-natural analogs of amino acids.
  • the mimetic can also incorporate any amount of natural amino acid conservative substitutions as long as such substitutions also do not substantially alter the mimetic' s structure and/or activity.
  • a mimetic composition is within the scope of the invention ifit is capable of carrying out the binding or other activities of an agonist or antagonist of a polypeptide of the invention.
  • the term "gene” means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons).
  • the term "isolated,” when applied to a nucleic acid or protein, denotes that the nucleic acid or protein is essentially free of other cellular components with which it is associated in the natural state. It is preferably in a homogeneous state although it can be in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography.
  • a protein that is the predominant species present in a preparation is substantially purified.
  • an isolated gene is separated from open reading frames that flank the gene and encode a protein other than the gene of interest.
  • the term “purified” denotes that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. Particularly, it means that the nucleic acid or protein is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure.
  • nucleic acid or “polynucleotide” refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double- stranded form.
  • nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides.
  • a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and or deoxyinosine residues (Batzer et ah, Nucleic Acid Res.
  • nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.
  • polypeptide amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g. , hydroxyproline, ⁇ -carboxyglutamate, and O-phosphoserine.
  • Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an ⁇ carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g. , norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but which functions in a manner similar to a naturally occurring amino acid.
  • Amino acids may be referred to herein by either the commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
  • "Conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, “conservatively modified variants” refers to those nucleic acids that encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein.
  • the codons GCA, GCC, GCG and GCU all encode the amino acid alanine.
  • the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide.
  • Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein that encodes a polypeptide also describes every possible silent variation of the nucleic acid.
  • each codon in a nucleic acid can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence.
  • amino acid sequences one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid.
  • Percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (e.g., a polypeptide of the invention), which does not comprise additions or deletions, for optimal alignment of the two sequences.
  • the percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • Sequences are "substantially identical” if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection, or across the entire sequence where not indicated.
  • the invention provides polypeptides or polynucleotides that are substantially identical to the polypeptides or polynucleotides, respectively, exemplified herein (e.g., SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, or 38).
  • This definition also refers to the complement of a test sequence.
  • the identity exists over a region that is at least about 50 nucleotides in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides in length.
  • For sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • a "comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch (1970) J Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988) Proc. Nat'l Acad. Sci.
  • HSPs high scoring sequence pairs
  • Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always ⁇ 0).
  • M forward score for a pair of matching residues; always > 0
  • N penalty score for mismatching residues; always ⁇ 0.
  • a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative- scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • W wordlength
  • E expectation
  • the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787).
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.
  • nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below.
  • a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions.
  • Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below.
  • Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.
  • sequenceselectively (or specifically) hybridizes to refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent hybridization conditions when that sequence is present in a complex mixture (e.g., total cellular or library DNA or RNA).
  • stringent hybridization conditions refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acid, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures.
  • T m thermal melting point
  • Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times background, optionally 10 times background hybridization.
  • Exemplary stringent hybridization conditions can be as following: 50% formamide, 5X SSC, and 1% SDS, incubating at 42°C, or 5X SSC, 1% SDS, incubating at 65°C, with wash in 0.2X SSC, and 0.1% SDS at 55°C, 60°C, or 65°C. Such washes can be performed for 5, 15, 30, 60, 120, or more minutes.
  • Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides that they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.
  • the nucleic acids typically hybridize under moderately stringent hybridization conditions.
  • exemplary “moderately stringent hybridization conditions” include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37°C, and a wash in IX SSC at 45°C. Such washes can be performed for 5, 15, 30, 60, 120, or more minutes. A positive hybridization is at least twice background. Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency.
  • a nucleic acid sequence encoding refers to a nucleic acid which contains sequence information for a structural RNA such as rRNA, a tRNA, or the primary amino acid sequence of a specific protein or peptide, or a binding site for a trans- acting regulatory agent. This phrase specifically encompasses degenerate codons (i.e., different codons which encode a single amino acid) of the native sequence or sequences that may be introduced to conform with codon preference in a specific host cell.
  • recombinant when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified.
  • recombinant cells express genes that are not found within the native (nonrecombinant) form of the cell or express native genes that are otherwise abnormally expressed, under-expressed or not expressed at all.
  • heterologous when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature.
  • the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source.
  • a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).
  • An "expression vector” is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a host cell.
  • the expression vector can be part of a plasmid, virus, or nucleic acid fragment.
  • the expression vector includes a nucleic acid to be transcribed operably linked to a promoter.
  • the specified antibodies bind to a particular protein and do not bind in a significant amount to other proteins present in the sample.
  • Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein.
  • antibodies raised against a protein having an amino acid sequence encoded by any of the polynucleotides of the invention can be selected to obtain antibodies specifically immunoreactive with that protein and not with other proteins, except for polymorphic variants.
  • a variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid- phase ELISA immunoassays, Western blots, or immunohistochemistry are routinely used to select monoclonal antibodies specifically immunoreactive with a protein.
  • Modulators encompass e.g., ligands, agonists, antagonists, their homologs and mimetics, as well as the polypeptides of the invention, or fragments thereof with antagonist activity or that act to increase overall polypeptide activity (i.e., fragments that have at least some of the activity of the full-length protein). In some cases, fragments of the polypeptides of the invention are at least 20, 50, 75 or 100 amino acids in length.
  • modulator includes inhibitors and activators.
  • Inhibitors are agents that, e.g., inhibit expression of a polypeptide of the invention or bind to, partially or totally block stimulation, decrease, prevent, delay activation, inactivate, desensitize, or down regulate the activity of a polypeptide of the invention, e.g., antagonists.
  • Activators are agents that, e.g., induce or activate the expression of a polypeptide of the invention or bind to, stimulate, increase, open, activate, facilitate, or enhance activation, sensitize or up regulate the activity of a polypeptide of the invention, e.g., agonists.
  • Modulators include naturally occurring and synthetic ligands, antagonists, agonists, small chemical molecules and the like.
  • Such assays for inhibitors and activators include, e.g., applying putative modulator compounds to cells . expressing a polypeptide of the invention and then determining the functional effects on a polypeptide of the invention activity, as described above.
  • Samples or assays comprising a polypeptide of the invention that are treated with a potential activator, inhibitor, or modulator are compared to control samples without the inhibitor, activator, or modulator to examine the extent of effect.
  • Control samples (untreated with modulators) are assigned a relative activity value of 100%.
  • Inhibition of a polypeptide of the invention is achieved when the polypeptide activity value relative to the control is less than about 80%, optionally 50% or 25, 10%, 5% or 1%.
  • Activation of the polypeptide is achieved when the polypeptide activity value relative to the control is 110%, optionally 150%, optionally 200, 300%, 400%, 500%, or 1000-3000% or more higher.
  • the present application demonstrates that, surprisingly, modulated levels of mRNA comprising sequences of the invention occur in muscle from tissue either insulin resistant obese non-diabetics or from type 2 diabetic individuals treated compared to levels of the mRNA in the lean, non-diabetic individuals. Additional muscle from tissue from type 2 diabetic individuals treated with thiazolidinedione (TZD) compared to levels of mRNA in the type 2 diabetic individuals before TZD treatment. Insulin resistant obese are generally pre-diposed to become type II diabetics. Therefore, the modulation of the sequences in the study described herein indicates the sequences' involvement in diabetes and pre- diabetes.
  • TZD thiazolidinedione
  • modulation of the expression or activity of the polypeptides or polynucleotides of the invention is beneficial in treating diabetic, pre-diabetic or obese insulin resistant, non- diabetic patients.
  • modulated levels of the polypeptides of the invention are indicative of insulin resistance.
  • the detection of a polypeptide of the invention is useful for diagnosis of diabetes and insulin resistance.
  • This invention also provides methods of using polypeptides of the invention and modulators of the polypeptides of the invention to diagnose and treat diabetes, pre-diabetes (including insulin resistant individuals) and related metabolic diseases.
  • the present method also provides methods of identifying modulators of expression or activity of the polypeptides of the invention. Such modulators are useful for treating Type 2 diabetes as well as the pathological aspects of diabetes (e.g., insulin resistance).
  • nucleic acids encoding a polypeptide of the present invention will be isolated and cloned using recombinant methods.
  • Such embodiments are used, e.g., to isolate polynucleotides identical or substantially identical to SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, or 37 for protein expression or during the generation of variants, derivatives, expression cassettes, or other sequences derived from an polypeptide or polynucleotide of the invention, to monitor gene expression, for the isolation or detection of sequences in different species, for diagnostic purposes in a patient, e.g., to detect mutations in a polypeptide or polynucleotide of the invention or to detect expression levels of nucleic acids or polypeptides.
  • sequences encoding the polypeptides of the invention are operably linked to a heterologous promoter.
  • fragments of the polypeptides of the invention are at least 20, 50, 75 or 100 amino acids in length.
  • the polypeptides of the invention can be linked to heterologous amino acid sequences using recombinant DNA technology.
  • the nucleic acids of the invention are from any mammal, including, in particular, e.g., a human, a mouse, a rat, etc.
  • Polynucleotides, including expression cassettes, encoding polypeptides of the invention can be introduced into cells and optionally expressed in the cells.
  • Polynucleotides of the invention can be introduced into eukaryotic or prokaryotic cells, including adipocyte or muscle cells.
  • the cells can be primary cells or cell lines.
  • oligonucleotides Purification of oligonucleotides is by either native acrylamide gel electrophoresis or by anion-exchange HPLC as described in Pearson & Rea ier, J Chrom. 255:137-149 (1983). [68] The sequence of the cloned genes and synthetic oligonucleotides can be verified after cloning using, e.g., the chain termination method for sequencing double- stranded templates of Wallace etal, Gene 16:21-26 (1981). B.
  • nucleic acids encoding the subject proteins are cloned from DNA sequence libraries that are made to encode cDNA or genomic DNA.
  • the particular sequences can be located by hybridizing with an oligonucleotide probe, the sequence of which can be derived from the sequences disclosed herein, which provide a reference for PCR primers and defines suitable regions for isolating probes pecif ⁇ c for the polypeptides or polynucleotides of the invention.
  • the sequence is cloned into an expression library, the expressed recombinant protein can be detected
  • the DNA is extracted from a suitable tissue and either mechanically sheared or enzymatically digested to yield fragments of preferably about 5-100 kb.
  • the fragments are then separated by gradient centrifugation from undesired sizes and are constructed in bacteriophage lambda vectors.
  • These vectors and phage are packaged in vitro, and the recombinant phages are analyzed by plaque hybridization. Colony hybridization is carried out as generally described in Grunstein etal., Proc. Natl. Acad. Sci. USA., 72:3961-3965 (1975).
  • An alternative method combines the use of synthetic oligonucleotide primers with polymerase extension on an mRNA or DNA template.
  • Suitable primers can be designed from specific sequences disclosed herein.
  • This polymerase chain reaction (PCR) method amplifies the nucleic acids encoding the protein of interest directly from mRNA, cDNA, genomic libraries or cDNA libraries. Restriction endonuclease sites can be incorporated into the primers.
  • Polymerase chain reaction or other in vitro amplification methods may also be useful, for example, to clone nucleic acids encoding specific proteins and express said proteins, to synthesize nucleic acids that will be used as probes for detecting the presence of mRNA encoding a polypeptide of the invention in physiological samples, for nucleic acid sequencing, or for other purposes (see, U.S. Patent Nos. 4,683,195 and
  • Genes amplified by a PCR reaction can be purified from agarose gels and cloned into an appropriate vector.
  • Appropriate primers and probes for identifying the genes encoding a polypeptide of the invention from mammalian tissues can be derived from the sequences provided herein. For a general overview of PCR, see, Innis et al PCR Protocols: A Guide to Methods and Applications, Academic Press, San Diego (1990).
  • Synthetic oligonucleotides can be used to construct genes. This is done using a series of overlapping oligonucleotides, usually 40-120 bp in length, representing both the sense and anti-sense strands of the gene.
  • a polynucleotide encoding a polypeptide of the invention can be cloned using intermediate vectors before transformation into mammalian cells for expression. These intermediate vectors are typically prokaryote vectors or shuttle vectors. The proteins can be expressed in either prokaryotes or eukaryotes, using standard methods well known to those of skill in the art.
  • polypeptides of the invention can be purified for use in functional assays.
  • Naturally occurring polypeptides of the invention can be purified from any source (e.g., tissues of an organism expressing an ortholog).
  • Recombinant polypeptides can be purified from any suitable expression system.
  • the polypeptides of the invention may be purified to substantial purity by standard techniques, including selective precipitation with such substances as ammonium sulfate; column chromatography, immunopurification methods, and others (see, e.g., Scopes, Protein Purification: Principles and Practice (1982); U.S. Patent No.
  • proteins having established molecular adhesion properties can be reversibly fused to a polypeptide of the invention.
  • either protein can be selectively adsorbed to a purification column and then freed from the column in a relatively pure form. The fused protein may be then removed by enzymatic activity.
  • polypeptides can be purified using immunoaffinity columns.
  • purification of Proteins from Recombinant Bacteria typically involves the extraction, separation and/or purification of inclusion bodies by disruption of bacterial cells typically, but not limited to, by incubation in a buffer of about 100-150 ⁇ g/ml lysozyme and 0.1% Nonidet P40, a non-ionic detergent.
  • the cell suspension can be ground using a Polytron grinder (Brinkman Instruments, Westbury, NY).
  • the cells can be sonicated on ice. Alternate methods of lysing bacteria are described in Ausubel et al. and Sambrook et al, both supra, and will be apparent to those of skill in the art. [80]
  • the cell suspension is generally centrifuged and the pellet containing the inclusion bodies resuspended in buffer which does not dissolve but washes the inclusion bodies, e.g., 20 mM Tris-HCl (pH 7.2), 1 mM EDTA, 150 mM NaCl and 2% Triton-X 100, a non-ionic detergent. It may be necessary to repeat the wash step to remove as much cellular debris as possible.
  • the remaining pellet of inclusion bodies may be resuspended in an appropriate buffer (e.g., 20 mM sodium phosphate, pH 6.8, 150 mM NaCl).
  • an appropriate buffer e.g. 20 mM sodium phosphate, pH 6.8, 150 mM NaCl.
  • appropriate buffers will be apparent to those of skill in the art.
  • the inclusion bodies are solubilized by the addition of a solvent that is both a strong hydrogen acceptor and a strong hydrogen donor (or a combination of solvents each having one of these properties).
  • the proteins that formed the inclusion bodies may then be renatured by dilution or dialysis with a compatible buffer.
  • Suitable solvents include, but are not limited to, urea (from about 4 M to about 8 M), formamide (at least about 80%, volume/volume basis), and guanidine hydrochloride (from about 4 M to about 8 M).
  • guanidine hydrochloride and similar agents are denaturants, this denaturation is not irreversible and renaturation may occur upon removal (by dialysis, for example) or dilution of the denaturant, allowing re-formation of the immunologically and/or biologically active protein of interest. After solubilization, the protein can be separated from other bacterial proteins by standard separation techniques. [82] Alternatively, it is possible to purify proteins from bacteria periplasm. Where the protein is exported into the periplasm of the bacteria, the periplasmic fraction of the bacteria can be isolated by cold osmotic shock in addition to other methods known to those of skill in the art (see, Ausubel et al. , supra).
  • the bacterial cells are centrifuged to form a pellet.
  • the pellet is resuspended in a buffer containing 20% sucrose.
  • the bacteria are centrifuged and the pellet is resuspended in ice-cold 5 mM MgSO 4 and kept in an ice bath for approximately 10 minutes.
  • the cell suspension is centrifuged and the supernatant decanted and saved.
  • the recombinant proteins present in the supernatant can be separated from the host proteins by standard separation techniques well known to those of skill in the art.
  • Proteins can also be purified from eukaryotic gene expression systems as described in, e.g., Fernandez and Hoeffler, Gene Expression Systems (1999).
  • baculovirus expression systems are used to isolate proteins of the invention.
  • Recombinant baculoviruses are generally generated by replacing the polyhedrin coding sequence of a baculovirus with a gene to be expressed (e.g., encoding a polypeptide of the invention).
  • Viruses lacking the polyhedrin gene have a unique plaque morphology making them easy to recognize.
  • a recombinant baculovirus is generated by first cloning a polynucleotide of interest into a transfer vector (e.g., a pUC based vector) such that the polynucleotide is operably linked to a polyhedrin promoter.
  • the transfer vector is transfected with wildtype DNA into an insect cell (e.g., Sf9, Sf21 or BT1-TN-5B1-4 cells), resulting in homologous recombination and replacement of the polyhedrin gene in the wildtype viral DNA with the polynucleotide of interest.
  • Virus can then be generated and plaque purified. Protein expression results upon viral infection of insect cells.
  • Expressed proteins can be harvested from cell supernatant if secreted, or from cell lysates if intracellular. See, e.g., Ausubel et al. and Fernandez and Hoeffler, supra.
  • C. Purification of secreted proteins from mammalian cells [84] Polypeptides of the invention, and in particular, secreted proteins of the invention can be readily purified from mammalian cells expressing the polypeptides. Expression of the polypeptides can be the result of either transient or stable expression of the protein from a recombinant expression cassette introduced into the cells. Secreted proteins can generally be isolated using standard procedures to purify the proteins from the cell culture medium. D. Standard Protein Separation Techniques For Purifying Proteins 1.
  • Solubility Fractionation Often as an initial step, and if the protein mixture is complex, an initial salt fractionation can separate many of the unwanted host cell proteins (or proteins derived from the cell culture media) from the recombinant protein of interest.
  • the preferred salt is ammonium sulfate. Ammonium sulfate precipitates proteins by effectively reducing the amount of water in the protein mixture. Proteins then precipitate on the basis of their solubility. The more hydrophobic a protein is, the more likely it is to precipitate at lower ammonium sulfate concentrations.
  • a typical protocol is to add saturated ammonium sulfate to a protein solution so that the resultant ammonium sulfate concentration is between 20- 30%.
  • the protein mixture is ultrafiltered through a membrane with a pore size that has a lower molecular weight cut-off than the molecular weight of the protein of interest.
  • the retentate of the ultrafiltration is then ultrafiltered against a membrane with a molecular cut off greater than the molecular weight of the protein of interest.
  • the recombinant protein will pass through the membrane into the filtrate.
  • the filtrate can then be chromatographed as described below. 3.
  • Column Chromatography [87]
  • the proteins of interest can also be separated from other proteins on the basis of their size, net surface charge, hydrophobicity and affinity for ligands.
  • antibodies raised against proteins can be conjugated to column matrices and the proteins immunopurified.
  • Immunoaffinity chromatography using antibodies raised to a variety of affinity tags such as hemagglutinin (HA), FLAG, Xpress, Myc, hexahistidine (His), glutathione S transferase (GST) and the like can be used to purify polypeptides.
  • His tag will also act as a chelating agent for certain metals (e.g., Ni) and thus the metals can also be used to purify His-containing polypeptides. After purification, the tag is optionally removed by specific proteolytic cleavage.
  • nucleic acid hybridization techniques A variety of methods of specific DNA and RNA measurement that use nucleic acid hybridization techniques are known to those of skill in the art (see, Sambrook, supra). Some methods involve an electrophoretic separation (e.g. , Southern blot for detecting DNA, and Northern blot for detecting RNA), but measurement of DNA and RNA can also be carried out in the absence of electrophoretic separation (e.g. , by dot blot). Southern blot of genomic DNA (e.g., from a human) can be used for screening for restriction fragment length polymorphism (RFLP) to detect the presence of a genetic disorder affecting a polypeptide of the invention.
  • RFLP restriction fragment length polymorphism
  • nucleic acid hybridization formats are known to those skilled in the art. For example, common formats include sandwich assays and competition or displacement assays. Hybridization techniques are generally described in Hames and Higgins Nucleic Acid Hybridization, A Practical Approach, IRL Press (1985); Gall and Pardue, Proc. Natl. Acad. Sci. U.S.A., 63:378-383 (1969); and John et al. Nature, 223:582-587 (1969). [93] Detection of a hybridization complex may require the binding of a signal-generating complex to a duplex of target and probe polynucleotides or nucleic acids.
  • the binding occurs through ligand and anti-ligand interactions as between a ligand-conjugated probe and an anti-ligand conjugated with a signal.
  • the binding of the signal generation complex is also readily amenable to accelerations by exposure to ultrasonic energy.
  • the label may also allow indirect detection of the hybridization complex.
  • the label is a hapten or antigen
  • the sample can be detected by using antibodies.
  • a signal is generated by attaching fluorescent or enzyme molecules to the antibodies or in some cases, by attachment to a radioactive label (see, e.g., Tijssen, "Practice and Theory of Enzyme Immunoassay s," Laboratory Techniques in
  • the probes are typically labeled either directly, as with isotopes, chromophores, lumiphores, chromogens, or indirectly, such as with biotin, to which a streptavidin complex may later bind.
  • the detectable labels used in the assays of the present invention can be primary labels (where the label comprises an element that is detected directly or that produces a directly detectable element) or secondary labels (where the detected label binds to a primary label, e.g., as is common in immunological labeling).
  • labeled signal nucleic acids are used to detect hybridization.
  • Complementary nucleic acids or signal nucleic acids may be labeled by any one of several methods typically used to detect the presence of hybridized polynucleotides. The most common method of detection is the use of autoradiography with 3 H, 125 1, 35 S, 14 C, or 32 P-labeled probes or the like. [96] Other labels include, e.g. , ligands that bind to labeled antibodies, fluorophores, chemiluminescent agents, enzymes, and antibodies that can serve as specific binding pair members for a labeled ligand.
  • a detector that monitors a particular probe or probe combination is used to detect the detection reagent label.
  • Typical detectors include spectrophotometers, phototubes and photodiodes, microscopes, scintillation counters, cameras, film and the like, as well as combinations thereof. Examples of suitable detectors are widely available from a variety of commercial sources known to persons of skill in the art.
  • an optical image of a substrate comprising bound labeling moieties is digitized for subsequent computer analysis.
  • the amount of, for example, an RNA is measured by quantitating the amount of label fixed to the solid support by binding of the detection reagent. Typically, the presence of a modulator during incubation will increase or decrease the amount of label fixed to the solid support relative to a control incubation that does not comprise the modulator, or as compared to a baseline established for a particular reaction type. Means of detecting and quantitating labels are well known to those of skill in the art.
  • the target nucleic acid or the probe is immobilized on a solid support.
  • Solid supports suitable for use in the assays of the invention are known to those of skill in the art. As used herein, a solid support is a matrix of material in a substantially fixed arrangement. [100] A variety of automated solid-phase assay techniques are also appropriate. For instance, very large scale immobilized polymer arrays (VLSIPSTM), i.e. Gene Chips or microarrays, available from Affymetrix, Inc. in Santa Clara, CA can be used to detect changes in expression levels of a plurality of genes involved in the same regulatory pathways simultaneously. See, Tijssen, supra., Fodor et al. (1991) Science, 251: 767- 777; Sheldon et al. (1993) Clinical Chemistry 39(4): 718-719, and Kozal et al. (1996) Nature
  • spotted cDNA arrays can also be used to monitor expression of a plurality of genes.
  • the array elements are organized in an ordered fashion so that each element is present at a specified location on the substrate. Because the array elements are at specified locations on the substrate, the hybridization patterns and intensities (which together create a unique expression profile) can be interpreted in terms of expression levels of particular genes and can be correlated with a particular disease or condition or treatment. See, e.g., Schena et al, Science 270: 467-470 (1995)) and (Lockhart et al, Nature Biotech. 14: 1675-1680 (1996)).
  • Hybridization specificity can be evaluated by comparing the hybridization of specificity-control polynucleotide sequences to specificity-control polynucleotide probes that are added to a sample in a known amount.
  • the specificity-control target polynucleotides may have one or more sequence mismatches compared with the corresponding polynucleotide sequences. In this manner, whether only complementary target polynucleotides are hybridizing to the polynucleotide sequences or whether mismatched hybrid duplexes are forming is determined.
  • Hybridization reactions can be performed in absolute or differential hybridization formats.
  • polynucleotide probes from one sample are hybridized to the sequences in a microarray format and signals detected after hybridization complex formation correlate to polynucleotide probe levels in a sample.
  • differential hybridization format the differential expression of a set of genes in two biological samples is analyzed.
  • polynucleotide probes from both biological samples are prepared and labeled with different labeling moieties.
  • the microarray is then examined under conditions in which the emissions from the two different labels are individually detectable. Sequences in the microarray that are hybridized to substantially equal numbers of polynucleotide probes derived from both biological samples give a distinct combined fluorescence (Shalon et al. PCT publication WO95/35505).
  • the labels are fluorescent labels with distinguishable emission spectra, such as Cy3 and Cy5 fluorophores.
  • the polynucleotide probes are labeled with a fluorescent label and measurement of levels and patterns of fluorescence indicative of complex formation is accomplished by fluorescence microscopy, such as confocal fluorescence microscopy.
  • fluorescence microscopy such as confocal fluorescence microscopy.
  • microarray fluorescence intensities can be normalized to take into account variations in hybridization intensities when more than one microarray is used under similar test conditions.
  • individual polynucleotide probe/target complex hybridization intensities are normalized using the intensities derived from internal normalization controls contained on each microarray.
  • Detection of nucleic acids can also be accomplished, for example, by using a labeled detection moiety that binds specifically to duplex nucleic acids (e.g., an antibody that is specific for RNA-DNA duplexes).
  • a labeled detection moiety that binds specifically to duplex nucleic acids
  • the antibody is detected when the enzyme reacts with its substrate, producing a detectable product.
  • RNA duplexes including homo and heteroduplexes. Kits comprising antibodies specific for DNA:RNA hybrids are available, e.g., from Digene Diagnostics, Inc. (Beltsville, MD). [108] In addition to available antibodies, one of skill in the art can easily make antibodies specific for nucleic acid duplexes using existing techniques, or modify those antibodies that are commercially or publicly available.
  • nucleic acids used in this invention can be either positive or negative probes. Positive probes bind to their targets and the presence of duplex formation is evidence of the presence of the target. Negative probes fail to bind to the suspect target and the absence of duplex formation is evidence of the presence of the target.
  • the use of a wild type specific nucleic acid probe or PCR primers may serve as a negative probe in an assay sample where only the nucleotide sequence of interest is present.
  • the sensitivity of the hybridization assays may be enhanced through use of a nucleic acid amplification system that multiplies the target nucleic acid being detected.
  • a nucleic acid amplification system that multiplies the target nucleic acid being detected.
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • Other methods recently described in the art are the nucleic acid sequence based amplification (NASBA, Cangene, Mississauga, Ontario) and Q Beta Replicase systems. These systems can be used to directly identify mutants where the PCR or LCR primers are designed to be extended or ligated only when a selected sequence is present.
  • the selected sequences can be generally amplified using, for example, nonspecific PCR primers and the amplified target region later probed for a specific sequence indicative of a mutation.
  • various detection probes including Taqman and molecular beacon probes can be used to monitor amplification reaction products, e.g., in real time.
  • An alternative means for determining the level of expression of the nucleic acids of the present invention is in situ hybridization. In situ hybridization assays are well known and are generally described in Angerer et al, Methods Enzymol 152:649-660 (1987).
  • cells In an in situ hybridization assay, cells, preferentially human cells from the cerebellum or the hippocampus, are fixed to a solid support, typically a glass slide. If DNA is to be probed, the cells are denatured with heat or alkali. The cells are then contacted with a hybridization solution at a moderate temperature to permit annealing of specific probes that are labeled. The probes are preferably labeled with radioisotopes or fluorescent reporters.
  • SNP Single nucleotide polymorphism
  • SNPs linked to genes encoding polypeptides of the invention are useful, for instance, for diagnosis of diseases (e.g., diabetes) whose occurrence is linked to the gene sequences of the invention.
  • diseases e.g., diabetes
  • the individual is likely predisposed for one or more of those diseases.
  • the individual is homozygous for a disease-linked SNP, the individual is particularly predisposed for occurrence of that disease (e.g., diabetes).
  • the SNP associated with the gene sequences of the invention is located within 300,000; 200,000; 100,000; 75,000; 50,000; or 10,000 base pairs from the gene sequence.
  • SNP detection methods include, e.g., DNA sequencing, sequencing by hybridization, dot blotting, oligonucleotide array (DNA Chip) hybridization analysis, or are described in, e.g., U.S. Patent No. 6,177,249; Landegren et al, Genome Research, 8:769-776 (1998); Botstein et al, Am J Human Genetics 32:314-331 (1980);
  • Immunoassays can be used to qualitatively or quantitatively analyze polypeptides of the invention. A general overview of the applicable technology can be found in Harlow & Lane, Antibodies: A Laboratory Manual (1988). A.
  • the protein of interest or an antigenic fragment thereof is isolated as described herein.
  • a recombinant protein is produced in a transformed cell line.
  • An inbred strain of mice or rabbits is immunized with the protein using a standard adjuvant, such as Freund's adjuvant, and a standard immunization protocol.
  • a synthetic peptide derived from the sequences disclosed herein is conjugated to a carrier protein and used as an immunogen.
  • Polyclonal sera are collected and titered against the immunogen in an immunoassay, for example, a solid phase immunoassay with the immunogen immobilized on a solid support.
  • Polyclonal antisera with a titer of 10 4 or greater are selected and tested for their crossreactivity against proteins other than the polypeptides of the invention or even other homologous proteins from other organisms, using a competitive binding immunoassay.
  • Specific monoclonal and polyclonal antibodies and antisera will usually bind with a KD of at least about 0.1 mM, more usually at least about 1 ⁇ M, preferably at least about 0.1 ⁇ M or better, and most preferably, 0.01 ⁇ M or better.
  • a number of proteins of the invention comprising immunogens may be used to produce antibodies specifically or selectively reactive with the proteins of interest. Recombinant protein is the preferred immunogen for the production of monoclonal or polyclonal antibodies.
  • Naturally occurring protein may also be used either in pure or impure form.
  • Synthetic peptides made using the protein sequences described herein may also be used as an immunogen for the production of antibodies to the protein.
  • Recombinant protein can be expressed in eukaryotic or prokaryotic cells and purified as generally described supra. The product is then injected into an animal capable of producing antibodies. Either monoclonal or polyclonal antibodies may be generated for subsequent use in immunoassays to measure the protein.
  • Methods of production of polyclonal antibodies are known to those of skill in the art. In brief, an immunogen, preferably a purified protein, is mixed with an adjuvant and animals are immunized.
  • the animal's immune response to the immunogen preparation is monitored by taking test bleeds and determining the titer of reactivity to polypeptides of the invention.
  • blood is collected from the animal and antisera are prepared. Further fractionation of the antisera to enrich for antibodies reactive to the protein can be done if desired (see, Harlow and Lane, supra).
  • Monoclonal antibodies may be obtained using various techniques familiar to those of skill in the art. Typically, spleen cells from an animal immunized with a desired antigen are immortalized, commonly by fusion with a myeloma cell (see, Kohler and Milstein, Eur. J. Immunol. 6:511-519 (1976)).
  • метод ⁇ immortalization include, e.g., transformation with Epstein Barr Virus, oncogenes, or retroviruses, or other methods well known in the art. Colonies arising from single immortalized cells are screened for production of antibodies of the desired specificity and affinity for the antigen, and yield of the monoclonal antibodies produced by such cells may be enhanced by various techniques, including injection into the peritoneal cavity of a vertebrate host. Alternatively, one may isolate DNA sequences that encode a monoclonal antibody or a binding fragment thereof by screening a DNA library from human B cells according to the general protocol outlined by Huse et ah, supra.
  • the immunogen can be measured by a variety of immunoassay methods with qualitative and quantitative results available to the clinician.
  • immunoassays can be performed in any of several configurations, which are reviewed extensively in Maggio Enzyme Immunoassay, CRC Press, Boca Raton, Florida (1980); Tijssen, supra; and Harlow and Lane, supra.
  • Immunoassays to measure target proteins in a human sample may use a polyclonal antiserum that was raised to full-length polypeptides of the invention or a fragment thereof. This antiserum is selected to have low cross-reactivity against other proteins and any such cross-reactivity is removed by immunoabso tion prior to use in the immunoassay.
  • a protein of interest is detected and/or quantified using any of a number of well-known immunological binding assays (see, e.g., U.S. Patents 4,366,241; 4,376,110; 4,517,288; and 4,837,168).
  • immunological binding assays see, e.g., U.S. Patents 4,366,241; 4,376,110; 4,517,288; and 4,837,168.
  • Immunological binding assays typically utilize a "capture agent" to specifically bind to and often immobilize the analyte (e.g., full-length polypeptides of the present invention, or antigenic subsequences thereof).
  • the capture agent is a moiety that specifically binds to the analyte.
  • Immunoassays also often utilize a labeling agent to bind specifically to and label the binding complex formed by the capture agent and the analyte.
  • the labeling agent may itself be one of the moieties comprising the antibody/analyte complex.
  • the labeling agent may be a third moiety, such as another antibody, that specifically binds to the antibody/protein complex.
  • the labeling agent is a second antibody bearing a label.
  • the second antibody may lack a label, but it may, in turn, be bound by a labeled third antibody specific to antibodies of the species from which the second antibody is derived.
  • the second antibody can be modified with a detectable moiety, such as biotin, to which a third labeled molecule can specifically bind, such as enzyme-labeled streptavidin.
  • Other proteins capable of specifically binding immunoglobulin constant regions such as protein A or protein G, can also be used as the label agents.
  • incubation and/or washing steps may be required after each combination of reagents. Incubation steps can vary from about 5 seconds to several hours, preferably from about 5 minutes to about 24 hours. The incubation time will depend upon the assay format, analyte, volume of solution, concentrations, and the like.
  • Non-Competitive Assay Formats Immunoassays for detecting proteins or analytes of interest from tissue samples may be either competitive or noncompetitive.
  • Noncompetitive immunoassays are assays in which the amount of captured protein or analyte is directly measured.
  • the capture agent e.g., antibodies specific for the polypeptides of the invention
  • the capture agent can be bound directly to a solid substrate where it is immobilized. These immobilized antibodies then capture the polypeptide present in the test sample.
  • the polypeptide of the invention thus immobilized is then bound by a labeling agent, such as a second labelled antibody specific for the polypeptide.
  • a labeling agent such as a second labelled antibody specific for the polypeptide.
  • the second antibody may lack a label, but it may, in turn, be bound by a labeled third antibody specific to antibodies of the species from which the second antibody is derived.
  • the second can be modified with a detectable moiety, such as biotin, to which a third labeled molecule can specifically bind, such as enzyme-labeled streptavidin.
  • the amount of protein or analyte present in the sample is measured indirectly by measuring the amount of an added (exogenous) protein or analyte displaced (or competed away) from a specific capture agent (e.g., antibodies specific for a polypeptide of the invention) by the protein or analyte present in the sample.
  • a specific capture agent e.g., antibodies specific for a polypeptide of the invention
  • the amount of immunogen bound to the antibody is inversely proportional to the concentration of immunogen present in the sample.
  • the antibody is immobilized on a solid substrate.
  • the amount of analyte may be detected by providing a labeled analyte molecule.
  • labels can include, e.g., radioactive labels as well as peptide or other tags that can be recognized by detection reagents such as antibodies.
  • Immunoassays in the competitive binding format can be used for cross- reactivity determinations.
  • the protein encoded by the sequences described herein can be immobilized on a solid support. Proteins are added to the assay and compete with the binding of the antisera to the immobilized antigen. The ability of the above proteins to compete with the binding of the antisera to the immobilized protein is compared to that of the protein encoded by any of the sequences described herein. The percent cross-reactivity for the above proteins is calculated, using standard calculations.
  • Those antisera with less than 10% cross-reactivity with each of the proteins listed above are selected and pooled.
  • the cross-reacting antibodies are optionally removed from the pooled antisera by immunoabsorption with the considered proteins, e.g., distantly related homologs.
  • the immunoabsorbed and pooled antisera are then used in a competitive binding immunoassay as described above to compare a second protein, thought to be perhaps a protein of the present invention, to the immunogen protein. In order to make this comparison, the two proteins are each assayed at a wide range of concentrations and the amount of each protein required to inhibit 50% of the binding of the antisera to the immobilized protein is determined.
  • the second protein is said to specifically bind to an antibody generated to an immtmogen consisting of the target protein.
  • western blot immunoblot analysis is used to detect and quantify the presence of a polypeptide of the invention in the sample.
  • the technique generally comprises separating sample proteins by gel electrophoresis on the basis of molecular weight, transferring the separated proteins to a suitable solid support (such as, e.g., a nitrocellulose filter, a nylon filter, or a derivatized nylon filter) and incubating the sample with the antibodies that specifically bind the protein of interest.
  • antibodies are selected that specifically bind to the polypeptides of the invention on the solid support. These antibodies may be directly labeled or alternatively may be subsequently detected using labeled antibodies (e.g., labeled sheep anti-mouse antibodies) that specifically bind to the antibodies against the protein of interest.
  • labeled antibodies e.g., labeled sheep anti-mouse antibodies
  • Other assay formats include liposome immunoassays (LIA), which use liposomes designed to bind specific molecules (e.g., antibodies) and release encapsulated reagents or markers. The released chemicals are then detected according to standard techniques (see, Monroe et al. (1986) Amer. Clin. Prod. Rev. 5:34-41). 4.
  • the particular label or detectable group used in the assay is not a critical aspect of the invention, as long as it does not significantly interfere with the specific binding of the antibody used in the assay.
  • the detectable group can be any material having a detectable physical or chemical property.
  • Such detectable labels have been well-developed in the field of immunoassays and, in general, most labels useful in such methods can be applied to the present invention.
  • a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
  • Useful labels in the present invention include magnetic beads (e.g., Dynabeads ), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like), radiolabels (e.g., 3 H, 125 1, 35 S, 14 C, or 32 P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads.
  • the label may be coupled directly or indirectly to the desired component of the assay according to methods well known in the art.
  • Non-radioactive labels are often attached by indirect means.
  • the molecules can also be conjugated directly to signal generating compounds, e.g., by conjugation with an enzyme or fluorescent compound.
  • a variety of enzymes and fluorescent compounds can be used with the methods of the present invention and are well-known to those of skill in the art (for a review of various labeling or signal producing systems which may be used, see, e.g., U.S. Patent No. 4,391,904).
  • Means of detecting labels are well known to those of skill in the art.
  • means for detection include a scintillation counter or photographic film as in autoradiography.
  • the label may be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence.
  • the fluorescence may be detected visually, by means of photographic film, by the use of electronic detectors such as charge coupled devices (CCDs) or photomultipliers and the like.
  • enzymatic labels may be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product.
  • simple colorimetric labels may be detected directly by observing the color associated with the label.
  • conjugated gold often appears pink, while various conjugated beads appear the color of the bead.
  • Some assay formats do not require the use of labeled components.
  • agglutination assays can be used to detect the presence of the target antibodies.
  • antigen-coated particles are agglutinated by samples comprising the target antibodies.
  • none of the components need to be labeled and the presence of the target antibody is detected by simple visual inspection.
  • Modulators of a polypeptide of the invention i.e. agonists or antagonists of a polypeptide's activity, or polypeptide's or polynucleotide' s expression of full-length polypetides of the invention or fragments thereof, are useful for treating a number of human diseases, including diabetes.
  • administration of modulators can be used to treat diabetic patients or prediabetic individuals to prevent progression, and therefore symptoms, associated with diabetes (including insulin resistance).
  • agents tested as modulators of polypeptides of the invention can be any small chemical compound, or a biological entity, such as a protein, sugar, nucleic acid or lipid.
  • any chemical compound can be used as a potential modulator or ligand in the assays of the invention, although most often compounds that can be dissolved in aqueous or organic (especially DMSO-based) solutions are used.
  • Modulators include agents designed to reduce the level of mRNA encoding a polypeptide of the invention (e.g.
  • Modulators of the invention also include antibodies that specific bind to and/or inhibit or activate the polypeptides of the invention.
  • Other modulators include the polypeptides of the invention themselves, fragments thereof, or fusion proteins comprising the polypeptides or fragments thereof (e.g., in some embodiments, comprising at least 25, 50, or 100 amino acids of the polypeptide).
  • polypeptides of the invention that are receptors
  • soluble fragments of the polypeptides i.e., lacking a transmembrane domain
  • polypeptides of the invention that are secreted both full length and fragments with biological activity can act as modulators.
  • Sigma Sigma (St. Louis, MO), Aldrich (St. Louis, MO), Sigma- Aldrich (St. Louis, MO), Fluka Chemika-Biochemica Analytika (Buchs, Switzerland) and the like.
  • high throughput screening methods involve providing a combinatorial chemical or peptide library containing a large number of potential therapeutic compounds (potential modulator compounds). Such "combinatorial chemical libraries” or “ligand libraries” are then screened in one or more assays, as described herein, to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds thus identified can serve as conventional "lead compounds” or can themselves be used as potential or actual therapeutics.
  • a combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis, by combining a number of chemical "building blocks” such as reagents.
  • a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks (amino acids) in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks.
  • Preparation and screening of combinatorial chemical libraries is well known to those of skill in the art.
  • Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Patent 5,010,175, Furka, Int. J. Pept. Prot. Res.
  • chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to: peptoids (e.g., PCT Publication No. WO 91/19735), encoded peptides (e.g., PCT Publication WO 93/20242), random bio-oligomers (e.g., PCT Publication No. WO 92/00091), benzodiazepines (e.g., U.S. Pat. No.
  • Patent 5,539,083) antibody libraries (see, e.g., Vaughn et al, Nature Biotechnology, 14(3):309-314 (1996) and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al, Science, 274:1520-1522 (1996) and U.S. Patent 5,593,853), small organic molecule libraries (see, e.g., benzodiazepines, Baum C&EN, Jan 18, page 33 (1993); isoprenoids, U.S. Patent 5,569,588; thiazolidinones and metathiazanones, U.S. Patent 5,549,974; pyrrolidines, U.S.
  • Devices for the preparation of combinatorial libraries are commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, Louisville KY, Symphony, Rainin, Woburn, MA, 433 A Applied Biosystems, Foster City, CA, 9050 Plus, Millipore, Bedford, MA).
  • numerous combinatorial libraries are themselves commercially available (.see, e.g., ComGenex, Princeton, N.J., Tripos, Inc., St.
  • the screening methods involve screening a plurality of agents to identify an agent that modulates the activity of a polypeptide of the invention by, e.g., binding to the polypeptide, preventing an inhibitor or activator from binding to the polypeptide, increasing association of an inhibitor or activator with the polypeptide, or activating or inhibiting expression of the polypeptide.
  • Any cell expressing a full-length polypeptide of the invention or a fragment thereof can be used to identify modulators.
  • the cells are eukaryotic cells lines (e.g., CHO or HEK293) transformed to express a heterologous polypeptide of the invention.
  • a cell expressing an endogenous polypeptide of the invention is used in screens.
  • modulators are screened for their ability to effect insulin responses. In other embodiments, modulators are screened for their ability to effect lipid metabolism.
  • modulators of HAK comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, or 8 may be identified using, e.g., expression assays, promoter-reporter assays, modulator binding assays, or kinase assays. Kinase assays can be carried out after contacting either purified recombinant HAK protein, or an intact cell with the modulator. It is possible to screen for modulators of the protein kinase activity, for example, by measuring substrate phosphorylation or ATP depletion.
  • modulators of Midori comprising the amino acid sequence of SEQ ID NO: 10, 12, or 14 may be identified using, e.g., expression assays, promoter-reporter assays, modulator binding assays, or kinase assays.
  • Kinase assays can be carried out after contacting either purified recombinant midori protein, or an intact cell with the modulator. It is possible to screen for modulators of the protein kinase activity, for example, by measuring substrate phosphorylation or ATP depletion.
  • modulators of MMD (Monocyte to macrophage differentiation-associated) comprising the amino acid sequence of SEQ ID NO: 16, 18, or 20 may be identified using, e.g., expression assays, modulator binding assays, or promoter- reporter assays.
  • modulators of PDE11 A (Phosphodiesterase 11A) comprising the amino acid sequence of SEQ ID NO: 22, 24, 26, 28, 30 or 32 may be identified using, e.g., expression assays, promoter-reporter assays, modulator binding assays, or screening methods that monitor modulator-induced fluctuation of either intracellular cAMP or cGMP concentrations.
  • Screening assay can be carried out after contacting either purified recombinant PDE11A protein, or an intact cell with the modulator.
  • modulators of PDE5A Phosphodiesterase 5A
  • modulator binding assays may be identified using, e.g., expression assays, promoter-reporter assays, modulator binding assays, or screening methods that monitor modulator-induced fluctuation of intracellular cGMP concentrations. Screening assay can be carried out after contacting either purified recombinant PDE5A protein, or an intact cell with the modulator. 1.
  • Binding assays are also useful, e.g., for identifying endogenous proteins that interact with polypeptides of the invention. For example, antibodies, receptors or other molecules that bind polypeptides of the invention can be identified in binding assays.
  • Binding assays usually involve contacting a polypeptide of the invention with one or more test agents and allowing sufficient time for the protein and test agents to form a binding complex. Any binding complexes formed can be detected using any of a number of established analytical techniques.
  • Protein binding assays include, but are not limited to, methods that measure co-precipitation or co-migration on non-denaturing SDS- polyacrylamide gels, and co-migration on Western blots (see, e.g., Bennet, J.P. and Yamamura, H.I. (1985) "Neurotransmitter, Hormone or Drug Receptor Binding Methods," in
  • binding assays involve the use of mass spectrometry or NMR techniques to identify molecules bound to a polypeptide of the invention or displacement of labeled substrates.
  • the polypeptides of the invention utilized in such assays can be naturally expressed, cloned or synthesized.
  • mammalian or yeast two-hybrid approaches see, e.g. ,
  • Barrel, P.L. et. al. Methods Enzymol, 254:241 (1995)) can be used to identify polypeptides or other molecules that interact or bind when expressed together in a host cell.
  • polypeptide Activity The activity of polypeptides of the invention can be assessed using a variety of in vitro and in vivo assays to determine functional, chemical, and physical effects, e.g., measuring ligand binding (e.g., radioactive or otherwise labeled ligand binding), second messengers (e.g., cAMP, cGMP, IP 3 , DAG, or Ca 2+ ), ion flux, phosphorylation levels, transcription levels, and the like. Furthermore, such assays can be used to test for inhibitors and activators of the polypeptides of the invention. Modulators can also be genetically altered versions of polypeptides of the invention.
  • ligand binding e.g., radioactive or otherwise labeled ligand binding
  • second messengers e.g., cAMP, cGMP, IP 3 , DAG, or Ca 2+
  • ion flux e.g., phosphorylation levels, transcription levels, and the like.
  • Modulators can also be
  • the polypeptide of the assay will be selected from a polypeptide with substantial identity to a sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 or conservatively modified variants thereof.
  • the amino acid sequence identity will be at least 70%, optionally at least 85%, optionally at least 90, or optionally at least 95% to the polypeptides exemplified herein.
  • the polypeptide of the assays will comprise a fragment of a polypeptide of the invention, such as an extracellular domain, transmembrane domain, cytoplasmic domain, ligand binding domain, subunit association domain, active site, and the like.
  • Either a polypeptide of the invention or a domain thereof can be covalently linked to a heterologous protein to create a chimeric protein used in the assays described herein.
  • Modulators of polypeptide activity are tested using either recombinant or naturally occurring polypeptides of the invention.
  • the protein can be isolated, expressed in a cell, expressed in a membrane derived from a cell, expressed in tissue or in an animal, either recombinant or naturally occurring.
  • tissue slices, dissociated cells, e.g., from tissues expressing polypeptides of the invention, transformed cells, or membranes can be used. Modulation is tested using one of the in vitro or in vivo assays described herein.
  • Modulator binding to polypeptides of the invention, a domain, or chimeric protein can be tested in solution, in a bilayer membrane, attached to a solid phase, in a lipid monolayer, or in vesicles. Binding of a modulator can be tested using, e.g., changes in spectroscopic characteristics (e.g., fluorescence, absorbance, refractive index), hydrodynamic (e.g., shape), chromatographic, or solubility properties.
  • Samples or assays that are treated with a potential moulator e.g., a "test compound" are compared to control samples without the test compound, to examine the extent of modulation.
  • Control samples (untreated with activators or inhibitors) are assigned a relative activity value of 100. Inhibition of the polypeptides of the invention is achieved when the activity value relative to the control is about 90%, optionally 50%, optionally 25- 0%. Activation of the polypeptides of the invention is achieved when the activity value relative to the control is 110%, optionally 150%, 200%, 300%, 400%, 500%, or 1000-2000%.
  • Screening for a compound that modulates the expression of a polynucleotide or a polypeptide of the invention is also provided. Screening methods generally involve conducting cell-based assays in which test compounds are contacted with one or more cells expressing a polynucleotide or a polypeptide of the invention, and then detecting an increase or decrease in expression (either transcript or translation product). Assays can be performed with any cells that express a polynucleotide or a polypeptide of the invention. [160] Expression can be detected in a number of different ways.
  • the expression level of a polynucleotide of the invention in a cell can be determined by probing the mRNA expressed in a cell with a probe that specifically hybridizes with a transcript (or complementary nucleic acid derived therefrom) of a polynucleotide of the invention. Probing can be conducted by lysing the cells and conducting Northern blots or without lysing the cells using in s z ' t ⁇ -hybridization techniques. Alternatively, a polypeptide of the invention can be detected using immunological methods in which a cell lysate is probed with antibodies that specifically bind to the polypeptide.
  • the level of expression or activity of a polynucleotide or a polypeptide of the invention can be compared to a baseline value.
  • the baseline value can be a value for a control sample or a statistical value that is representative of expression levels of a polynucleotide or a polypeptide of the invention for a control population (e.g., lean individuals as described herein) or cells (e.g., tissue culture cells not exposed to a modulator). Expression levels can also be determined for cells that do not express the polynucleotide or a
  • polypeptide of the invention as a negative control. Such cells generally are otherwise substantially genetically the same as the test cells.
  • Promoter-reporter assays can be carried out using mammalian cells transfected with a reporter gene operably linked to sequences derived from the promoter regions of the polypeptides of the invention. The increased or decreased expression of the reporter gene can be detected in the presence and absence of the modulator. Expression of reporter genes may be detected by hybridization to a complementary nucleic acid, by using an immunological reagent, by assaying for an activity of the reporter gene product or other methods known to those in the art . [163] A variety of different types of cells can be utilized in the reporter assays.
  • Cells that do not endogenously express a polypeptide of the invention can be prokaryotic, but are preferably eukaryotic.
  • the eukaryotic cells can be any of the cells typically utilized in generating cells that harbor recombinant nucleic acid constructs.
  • Exemplary eukaryotic cells include, but are not limited to, yeast, and various higher eukaryotic cells such as the HEK293, HepG2, COS, CHO and HeLa cell lines.
  • Narious controls can be conducted to ensure that an observed activity is authentic including running parallel reactions with cells that lack the reporter construct or by not contacting a cell harboring the reporter construct with test compound. Compounds can also be further validated as described below.
  • Agents that are initially identified by any of the foregoing screening methods can be further tested to validate the apparent activity.
  • Modulators that are selected for further study can be tested on the "classic" insulin responsive cell line, mouse 3T3-L1 adipocytes, muscle cells such as L6 cells and the like. Cells (e.g., adipocytes or muscle cells) are pre-incubated with the modulators and tested for acute (up to 4 hours) and chronic (overnight) effects on basal and insulin-stimulated GLUT4 translocation and glucose uptake.
  • validity of the modulators is tested in suitable animal models.
  • the basic format of such methods involves administering a lead compound identified during an initial screen to an animal that serves as a model for humans and then determining if expressionof activity of a polypeptide of the invention is in fact modulated.
  • the effect of the compound will be assessed in either diabetic animals or in diet induced insulin resistant animals. The blood glucose and insulin levels will be determined.
  • the animal models utilized in validation studies generally are mammals of any kind. Specific examples of suitable animals include, but are not limited to, primates, mice and rats.
  • monogenic models of diabetes e.g., ob/ob and db/db mice, Zucker rats and Zucker Diabetic Fatty rats etc
  • polygenic models of diabetes e.g., OLETF rats, GK rats, NSY mice, and KK mice
  • transgenic animals expressing human polypeptides of the invention can be used to further validate drug candidates.
  • each well of a microtiter plate can be used to run a separate assay against a selected potential modulator, or, if concentration or incubation time effects are to be observed, every 5-10 wells can test a single modulator.
  • a single standard microtiter plate can assay about 100 (e.g., 96) modulators. If 1536 well plates are used, then a single plate can easily assay from about 100 to about 1500 different compounds.
  • a molecule of interest e.g., a polypeptide or polynucleotide of the invention, or a modulator thereof
  • a molecule of interest can be bound to the solid-state component, directly or indirectly, via covalent or non-covalent linkage, e.g. , via a tag.
  • the tag can be any of a variety of components.
  • a molecule that binds the tag (a tag binder) is fixed to a solid support, and the tagged molecule of interest is attached to the solid support by interaction of the tag and the tag binder.
  • tags and tag binders can be used, based upon known molecular interactions well described in the literature.
  • a tag has a natural binder, for example, biotin, protein A, or protein G
  • tag binders avidin, streptavidin, neutravidin, the Fc region of an immunoglobulin, poly-His, etc.
  • Antibodies to molecules with natural binders such as biotin are also widely available and appropriate tag binders (see, SIGMA Immunochemicals 1998 catalogue SIGMA, St. Louis MO).
  • any haptenic or antigenic compound can be used in combination with an appropriate antibody to form a tag/tag binder pair. Thousands of specific antibodies are commercially available and many additional antibodies are described in the literature.
  • the tag is a first antibody and the tag binder is a second antibody that recognizes the first antibody.
  • receptor-ligand interactions are also appropriate as tag and tag-binder pairs, such as agonists and antagonists of cell membrane receptors (e.g., cell receptor-ligand interactions such as transferrin, c-kit, viral receptor ligands, cytokine receptors, chemokine receptors, interleukin receptors, immunoglobulin receptors and antibodies, the cadherin family, the integrin family, the selectin family, and the like; see, e.g., Pigott & Power, The Adhesion Molecule Facts Book I (1993)).
  • toxins and venoms can all interact with various cell receptors.
  • hormones e.g., opiates, steroids, etc.
  • intracellular receptors e.g., which mediate the effects of various small ligands, including steroids, thyroid hormone, retinoids and vitamin D; peptides
  • lectins e.g., which mediate the effects of various small ligands, including steroids, thyroid hormone, retinoids and vitamin D; peptides
  • drugs lectins
  • sugars e.g., nucleic acids (both linear and cyclic polymer configurations), oligosaccharides, proteins, phospholipids and antibodies
  • nucleic acids both linear and cyclic polymer configurations
  • oligosaccharides oligosaccharides
  • proteins e.g.
  • Synthetic polymers such as polyurethanes, polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylene sulfides, polysiloxanes, polyimides, and polyacetates can also form an appropriate tag or tag binder. Many other tag/tag binder pairs are also useful in assay systems described herein, as would be apparent to one of skill upon review of this disclosure.
  • Common linkers such as peptides, polyethers, and the like can also serve as tags, and include polypeptide sequences, such as poly-gly sequences of between about 5 and 200 amino acids. Such flexible linkers are known to those of skill in the art. For example, poly(ethelyne glycol) linkers are available from Shearwater Polymers, Inc.,
  • Tag binders are fixed to solid substrates using any of a variety of methods currently available. Solid substrates are commonly derivatized or functionalized by exposing all or a portion of the substrate to a chemical reagent that fixes a chemical group to the surface that is reactive with a portion of the tag binder. For example, groups that are suitable for attachment to a longer chain portion would include amines, hydroxyl, thiol, and carboxyl groups. Aminoalkylsilanes and hydroxyalkylsilanes can be used to functionalize a variety of surfaces, such as glass surfaces.
  • Non-chemical approaches for fixing tag binders to substrates include other common methods, such as heat, cross-linking by UV radiation, and the like.
  • the invention provides in vitro assays for identifying, in a high throughput format, compounds that can modulate the expression or activity of a polypeptide of the invention.
  • Control reactions that measure activity of a polypeptide of the invention in a cell in a reaction that does not include a potential modulator are optional, as the assays are highly uniform. Such optional control reactions are appropriate and increase the reliability of the assay. Accordingly, in some embodiments, the methods of the invention include such a control reaction.
  • "no modulator" control reactions that do not include a modulator provide a background level of binding activity.
  • a known activator of a polypeptide or a polynucleotide of the invention can be incubated with one sample of the assay, and the resulting increase in signal resulting from an increased expression level or activity of a polypeptide or a polynucleotide of the invention are determined according to the methods herein.
  • a known inhibitor of a polypeptide or a polynucleotide of the invention can be added, and the resulting decrease in signal for the expression or activity of a polypeptide or a polynucleotide of the invention can be similarly detected.
  • modulators can also be combined with activators or inhibitors to find modulators that inhibit the increase or decrease that is otherwise caused by the presence of the known modulator of a polypeptide or a polynucleotide of the invention.
  • the invention provides compositions, kits and integrated systems for practicing the assays described herein using nucleic acids or polypeptides of the invention, antibodies, etc.
  • the invention provides assay compositions for use in solid phase assays; such compositions can include, for example, one or more nucleic acids encoding a polypeptide of the invention immobilized on a solid support, and a labeling reagent. In each case, the assay compositions can also include additional reagents that are desirable for hybridization. Modulators of expression or activity of a polypeptide of the invention can also be included in the assay compositions.
  • kits for carrying out the assays of the invention.
  • the kits typically include a probe that comprises an antibody that specifically binds to a polypeptide of the invention or a polynucleotide sequence encoding such polypeptides, and a label for detecting the presence of the probe.
  • the kits may include at least one polynucleotide sequence encoding a polypeptide of the invention.
  • Kits can include any of the compositions noted above, and optionally further include additional components such as instructions to practice a high-throughput method of assaying for an effect on expression of the genes encoding a polypeptide of the invention, or on activity of a polypeptide of the invention, one or more containers or compartments (e.g., to hold the probe, labels, or the like), a control modulator of the expression or activity of a polypeptide of the invention, a robotic armature for mixing kit components or the like.
  • the invention also provides integrated systems for high-throughput screening of potential modulators for an effect on the expression or activity of a polypeptide of the invention.
  • the systems can include a robotic armature which transfers fluid from a source to a destination, a controller which controls the robotic armature, a label detector, a data storage unit which records label detection, and an assay component such as a microtiter dish comprising a well having a reaction mixture or a substrate comprising a fixed nucleic acid or immobilization moiety.
  • a robotic fluid transfer system is available, or can easily be made from existing components.
  • a Zymate XP (Zymark Corporation; Hopkinton, MA) automated robot using a Microlab 2200 (Hamilton; Reno, NV) pipetting station can be used to transfer parallel samples to 96 well microtiter plates to set up several parallel simultaneous binding assays.
  • Optical images viewed (and, optionally, recorded) by a camera or other recording device are optionally further processed in any of the embodiments herein, e.g., by digitizing the image and storing and analyzing the image on a computer.
  • a variety of commercially available peripheral equipment and software is available for digitizing, storing and analyzing a digitized video or digitized optical image.
  • One conventional system carries light from the specimen field to a cooled charge-coupled device (CCD) camera, in common use in the art.
  • a CCD camera includes an array of picture elements (pixels). The light from the specimen is imaged on the CCD.
  • Particular pixels corresponding to regions of the specimen are sampled to obtain light intensity readings for each position. Multiple pixels are processed in parallel to increase speed.
  • the apparatus and methods of the invention are easily used for viewing any sample, e.g., by fluorescent or dark field microscopic techniques.
  • Modulators of the polypeptides of the invention can be administered directly to the mammalian subject for modulation of activity of a polypeptide of the invention in vivo. Administration is by any of the routes normally used for introducing a modulator compound into ultimate contact with the tissue to be treated and is well known to those of skill in the art. Although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route.
  • the pharmaceutical compositions of the invention may comprise a pharmaceutically acceptable carrier.
  • compositions of the present invention are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there are a wide variety of suitable formulations of pharmaceutical compositions of the present invention (see, e.g., Remington 's Pharmaceutical Sciences, 17 th ed. 1985)).
  • modulators e.g., agonists or antagonists
  • the modulators can be prepared for injection or for use in a pump device.
  • Pump devices also known as "insulin pumps” are commonly used to administer insulin to patients and therefore can be easily adapted to include compositions of the present invention. Manufacturers of insulin pumps include Animas, Disetronic and MiniMed.
  • the modulators e.g., agonists or antagonists of the expression or activity of a polypeptide of the invention, alone or in combination with other suitable components, can be made into aerosol formulations (i.e., they can be "nebulized") to be administered via inhalation. Aerosol formulations can be placed into pressurized acceptable propellents, such as dichlorodifluoromethane, propane, nitrogen, and the like.
  • Formulations suitable for administration include aqueous and non- aqueous solutions, isotonic sterile solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • compositions can be administered, for example, orally, nasally, topically, intravenously, intraperitoneally, or intrathecally.
  • the formulations of compounds can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials.
  • Solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
  • the modulators can also be administered as part of a prepared food or drug.
  • the dose administered to a patient, in the context of the present invention should be sufficient to induce a beneficial response in the subject over time.
  • the optimal dose level for any patient will depend on a variety of factors including the efficacy of the specific modulator employed, the age, body weight, physical activity, and diet of the patient, on a possible combination with other drugs, and on the severity of the case of diabetes. It is recommended that the daily dosage of the modulator be determined for each individual patient by those skilled in the art in a similar way as for known insulin compositions.
  • the size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of a particular compound or vector in a particular subject.
  • a physician may evaluate circulating plasma levels of the modulator, modulator toxicity, and the production of anti-modulator antibodies.
  • the dose equivalent of a modulator is from about 1 ng/kg to 10 mg/kg for a typical subject.
  • modulators of the present invention can be administered at a rate determined by the LD-50 of the modulator, and the side-effects of the modulator at various concentrations, as applied to the mass and overall health of the subject. Administration can be accomplished via single or divided doses.
  • the compounds of the present invention can also be used effectively in combination with one or more additional active agents depending on the desired target therapy (see, e.g., Turner, N. et al. Prog. Drug Res. (1998) 51: 33-94; Haffher, S. Diabetes Care (1998) 21 : 160-178; and DeFronzo, R. et al. (eds.), Diabetes Reviews (1997) Vol. 5 No. 4).
  • a number of studies have investigated the benefits of combination therapies with oral agents (see, e.g., Mahler, R., J Clin. Endocrinol Metab. (1999) 84: 1165-71; United Kingdom Prospective Diabetes Study Group: UKPDS 28, Diabetes Care (1998) 21: 87-92; Bardin, C.
  • Combination therapy includes administration of a single pharmaceutical dosage formulation that contains a modulator of the invention and one or more additional active agents, as well as administration of a modulator and each active agent in its own separate pharmaceutical dosage formulation.
  • a modulator and a thiazolidinedione can be administered to the human subject together in a single oral dosage composition, such as a tablet or capsule, or each agent can be administered in separate oral dosage formulations.
  • a modulator and one or more additional active agents can be administered at essentially the same time (i.e., concurrently), or at separately staggered times (i.e., sequentially).
  • Combination therapy is understood to include all these regimens.
  • One example of combination therapy can be seen in treating prediabetic individuals (e.g., to prevent progression into type 2 diabetes) or diabetic individuals (or treating diabetes and its related symptoms, complications, and disorders), wherein the modulators can be effectively used in combination with, for example, sulfonylureas (such as chlorpropamide, toibutamide, acetohexamide, tolazamide, glyburide, gliclazide, glynase, glimepiride, and glipizide); biguanides (such as metformin); a PPAR beta delta agonist; a ligand or agonist of PPAR gamma such as thiazolidinediones (such as ciglitazone, pioglitazone (see, e.g., U.S.
  • sulfonylureas such as chlorpropamide, toibutamide, acetohexamide, tola
  • Patent No. 6,218,409 troglitazone, and rosiglitazone (see, e.g., U.S. Patent No. 5,859,037)); PPAR alpha agonists such as clofibrate, gemf ⁇ brozil, fenofibrate, ciprofibrate, and bezafibrate; dehydroepiandrosterone (also referred to as DHEA or its conjugated sulphate ester, DHEA-SO4); antiglucocorticoids; TNF ⁇ inhibitors; ⁇ - glucosidase inhibitors (such as acarbose, miglitol, and voglibose); amylin and amylin derivatives (such as pramlintide, (see, also, U.S.
  • O IX. GENE THERAPY Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids encoding engineered polypeptides of the invention in mammalian cells or target tissues. Such methods can be used to administer nucleic acids encoding polypeptides of the invention to cells in vitro.
  • nucleic acids encoding polypeptides of the invention are administered for in vivo or ex vivo gene therapy uses.
  • Non- viral vector delivery systems include DNA plasmids, naked nucleic acid, and nucleic acid complexed with a delivery vehicle such as a liposome.
  • Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell.
  • Methods of non- viral delivery of nucleic acids encoding engineered polypeptides of the invention include lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid ucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA.
  • Lipofection is described in e.g., US 5,049,386, US 4,946,787; and US 4,897,355) and lipofection reagents are sold commercially (e.g., TransfectamTM and LipofectinTM).
  • Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Feigner, WO 91/17424, WO 91/16024. Delivery can be to cells (ex vivo administration) or target tissues (in vivo administration).
  • Delivery can be to cells (ex vivo administration) or target tissues (in vivo administration).
  • target tissues in vivo administration.
  • lipidmucleic acid complexes including targeted liposomes such as immunolipid complexes, is well known to one of skill in the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese et al, Cancer Gene Ther. 2:291-297 (1995); Behr et al, Bioconjugate Chem.
  • RNA or DNA viral based systems for the delivery of nucleic acids encoding engineered polypeptides of the invention take advantage of highly repetitive DNA sequences.
  • Viral vectors can be administered directly to patients (in vivo) or they can be used to treat cells in vitro and the modified cells are administered to patients (ex vivo).
  • Conventional viral based systems for the delivery of polypeptides of the invention could include retro viral, lentivirus, adeno viral, adeno-associated and -herpes simplex virus vectors for gene transfer.
  • Viral vectors are currently the most efficient and versatile method of gene transfer in target cells and tissues. Integration in the host genome is possible with the retrovirus, lentivirus, and adeno-associated virus gene transfer methods, often resulting in long term expression of the inserted transgene.
  • Retroviral vectors are retroviral vectors that are able to transduce or infect non-dividing cells and typically produce high viral titers. Selection of a retroviral gene transfer system would therefore depend on the target tissue. Retroviral vectors are comprised of cis -acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum exacting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the therapeutic gene into the target cell to provide permanent transgene expression.
  • Widely used retroviral vectors include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immuno deficiency virus (SIN), human immuno deficiency virus (HIN), and combinations thereof (see, e.g., Buchscher et al, J. Virol. 66:2731-2739 (1992); Johann et al, J. Virol 66:1635-1640 (1992); Sommerf ⁇ lt et al, Virol. 176:58-59 (1990); Wilson et al, J. Virol 63:2374-2378 (1989); Miller et al, J. Virol. 65:2220-2224 (1991); PCT/US94/05700).
  • MiLV murine leukemia virus
  • GaLV gibbon ape leukemia virus
  • SIN Simian Immuno deficiency virus
  • HIN human immuno deficiency virus
  • Adenoviral based systems are typically used.
  • Adenoviral based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. With such vectors, high titer and levels of expression have been obtained. This vector can be produced in large quantities in a relatively simple system.
  • Adeno-associated virus (“AAV”) vectors are also used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures (see, e.g., West et al, Virology 160:38-47 (1987); U.S. Patent No.
  • PA317/pLASN was the first therapeutic vector used in a gene therapy trial. (Blaese et al, Science 270:475-480 (1995)). Transduction efficiencies of 50% or greater have been observed for MFG-S packaged vectors. (Ellem et al, Immunol Immunother. 44(l):10-20 (1997); Dranoff et al, Hum. Gene Ther. 1:111-2 (1997).
  • Recombinant adeno-associated virus vectors are a promising alternative gene delivery systems based on the defective and nonpathogenic parvovirus adeno-associated type 2 virus.
  • All vectors are derived from a plasmid that retains only the AAV 145 bp inverted terminal repeats flanking the transgene expression cassette. Efficient gene transfer and stable transgene delivery due to integration into the genomes of the transduced cell are key features for this vector system. (Wagner et al, Lancet 351:9117 1702- 3 (1998), Kearns et al, Gene Ther. 9:748-55 (1996)). [202] Replication-deficient recombinant adenoviral vectors (Ad) can be engineered such that a transgene replaces the Ad El a, El b, and E3 genes; subsequently the replication defector vector is propagated in human 293 cells that supply deleted gene function in trans.
  • Ad vectors can transduce multiply types of tissues in vivo, including nondividing, differentiated cells such as those found in the liver, kidney and muscle system tissues. Conventional Ad vectors have a large carrying capacity.
  • An example of the use of an Ad vector in a clinical trial involved polynucleotide therapy for antitumor immunization with intramuscular injection (Sterman et al, Hum. Gene Ther. 7:1083-9 (1998)). Additional examples of the use of adenovirus vectors for gene transfer in clinical trials include Rosenecker et al, Infection 24:1 5-10 (1996); Sterman et al, Hum. Gene Ther. 9:7 1083- 1089 (1998); Welsh et al, Hum. Gene Ther.
  • Packaging cells are used to form virus particles that are capable of infecting a host cell. Such cells include 293 cells, which package adenovirus, and ⁇ 2 cells or PA317 cells, which package retrovirus. Viral vectors used in gene therapy are usually generated by producer cell line that packages a nucleic acid vector into a viral particle.
  • the vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host, other viral sequences being replaced by an expression cassette for the protein to be expressed.
  • the missing viral functions are supplied in trans by the packaging cell line.
  • AAV vectors used in gene therapy typically only possess ITR sequences from the AAV genome which are required for packaging and integration into the host genome.
  • Viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences.
  • the cell line is also infected with adenovirus as a helper.
  • the helper virus promotes replication of the AAV vector and expression of AAV genes from the helper plasmid.
  • the helper plasmid is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV.
  • a viral vector is typically modified to have specificity for a given cell type by expressing a ligand as a fusion protein with a viral coat protein on the viruses outer surface.
  • the ligand is chosen to have affinity for a receptor known to be present on the cell type of interest. For example, Han et al.
  • Moloney murine leukemia virus can be modified to express human heregulin fused to gp70, and the recombinant virus infects certain human breast cancer cells expressing human epidermal growth factor receptor.
  • This principle can be extended to other pairs of virus expressing a ligand fusion protein and target cell expressing a receptor.
  • filamentous phage can be engineered to display antibody fragments (e.g., FAB or Fv) having specific binding affinity for virtually any chosen cellular receptor.
  • Gene therapy vectors can be delivered in vivo by administration to an individual patient, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subdermal, or intracranial infusion) or topical application, as described below.
  • vectors can be delivered to cells ex vivo, such as cells explanted from an individual patient (e.g., lymphocytes, bone marrow aspirates, tissue biopsy) or universal donor hematopoietic stem cells, followed by reimplantation of the cells into a patient, usually after selection for cells which have incorporated the vector.
  • Ex vivo cell transfection for diagnostics, research, or for gene therapy (e.g., via re-infusion of the transfected cells into the host organism) is well known to those of skill in the art.
  • cells are isolated from the subject organism, transfected with a nucleic acid (gene or cDNA) encoding a polypeptides of the invention, and re-infused back into the subject organism (e.g., patient).
  • Various cell types suitable for ex vivo transfection are well known to those of skill in the art (see, e.g., Freshney et al, Culture of Animal Cells, A Manual of Basic Technique (3rd ed.
  • stem cells are used in ex vivo procedures for cell transfection and gene therapy.
  • the advantage to using stem cells is that they can be differentiated into other cell types in vitro, or can be introduced into a mammal (such as the donor of the cells) where they will engraft in the bone marrow.
  • Methods for differentiating CD34+ cells in vitro into clinically important immune cell types using cytokines such as a GM- CSF, IFN- ⁇ and TNF- ⁇ are known (see Inaba et al, J. Exp. Med. 176:1693-1702 (1992)).
  • Stem cells are isolated for transduction and differentiation using known methods.
  • stem cells are isolated from bone marrow cells by panning the bone marrow cells with antibodies which bind unwanted cells, such as CD4+ and CD 8+ (T cells), CD45+ (panB cells), GR-1 (granulocytes), and lad (differentiated antigen presenting cells) (see Inaba et al, J. Exp. Med. ⁇ 16:1693-1102 (1992)).
  • Vectors e.g., retroviruses, adenoviruses, liposomes, etc.
  • therapeutic nucleic acids can be also administered directly to the organism for transduction of cells in vivo.
  • naked DNA can be administered.
  • Administration is by any of the routes normally used for introducing a molecule into ultimate contact with blood or tissue cells. Suitable methods of administering such nucleic acids are available and well known to those of skill in the art, and, although more than one route can be used to admimster a particular composition, a particular route can often provide a more immediate and more effective reaction than another route.
  • Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions of the present invention, as described below (see, e.g., Remington 's Pharmaceutical Sciences, 17th ed., 1989). X.
  • the present invention also provides methods of diagnosing diabetes or a predisposition of at least some of the pathologies of diabetes. Diagnosis can involve determination of a genotype of an individual (e.g., with SNPs) and comparison of the genotype with alleles known to have an association with the occurrence of diabetes. Alternatively, diagnosis also involves determining the level of a polypeptide or polynucleotide of the invention in a patient and then comparing the level to a baseline or range. Typically, the baseline value is representative of a polypeptide or polynucleotide of the invention in a healthy (e.g., lean) person.
  • a healthy e.g., lean
  • level of a polypeptide or polynucleotide of the invention indicates that the patient is either diabetic or at risk of developing at least some of the pathologies of diabetes (e.g., pre-diabetic).
  • the level of a polypeptide in a lean individual can be a reading from a single individual, but is typically a statistically relevant average from a group of lean individuals.
  • the level of a polypeptide in a lean individual can be represented by a value, for example in a computer program.
  • the level of polypeptide or polynucleotide of the invention is measured by taking a blood, urine or tissue sample from a patient and measuring the amount of a polypeptide or polynucleotide of the invention in the sample using any number of detection methods, such as those discussed herein. For instance, fasting and fed blood or urine levels can be tested.
  • the baseline level and the level in a lean sample from an individual, or at least two samples from the same individual differ by at least about 5%, 10%, 20%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, 500%, 1000% or more.
  • the sample from the individual is greater by at least one of the above- listed percentages relative to the baseline level. In some embodiments, the sample from the individual is lower by at least one of the above-listed percentages relative to the baseline level. [215] In some embodiments, the level of a polypeptide or polynucleotide of the invention is used to monitor the effectiveness of antidiabetic therapies such as thiazolidinediones, metformin, sulfonylureas and other standard therapies.
  • antidiabetic therapies such as thiazolidinediones, metformin, sulfonylureas and other standard therapies.
  • the activity or expression of a polypeptide or polynucleotide of the invention will be measured prior to and after treatment of diabetic or pre-diabetic patients with antidiabetic therapies as a surrogate marker of clinical effectiveness.
  • the greater the reduction in expression or activity of a polypeptide of the invention indicates greater effectiveness.
  • Glucose/insulin tolerance tests can also be used to detect the effect of glucose levels on levels of a polypeptide or polynucleotide of the invention. In glucose tolerance tests, the patient's ability to tolerate a standard oral glucose load is evaluated by assessing serum and urine specimens for glucose levels.
  • Blood samples are taken before the glucose is ingested, glucose is given by mouth, and blood or urine glucose levels are tested at set intervals after glucose ingestion. Similarly, meal tolerance tests can also be used to detect the effect of insulin or food, respectively, on levels of a polypeptide or polynucleotide of the invention.
  • peripheral tissues especially muscle and fat
  • This defect in glucose metabolism is usually compensated for by increased secretion of insulin from the pancreas, thereby maintaining normal glucose levels.
  • the majority of glucose disposal occurs in the muscle.
  • a number of obese insulin resistant patients will progress to overt diabetics with time.
  • the molecular defects underlying this peripheral insulin resistance in (obese individuals) diabetics are not well defined. Genes in muscle or fat whose expression is altered in (obese individuals) diabetics when compared to lean individuals may be causative genes for insulin resistance and also may predict the transition to diabetes.
  • Modulators of such genes have the ability to reverse insulin resistance and restore normal insulin sensitivity, thereby improving whole body glucose homeostasis including for example insulin secretion. Modulators of such genes also have the ability to be used to pre-empt the transition from obesity-induced insulin resisatnce to diabetes. The majority of glucose disposal occurs in the muscle. For these reasons, gene expression profiling was performed in muscle from lean, obese and diabetic individuals. [221] Gene expression profiling was performed on tissue samples (muscle) obtained from lean, obese and diabetic individuals. Two studies were performed. In the first study, basal samples were isolated from all individuals at the beginning of a 5 hour hyperinsulinemic euglycemic clamp. Clamp samples were isolated at the end of this procedure.
  • the mean 'average difference score' was then calculated from the average difference scores for all individuals in a particular treatment group.
  • Genes were determined to be changed in condition 1 (such as basal diabetics before frogliazone (fro) treatment) versus condition 2 (such as basal diabetics after troglitazone treatment), condition 1 (such as basal leans) versus condition 2 (such as basal diabetics) or condition 1 (such as basal leans) versus condition 2 (such as basal obese) by calculating the Students t test statistic between the two conditions and selecting those with t less than or equal to 0.05.
  • Fold change was determined as the ratio of mean average difference score in condition 2 to the mean average difference score in condition 1.
  • HAK [01] Probe set MBXHUMMUS 12886 detects HAK nucleic acid sequences. Expression of HAK transcripts was increased in obese compared to lean patients in the gene profiling experiment.
  • B/C indicates sample is from Basal or Clamp; "Mean Expr” indicates mean expression; “SEM” indicates standard error of mean; “n” indicates number of patient samples; “Fold Change” indicates fold change of obese in comparison to lean patients.
  • HAK was also evaluated using real-time PCR. The results further show that HAK is significantly over-expressed in muscle from obese individuals when compared to muscle from lean individuals. "Fold Change” indicates the fold expression calculated as the ratio of the mean obese expression/ mean lean expression. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR.
  • HAK contains the following protein domains (designated with reference to SEQ ID NO:2): Alpha-kinase family (PF02816) at amino acids 1266 to 1486; Origin of replication binding protein (PF02217) at amino acids 1120 to 1233; and Immunoglobulin domain (PF00047) at amino acids 1162 to 1221.
  • HAK is a member of the alpha-kinase family whose catalytic domain may be adapted to recognize and phosphorylate amino acids located within ⁇ helices (Ryazanov et al, Curr.Biol. 9, R43-45 (1999)).
  • MIDORI Probe set MBXHUMMUS06524 detects MIDORI nucleic acid sequences.
  • MIDORI transcripts were increased in diabetic compared to obese patients in the gene profiling experiment.
  • DIABETIC OBESE B/C Mean SEM n Mean SEM n Fold Students t Gene Expr Expr Change test Name B 711.89 93.27 19 438 70.74 16 1.63 0.025 MIDORI B/C indicates sample is from Basal or Clamp; "Mean Expr” indicates mean expression; “SEM” indicates standard error of mean; “n” indicates number of patient samples; "Fold Change” indicates fold change of diabetic in comparison to obese patients. [05] Probe set MBXHUMMUS 06524 detects MIDORI nucleic acid sequences. Expression of MIDORI transcripts was increased in obese post trog compared to obese before trog patients in the gene profiling experiment.
  • B/C indicates sample is from Basal or Clamp; "Pre-Trog” and “Post-Trog” indicates sample was taken before or after 3 months of troglitazone treatment; "Mean Expr” indicates mean expression; “SEM” indicates standard error of mean; “n” indicates number of patient samples; “Fold Change” indicates fold change of obese post-frog in comparison to obese pre- trog patients.
  • MIDORI was also evaluated using real-time PCR. The results further show that MIDORI is significantly over-expressed in muscle from obese post trog individuals when compared to muscle from obese before trog individuals.
  • Midori is a member of the alpha-kinase family containing a MHCK (myosin heavy chain kinase) or EF2 (elongation factor 2) kinase domain and two immunoglobulin (Ig) domains, has a region of low similarity to a region of myosin light polypeptide kinase, and may promote the differentiation of P19CL6 into cardiomyocytes (Ryazanov et al, Curr.Biol. 9, R43-45 (1999); Hosoda et al, J. Biol. Chem. 276, 35978-35989 (2001)).
  • MHCK myosin heavy chain kinase
  • EF2 elongation factor 2
  • Ig immunoglobulin
  • Probe set MBXHUMMUS03231 detects MMD nucleic acid sequences. Expression of MMD transcripts was increased in diabetic compared to lean patients in the gene profiling experiment.
  • B/C indicates sample is from Basal or Clamp; "Mean Expr” indicates mean expression; “SEM” indicates standard error of mean; “n” indicates number of patient samples; “Fold Change” indicates fold change of diabetic in comparison to lean patients. [09] MMD was also evaluated using real-time PCR. The results further show that MMD is significantly over-expressed in muscle from diabetic individuals when compared to muscle from lean individuals. “Fold Change” indicates the fold expression calculated as the ratio of the mean diabetic expression/ mean lean expression. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR.
  • MMD contains the following protein domains (designated with reference to SEQ ID NO: 16): Haemolysin-III related (PF03006) at amino acids 23 to 220; and 7 transmembrane domains (TMHMM2.0) at amino acids 28 to 50, 60 to 82, 102 to 119, 129 to 146, 153 to 172, 175 to 195, 201 to 223.
  • MMD (Monocyte to macrophage differentiation-associated) contains seven transmembrane domains and is found to be upregulated during diiferentiation of monocytes to macrophages. MMD may function as a receptor or an ion channel (Rehli et al, Biochem. Biophys. Res. Commun.
  • PDE11A Probe set MBXHUMMUS08148 detects PDE11 A nucleic acid sequences. Expression of PDE11 A transcripts was increased in diabetic compared to lean patients in the gene profiling experiment. B/C indicates sample is from Basal or Clamp; “Mean Expr” indicates mean expression; “SEM” indicates standard error of mean; “n” indicates number of patient samples; “Fold Change” indicates fold change of diabetic in comparison to lean patients. [12] PDEl 1 A was also evaluated using real-time PCR. The results further show that PDEl 1 A is significantly over-expressed in muscle from diabetic individuals when compared to muscle from lean individuals.
  • PDEl 1 A contains the following protein domains (designated with reference to SEQ ID NO:22): HD domain (PF01966) at amino acids 719 to 838; GAF domain (PF01590) at amino acids 271 to 424, 456 to 612; and 3'5*-cyclic nucleotide phosphodiesterase (PF00233) at amino acids 717 to 956.
  • PDEl 1 A Phosphodiesterase 11 A is a dual-substrate phosphodiesterase that hydrolyzes both cGMP and cAMP.
  • PDE5A [16] Probe set MBXHUMMUS32957 detects PDE5A nucleic acid sequences. Expression of PDE5A transcripts was increased in diabetic compared to lean patients in the gene profiling experiment. DIABETIC LEAN
  • “Fold Change” indicates the fold expression calculated as the ratio of the mean diabetic expression/ mean lean expression. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR. [18] Probe set MBXHUMMUS32957 detects PDE5A nucleic acid sequences. Expression of PDE5A transcripts was increased in obese compared to lean patients in the gene profiling experiment. OBESE LEAN
  • B/C indicates sample is from Basal or Clamp; "Mean Expr” indicates mean expression; “SEM” indicates standard error of mean; “n” indicates number of patient samples; “Fold Change” indicates fold change of obese in comparison to lean patients.
  • PDE5 A was also evaluated using real-time PCR. The results further show that PDE5 A is significantly over-expressed in muscle from obese individuals when compared to muscle from lean individuals. "Fold Change” indicates the fold expression calculated as the ratio of the mean obese expression/ mean lean expression. Numbers in parentheses indicates the number of patient samples analyzed by real-time PCR.
  • PDE5 A contains the following protein domains (designated with reference to SEQ ID NO:34): GAF domain (PF01590) at amino acids 164 to 314, 346 to 503; and 3'5'-cyclic nucleotide phosphodiesterase (PF00233) at amino acids 612 to 850.
  • PDE5A Phosphodiesterase 5 A
  • Alternative splicing gives rise to at least three unique variants with differing biochemical properties (Lin, C.S, etal, Int J Impot Res. 14: 15-24 (2002)).

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Abstract

L’invention concerne des compositions et des procédés de diagnostic et de traitement du diabète et de l’insulinoresistance. En particulier, l’invention concerne des procédés d’identification des modulateurs des polynucléotides ou des polypeptides de l’invention, ainsi que l’utilisation de ces modulateurs pour traiter le diabète. L’invention concerne en outre des procédés de diagnostic du diabète qui consistent à mesurer les taux de polynucléotides ou de polypeptides de l’invention chez le patient.
PCT/US2005/021296 2004-06-15 2005-06-15 Procede de diagnostic et de traitement du diabete et de l’insulinoresistance WO2005124359A2 (fr)

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WO2024026454A1 (fr) * 2022-07-28 2024-02-01 University Of Maryland, Baltimore Inhibiteurs de pde11a4 et leurs méthodes d'utilisation
US11976282B2 (en) 2020-06-23 2024-05-07 Qatar University GATA3 inhibitors for the promotion of subcutaneous fat deposition

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WO2003077949A2 (fr) * 2002-03-14 2003-09-25 Bayer Pharmaceuticals Corporation Procedes relatifs au traitement du diabete par administration d'inhibiteurs de pde11a

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Publication number Priority date Publication date Assignee Title
WO2003077949A2 (fr) * 2002-03-14 2003-09-25 Bayer Pharmaceuticals Corporation Procedes relatifs au traitement du diabete par administration d'inhibiteurs de pde11a

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11976282B2 (en) 2020-06-23 2024-05-07 Qatar University GATA3 inhibitors for the promotion of subcutaneous fat deposition
WO2024026454A1 (fr) * 2022-07-28 2024-02-01 University Of Maryland, Baltimore Inhibiteurs de pde11a4 et leurs méthodes d'utilisation

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