WO2000065091A2 - Method of identifying ligands for the peroxisome proliferator activated receptor gamma using differential gene expression - Google Patents

Method of identifying ligands for the peroxisome proliferator activated receptor gamma using differential gene expression Download PDF

Info

Publication number
WO2000065091A2
WO2000065091A2 PCT/US2000/010757 US0010757W WO0065091A2 WO 2000065091 A2 WO2000065091 A2 WO 2000065091A2 US 0010757 W US0010757 W US 0010757W WO 0065091 A2 WO0065091 A2 WO 0065091A2
Authority
WO
WIPO (PCT)
Prior art keywords
nucleic acid
adipo
sequence
expression
cell population
Prior art date
Application number
PCT/US2000/010757
Other languages
French (fr)
Other versions
WO2000065091A3 (en
Inventor
Bonnie Gould-Rothberg
Original Assignee
Curagen Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Curagen Corporation filed Critical Curagen Corporation
Priority to AU46526/00A priority Critical patent/AU4652600A/en
Publication of WO2000065091A2 publication Critical patent/WO2000065091A2/en
Publication of WO2000065091A3 publication Critical patent/WO2000065091A3/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the invention relates generally to nucleic acids and polypeptides and in particular to the identification of ligands for the Peroxisome Proliferator Activated Receptor Gamma (PPAR ⁇ ) using differential gene expression.
  • PPAR ⁇ Peroxisome Proliferator Activated Receptor Gamma
  • NIDDM noninsulin-dependent diabetes mellitus
  • NIDDM is associated with functional and biochemical abnormalities in the pancreas, liver and peripheral insulin-sensitive tissues such as skeletal muscle and adipose tissue.
  • the abnormalities can include, e.g. relative, but not absolute deficiency of pancreatic insulin secretion, an increased rate of hepatic glucose production and extreme insulin resistance in peripheral tissues such as adipose and skeletal muscle.
  • PPAR ⁇ Peroxisome Proliferator Activated Receptor ⁇
  • PPAR ⁇ receptors exist in three forms, which have been named ⁇ l and ⁇ 2 and ⁇ 3.
  • PPAR ⁇ 1 has a ubiquitous tissue distribution with increased expression levels in the heart, liver and kidney. Its function in these tissues is largely uncharacterized.
  • PPAR ⁇ 2 expression however, is reported to be almost exclusively expressed within the white adipocyte. Activation of this receptor in adipose tissue has been associated with adipocyte differentiation and improved glycemic control.
  • a natural ligand for PPAR ⁇ is 15-deoxy- ⁇ -J2 prostaglandin.
  • the invention is based in part on the discovery that certain nucleic acids are differentially expressed in adipose tissue of animals treated with N-(2-benzoylphenyl)-L-tyrosine, a synthetic Peroxisome Proliferator Activated Receptor Gamma ligand (PPAR ⁇ L).
  • PPAR ⁇ L Peroxisome Proliferator Activated Receptor Gamma ligand
  • the invention includes methods of identifying PPAR ⁇ ligands, methods of diagnosing PPAR ⁇ pathophysiologies, and methods of treating those pathophysiologies.
  • the invention provides a method of identifying a PPAR ⁇ ligand by providing a test cell population that includes one or more cells capable of expressing one or more nucleic acids sequences responsive to PPAR ⁇ ligands and contacting the test cell population with the test agent. Levels of expression of one or more sequences, termed ADIPO sequences, are then compared to the levels of expression of the corresponding nucleic acids in a reference cell population.
  • the reference cell population contains cells whose PPAR ⁇ ligand exposure status is known, i.e., the reference cells are known to have been exposed to a PPAR ⁇ ligand, or are known not to have been exposed to the ligand.
  • the former type of reference cell population is used, an similar expression profiles of the ADIPO nucleic acids sequences in the test cell population and the reference cell population indicates that the test agent is a ligand for PPAR ⁇ .
  • the invention in a further aspect includes a method of selecting an individualized therapeutic agent appropriate for a particular subject.
  • the method includes providing from the subject a test cell population comprising a cell capable of expressing one or more nucleic acids sequences responsive to PPAR ⁇ ligands, contacting the test cell population with the therapeutic agent, and comparing the expression of the nucleic acids sequences in the test cell population to the expression of the nucleic acids sequences in a reference cell population.
  • the invention provides a method of diagnosing or determining susceptibility to a PPAR ⁇ mediated pathophysiology, e.g., noninsulin-dependent diabetes mellitus, or adipocyte differentiation.
  • the method includes providing from the subject a cell population comprising a cell capable of expressing one or more PPAR ⁇ -responsive genes, and comparing the expression of the nucleic acids sequences to the expression of the nucleic acids sequences in a reference cell population that includes cells from a subject not suffering from a PPAR ⁇ mediated pathophysiology.
  • novel nucleic acids whose expression is responsive to the effects of N-(2-benzoylphenyl)-L-tyrosine, as well as single nucleotide polymorphisms in ADIPO sequences, as well as methods of using the ADIPO single nucleotide polymorphisms.
  • the present invention is based in part on the discovery of changes in expression patterns of multiple nucleic acid sequences in rodent adipose cells following exposure to a ligand for the Peroxisome Proliferator Activated Receptor Gamma (PPAR ⁇ ).
  • PPAR ⁇ Peroxisome Proliferator Activated Receptor Gamma
  • the differences in gene expression were identified following administration of a PPAR ⁇ -specific ligand, N- (2-benzoylphenyl)-L-tyrosine, referred to herein as PPAR ⁇ L.
  • PPAR ⁇ L N- (2-benzoylphenyl)-L-tyrosine
  • the differentially expressed nucleic acids were identified by administering PPAR ⁇ L to male 10-14 week old Sprague Dawley rats at 5.0 mg/kg/day b.i.d. dosing for 3 days. Control animals received N-methylglucamine. The animals were sacrificed 24 hours following the last dose. White adipose tissue was dissected from the animals, and total RNA was recovered from the dissected tissue and cDNA prepared. Genes whose transcript levels varied between the two tissue types were identified using GENECALLINGTM differential expression analysis as described in U. S. Patent No. 5,871,697 and in Shimkets et al., Nature Biotechnology 17:798- 803 (1999). The contents of these patents and publications are inco ⁇ orated herein by reference in their entirety.
  • ADIPO 1-43 43 single copy nucleic acid sequences whose expression levels differed in PPAR ⁇ L- treated tissue and tissue treated with vehcicle alone were chosen for further characterization. These sequences are referred to herein as ADIPO 1-43. A summary of the ADIPO sequences analyzed is presented in Table 1.
  • the 28 other sequenced identified have been previously described. 17 of the sequences (ADIPO 16-32) are newly shown to be PPAR ⁇ responsive. 1 1 sequences have been previously recognized as being differentially expressed as part of the PPAR ⁇ response (ADIPO: 33-43). Six of these 11 sequences have been shown to be differentially expressed as part of the PPAR ⁇ response specifically in adipose tissue (ADIPO: 38-43), while the other 5 have been shown to be differentially expressed as part of the PPAR ⁇ response in liver tissue (ADIPO: 33-37).
  • a cloned sequence is provided along with one or more additional sequence fragments (e.g., ESTs or contigs) which contain sequences identical to, or substantially identical to, the cloned sequence.
  • additional sequence fragments e.g., ESTs or contigs
  • a consensus sequences which includes a composite sequence assembled from the cloned and additional fragments.
  • ADIPO sequence its expression can be measured using any of the associated nucleic acid sequence in the methods described herein.
  • database accession numbers are provided. This information allows for one of ordinary skill in the art to deduce information necessary for detecting and measuring expression of the ADIPO nucleic acid sequences.
  • PPAR ⁇ -responsive nucleic acids discussed herein include the following:
  • ADIPO 1 is a novel 744 bp gene fragment.
  • the nucleic acid has the following sequence:
  • ADIPO2 is a novel 434 bp gene fragment.
  • the nucleic acid was initially identified in a cloned fragment having the following sequence:
  • the cloned sequence was assembled into a contig that includes: gaatt ct ctgct aggttggcaatgtctgctcggcctgtcttgctcaggt act ttt teat cctgtggttgtaggggaacactgatgtagtggccccaatttctgcacccatgttgcaaat tgttgccatgccagtgcaggagatggagtcgacaccaggtccatggtattccacaatggc acctgtgcacctttcactgtgaggatgccggc, (SEQ ID NO : 3 )
  • ADIPO3 ADIPO3 is a novel 181 bp gene fragment.
  • the cloned sequence is:
  • ADIPO4 is a novel 63 bp gene fragment.
  • the cloned sequence is:
  • ADIPO5 is a novel 317 bp gene fragment.
  • the nucleic acid has the following sequence:
  • ADIPO6 is a novel 106 bp gene fragment.
  • the cloned sequence is:
  • ADIPO7 is a novel 258 bp gene fragment.
  • the cloned sequence is:
  • ADIPO8 ADIPO8 is a novel 531 bp gene fragment.
  • the nucleic acid was initially identified in a cloned fragment having the following sequence:
  • ADIPO9 ADIPO9 is a novel 316 bp gene fragment.
  • the nucleic acid has the following sequence:
  • ADIPOIO is a novel 713 bp gene fragment.
  • the nucleic acid has the following sequence: 1 TCTGAACGTTCTACAGCAGAAGCAGAAGGCTCTCAATGCAGGTTACATCCTAAACGGTCTGACCGTGTCCATCCCTGGAC
  • ADIPO 1 1 is a novel 407 bp gene fragment.
  • the cloned sequence is:
  • ADIPO 12 is a novel 120 bp gene fragment.
  • the cloned sequence is: 1 actagtgaaa aggcctttgt cctcaggtct tcgctccctg gtataggtgg ggtgcagggc
  • ADIPO13 ADIPO 13 is a novel 231 bp gene fragment.
  • the nucleic acid has the following sequence:
  • ADIPO14 is a novel 315 bp gene fragment.
  • the nucleic acid has the following sequence:
  • ADIPO 15 is a novel 178 bp gene fragment.
  • the cloned sequence is:
  • ADIPO 16 corresponds to the gene encoding tricarboxylate transport protein (rat; L12016, human; U25147). Its transcription is increased 4-fold in PPAR ⁇ L treated rats.
  • Tricarboxylate transport protein functions in fatty acid synthesis.
  • the protein transports citrate synthesized in the mitochondria from oxaloacetate and acetyl-CoA across the mitochondrial membrane to the cytosol where the citrate is reconverted to oxaloacetate and acetyl-CoA.
  • ADIPO 17 corresponds to the gene encoding ATP-citrate lyase (rat; J05210, human; X64330). Its transcription is increased 90-fold in PPAR ⁇ L treated rats.
  • Gene encoding ATP-citrate lyase cleaves off acetyl-CoA from cytosolic citrate to allow acetyl-CoA to enter the fatty acid synthesis cycle.
  • ADIPO 18 corresponds to the gene encoding 12-lipoxygenase (rat; L06040, human; M58704). Its transcription is decreased 5-fold in PPAR ⁇ L treated rats.
  • the major pathway of arachidonic acid metabolism in human platelets proceeds via a 12- lipoxygenase enzyme.
  • the enzyme introduces molecular oxygen into arachidonic acid in the C- 12 position to create 12(S)-hydroperoxy-5,8,10,14-eicosatetraenoic acid (12-S HETE).
  • ADIPO19 corresponds to a gene encoding rhodanese (rat; X56228, human; D87292). Its transcription is decreased 2.0-fold in PPAR ⁇ L treated rats. It is also known as nuclear-encoded mitochondrial thiosulfate sulfotransferase. Rhodanese is involved in forming iron-sulfur compounds,cyanide detoxification, and modification of sulfur containing enzymes. Rhodanese converts cyanide to thiocyanate.
  • ADIPO20 corresponds to a gene encoding NADH-ubiquinone oxidoreductase chain 4 (rat ; P05508, C06662, human ; P03905, V00662, D381120). Its expression is increased 80-fold in PPAR ⁇ L treated rats.
  • the NADH-ubiquinone oxidoreductase chain 4 protein is one of the proteins involed in the one of the first steps of the electron transport chain.
  • ADIPO21 ADIPO21 corresponds to cytochrome C oxidase polypeptide I (rat; S79304, human;
  • Cytochrome C oxidase polypeptide I is one of 3 cytochrome C oxidase subunits encoded in the mitochondrial genome. Cytochrome C oxidase is the last step in the electron transport chain and is responsible for collecting electrons from reduced cytochrome C and then transfering the electrons to oxygen to generate water.
  • ADIPO22 corresponds to adenine nucleotide translocator (rat; D12771, human; J02683). Its expression is increased 2.0-fold PPAR ⁇ L treated rats.
  • Adenine nucleotide translocator is the most abundant mitochondrial protein.
  • the protein exists as a homodimer of two 30 kDa subunits.
  • the dimer forms a gated pore that shuttles ADP across the mitochondrial membrane.
  • the pore controls the rate of ADP/ATP flux between the mitochondrion and the cytosol.
  • ADIPO23 corresponds to ⁇ 4-3-ketosteroid 5 ⁇ reductase (rat; D 17309, human; Z28339). Its expresssion is decreased 50 fold in PPAR ⁇ L treated rats.
  • ⁇ 4-3-Ketosteroid 5 ⁇ reductase is a key enzyme in bile acid synthesis. It catalyzes the reduction of ⁇ -4-3-oxosteroids to give the cis conformation and is required for synthesis of chenodeoxycholic acid and cholic acid.
  • ADIPO24 corresponds to major ⁇ -globin (rat; Ml 7083, human; N00493). Its expression is decreased 10-fold in PPAR ⁇ L treated rats.
  • Major ⁇ -globin is one of the two protein constituents of adult major hemoglobin, fetal hemoglobin and A2 hemoglobin. Hemoglobin forms a tetramer of two alpha-class chains and two beta-class chains and serves as the major carrier of molecular oxygen in the blood to peripheral tissues. Hemoglobin has also been shown to have minor transport functions for some essential cofactors, including folate. ADIPO25
  • ADIPO25 corresponds to ⁇ -globin (rat; X05080, human; N00497). Its expresssion is decreased 7.0-fold in PPAR ⁇ L treated rats.
  • ⁇ globin forms the ⁇ -class component of adult major hemoglobin, where two beta chains heterodimerize with two alpha chains ( ⁇ -1 chains for major hemoglobin and ⁇ 2 chains for minor hemoglobin).
  • ADIPO26 corresponds to keratin 19 (rat; X81449, human; Y00503). Its expression is decreased 15.0-fold in PPAR ⁇ L treated rats.
  • ADIPO27 corresponds to brain S-100 ⁇ subunit (rat; X01090, human; M59488). Its expression is increased 90-fold in ⁇ -(2-benzoylphenyl)-L-tyrosine teated rats.
  • Brain S-100 ⁇ subunit is abundantly expressed in glial cells and other tissues.
  • the protein contains two calcium-binding domains. It is thought to be involved in signaling to cause axonal growth in nervous tissue and mediate neuron/glial communication, S-100 proteins bind glial fibrillary acidic proteins and desmin intermediate filaments in the N-terminal head domains. This binding both causes disassembly of existing polymers and inhibits reassemblies of these intermediate filaments, thereby antagonizing the effects of annexin-II2 in micro filament assembly.
  • ADIPO28 ADIPO28
  • ADIPO28 corresponds to SPI-3 serine protease inhbitor (rat; XI 6539, human; KOI 500). Its expression is decreased 5-fold in PPAR ⁇ L-treated rats.
  • SPI-3 serine protease inhbitor is a liver acidic glycoprotein. Under normal conditions, expression of the protein in rats is minimal. Its transcription is regulated by IL-6 and glucocorticoids. Thus, expression levels are correlated with inflammation.
  • ADIPO29 corresponds to WAP four disulfide core domain protein (rat; AF037272, human; ESTAA 005075). Its expression is increased 1.5-fold in PPAR ⁇ L-treated rats.
  • WAP four disulfide core domain protein member of the class of WAP serine protease inhibitors.
  • the gene was initially isolated from a urogenital sinusmesenchymal cell line and is shown to have growth-inhibitory properties.
  • ADIPO30 corresponds to ⁇ 3 adrenergic receptor (rat; M74716, human; X70811). Its expresssion increases 2-fold in PPAR ⁇ L treated rats.
  • ⁇ 3 adrenergic receptor is a G-protein that is involved in thermoregulation and in lipolysis. Evolutionarily, the protein is linked to brown fat and is used in uncoupled Ox-Phos to generate heat in hibernating animals. There is also evidence that the responsiveness of the receptor decreases with age. This relative lack of responsiveness may be related to a decrease in B-3- ADR which can be restored by exposing older animals to the cold. Additionally, it may be involved in regulating intestinal absorption of foodstuffs. Correlation of a mutation of this gene with an increased incidence of NIDDM onset has been demonstrated in some populations.
  • ADIPO31 corresponds to GTP binding protein (rat; Ml 7528, human; X04828). Its expresssion decreases 1.5-fold in PPAR ⁇ L treated rats.
  • GTP binding protein was originally cloned out of olfactory epithelium. The protein consists of three subunits; alpha, beta and gamma. The alpha subunit tends to be the effector subunit. When no ligand is bound the associated receptor, the alpha subunit stays bound to the beta/gamma complex and is complexed with GDP. The receptor, when bound to its ligand then associates with the beta/gamma complex and dissociates from the alpha subunit. The free alpha subunit exchanges GDP for GTP and goes to adenylate cyclase.
  • ADIPO32 corresponds to insulin induced growth response protein (rat; L13619, human; U96876).
  • the protein is also known as insulin-induced-gene- 1 (INSIG1). Its expresssion increases 30-fold in PPAR ⁇ L treated rats.
  • Insulin induced growth response protein is the most abundantly induced insulin-responsive gene and is highly hydrophobic In addition to hepatic expression, it has been induced in a murine model of adipocyte differentiation suggesting that it may play a role in regulating growth/differentiation of tissues involved in metabolic control.
  • ADIPO33 corresponds to malic enzyme (rat; M26581, M30596, M26594, M26585, human; X77244), whose expression has previously been reported to be differentially regulated by PPAR ⁇ in liver tissue. Its expression increased 100-fold in PPAR ⁇ L treated rats.
  • ADIPO34 corresponds to long chain 3-ketoacyl-CoA thiolase (rat; D 16479, human; D 16481), whose expression has been previously reported to be differentially regulated by PPAR ⁇ in liver tissue. In the present studies, its expresssion increased 15-fold in PPAR ⁇ L treated rats.
  • ADIPO35 ADIPO35 corresponds to catalase (rat; Ml 6670, human; X04076), whose expression has been previously reported to be differentially regulated by PPAR ⁇ in liver tissue. In the present studies, its expression increased 8-fold in PPAR ⁇ L treated rats.
  • ADIPO36 correponds to carnitine/acylcarnitine carrier protein (rat; X97831, human; Y 10319), whose expression has been previously reported to be differentially regulated by PPAR ⁇ in liver tissue. In the present studies, its expression increased 1.5-fold in PPAR ⁇ L treated rats.
  • ADIPO37 corresponds to fatty acid synthase (rat; M76767, human; U26644), whose expression has previously been reported to be differentially regulated by PPAR ⁇ in liver tissue. In the present studies, its expression increased 40fold in PPAR ⁇ L treated rats.
  • ADIPO38 corresponds to stearyl-CoA desaturase (rat; J02585, human; Y13647), whose expression has been previously reported to be differentially regulated by PPAR ⁇ in adipose tissue. In the present studies, its expresssion increased 20-fold in PPAR ⁇ L treated rats.
  • ADIPO39 corresponds to phosphoenolpyruvate carboxykinase (rat; K03243, human;
  • ADIPO40 correponds to glycogen synthase (rat; J05446, human; D29685), whose expression has been previously reported to be differentially regulated by PPAR ⁇ in adipose tissue. In the present studies, its expression increased 100-fold in PPAR ⁇ L treated rats.
  • ADIPO41 corresponds to uncoupling protein (rat; AB010743, human; U76367), whose expression has been previously reported to be differentially regulated by PPAR ⁇ in adipose tissue. In the present studies, its expresssion increased 1.5-fold in PPAR ⁇ L treated rats.
  • ADIPO42 corresponds to fatty acid transport protein (rat; U89529, human; AF055899), whose expression has been previously reported to be differentially regulated by PPAR ⁇ in adipose tissue. In the present studies, its expression increased 1.5-fold in PPAR ⁇ L treated rats.
  • ADIPO43 correponds to adipocyte fatty acid binding protein (rat; U75581, human; X56549), whose expression has been previously reported to be differentially regulated by PPAR ⁇ in adipose tissue.
  • ADIPO nucleic acids and polypeptide correspond to nucleic acids or polypeptides which include the various sequences (referenced by SEQ ID NOs) disclosed for each ADIPO nucleic acid sequence.
  • the invention includes providing a test cell population which includes at least one cell that is capable of expressing one or more of the sequences ADIPO 1-32, or any combination of ADIPO sequences thereof.
  • capable of expressing is meant that the gene is present in an intact form in the cell and can be expressed. Expression of one, some, or all of the ADIPO sequences is then detected, if present, and, preferably, measured. Using sequence information provided by the database entries for the known sequences, or the sequence information for the newly described sequences, expression of the ADIPO sequences can be detected (if expressed) and measured using techniques well known to one of ordinary skill in the art.
  • sequences within the sequence database entries corresponding to ADIPO sequences, or within the sequences disclosed herein can be used to construct probes for detecting ADIPO RNA sequences in, e.g., northern blot hybridization analyses or methods which specifically, and, preferably, quantitatively amplify specific nucleic acid sequences.
  • the sequences can be used to construct primers for specifically amplifying the ADIPO sequences in, e.g., amplification-based detection methods such as reverse-transcription based polymerase chain reaction.
  • sequence comparisons in test and reference populations can be made by comparing relative amounts of the examined DNA sequences in the test and reference cell populations.
  • expression can be also measured at the protein level, i.e., by measuring the levels of polypeptides encoded by the gene products described herein.
  • Such methods are well known in the art and include, e.g., immunoassays based on antibodies to proteins encoded by the genes.
  • Expression level of one or more of the ADIPO sequences in the test cell population is then compared to expression levels of the sequences in one or more cells from a reference cell population.
  • Expression of sequences in test and control populations of cells can be compared using any art-recognized method for comparing expression of nucleic acid sequences. For example, expression can be compared using GENECALLING ® methods as described in US Patent No. 5,871,697 and in Shimkets et al., Nat. Biotechnol. 17:798-803.
  • the expression of 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, 25, 28, 30, 35, 40, or all of the sequences represented by ADIPO 1-32 are measured. If desired, expression of these sequences can be measured along with other sequences whose expression is known to be altered according to one of the herein described parameters or conditions. For example, expression of one or more of sequences represented by ADIPO 1-32 can be compared with sequences from ADIPO33-43, which have been previously shown to be response to PPAR ⁇ ligands.
  • the latter genes correspond to adipocyte fatty acid binding protein, phosphoenolpyruvate carboxykinase, stearyl-CoA desaturase, glycogen sythatase, uncoupling protein-2, fatty acid transport protein, long chain acyl-CoA sythatase and leptin.
  • the reference cell population includes cells one or more cells capable of expressing the measured ADIPO sequences and for which the compared parameter is known, e.g., PPAR ⁇ expression status.
  • PPAR ⁇ expression status is meant that is known whether the reference cell has been exposed to a PPAR ⁇ ligand, e.g. N-(2-benzoylphenyl)-L-tyrosine.
  • Whether or not comparison of the gene expression profile in the test cell population to the reference cell population reveals the presence, or degree, of the measured parameter depends on the composition of the reference cell population. For example, if the reference cell population is composed of cells that have not been treated with a known PPAR ⁇ ligand, a similar gene expression level in the test cell population and a reference cell population indicates the test agent is not a PPAR ⁇ ligand. Conversely, if the reference cell population is made up of cells that have been treated with a known PPAR ⁇ ligand , a similar gene expression profile between the test cell population and the reference cell population indicates the test agent is a PPAR ⁇ ligand.
  • a ADIPO sequence in a test cell population is considered comparable in expression level to the expression level of the ADIPO sequence in the reference cell population if its expression level varies within a factor of 2.0, 1.5, or 1.0 fold to the level of the ADIPO transcript in the reference cell population.
  • a ADIPO sequence in a test cell population can be considered altered in levels of expression if its expression level varies from the reference cell population by more than 1.0, 1.5, 2.0 or more fold from the expression level of the corresponding ADIPO sequence in the reference cell population.
  • the variation in expression of a particular ADIPO sequence corresponds to the change in expression level observed for the ADIPO sequence in the presence and absence of the PPAR ⁇ ligand as shown in Table 1.
  • test cell population is compared to multiple reference cell populations. Each of the multiple reference populations may differ in the known parameter. Thus, a test cell population may be compared to a first reference cell population known to have been exposed to a PPAR ⁇ ligand, as well as a second reference population known have not been exposed to a PPARy ligand.
  • test cell population that is exposed to, i.e., contacted with, the test PPAR ⁇ ligand can be any number of cells, i.e., one or more cells, and can be provided in vitro, in vivo, or ex vivo.
  • the test cell population can be divided into two or more subpopulations.
  • the subpopulations can be created by dividing the first population of cells to create as identical a subpopulation as possible. This will be suitable, in, for example, in vitro or ex vivo screening methods.
  • various sub populations can be exposed to a control agent, and/or a test agent, multiple test agents, or, e.g., varying dosages of one or multiple test agents administered together, or in various combinations.
  • cells in the reference cell population are derived from a tissue type as similar as possible to test cell, e.g., adipose tissue.
  • the control cell is derived from the same subject as the test cell, e.g., from a region proximal to the region of origin of the test cell.
  • the reference cell population is derived from a plurality of cells.
  • the reference cell population can be a database of expression patterns from previously tested cells for which one of the herein-described parameters or conditions (e.g., PPAR ⁇ status, screening, diagnostic, or therapeutic claims) is known.
  • the subject is preferably a mammal.
  • the mammal can be, e.g., a human, non-human primate, mouse, rat, dog, cat, horse, or cow.
  • the invention provides a method of identifying PPAR ⁇ ligands.
  • the PPAR ⁇ ligand can be identified by providing a cell population that includes cells capable of expressing one or more nucleic acid sequences homologous to those listed in Table 1 as ADIPO 1-32 and optionally 33-43.
  • the sequences need not be identical to sequences including ADIPO 1-32 and optionally 33-43, as long as the sequence is sufficiently similar that specific hybridization can be detected.
  • the cell includes sequences that are identical, or nearly identical to those identifying the ADIPO nucleic acids shown in Table 1.
  • Expression of the nucleic acid sequences in the test cell population is then compared to the expression of the nucleic acid sequences in a reference cell population, which is a cell population that has not been exposed to the test agent, or, in some embodiments, a cell population exposed the test agent. Comparison can be performed on test and reference samples measured concurrently or at temporally distinct times. An example of the latter is the use of compiled expression information, e.g., a sequence database, which assembles information about expression levels of known sequences following administration of various agents. For example, alteration of expression levels following administration of test agent can be compared to the expression changes observed in the nucleic acid sequences following administration of a control agent, such as N-(2-benzoylphenyl)-L-tyrosine.
  • a control agent such as N-(2-benzoylphenyl)-L-tyrosine.
  • test agent is a PPAR ⁇ ligand.
  • the invention also includes a PPAR ⁇ ligand identified according to this screening method, and a pharmaceutical composition which includes the PPAR ⁇ ligands.
  • the differentially expressed sequences disclosed herein can also be used to identify candidate therapeutic agents pathophysiologies associated with the PPAR ⁇ mediated pathway.
  • the method is based on screening a candidate therapeutic agent to determine if it induces an expression profile of one or more ADIPO 1-32 sequences, and, optionally, ADIPO33-43 sequences in a test cell population that is characteristic of a PPARXresponse.
  • a test cell population is exposed to a test agent or a combination of test agents (sequentially or consequentially), and the expression of one or more of the ADIPO sequences is measured.
  • the expression of the ADIPO sequences in the test population is compared to expression level of the ADIPO sequences in a reference cell population whose PPAR ⁇ status is known. If the reference cell population contains cells that have not been exposed to a PPAR ⁇ ligand, alteration of expression of the nucleic acids in the test cell population as compared to the reference cell population indicates that the test agent is a candidate therapeutic agent.
  • the reference cell population includes cells that have been exposed to a test agent.
  • a test agent When this cell population is used, an alteration in expression of the nucleic acid sequences in the presence of the agent from the expression profile of the cell population in the absence of the agent indicates the agent is a candidate therapeutic agent.
  • the test cell population includes cells that have not been exposed to a PPAR ⁇ ligand. For this cell population, a similarity in expression of the ADIPO sequences in the test and control cell populations indicates the test agent is not a candidate therapeutic agent, while a difference suggests it is a candidate.
  • test agent can be a compound not previously described or can be a previously known compound but which is not known to be a PPAR ⁇ ligand
  • An agent effective in stimulating expression of underexpressed genes, or in suppressing expression of overexpressed genes can be further tested for its ability to prevent the PPAR ⁇ mediated pathophysiology, e.g. NIDDM, and as a potential therapeutic useful for the treatment of such pathophysiology. Further evaluation of the clinical usefulness of such a compound can be performed using standard methods of evaluating toxicity and clinical effectiveness of anti-diabetic agents. SELECTING A THERAPEUTIC AGENT FOR TREATING A PATHOPHYSIOLOGY ASSOCIATED WITH THE PPAR ⁇ MEDIATED PATHWAY THAT IS APPROPRIATE FOR A PARTICULAR INDIVIDUAL
  • differentially expressed ADIPO sequences disclosed herein allow for a putative therapeutic or prophylactic agent to be tested in a test cell population from a selected subject in order to determine if the agent is a suitable PPAR ⁇ ligand in the subject.
  • a test cell population from the subject is exposed to a therapeutic agent, and the expression of one or more of ADIPO 1-32 and, optionally, ADIPO33-43 sequences is measured.
  • the test cell population contains an adipocyte.
  • the agent is first mixed with a cell extract, e.g., an adipose cell extract, which contains enzymes that metabolize drugs into an active form.
  • the activated form of the therapeutic agent can then be mixed with the test cell population and gene expression measured.
  • the cell population is contacted ex vivo with the agent or activated form of the agent.
  • the reference cell population includes at least one cell whose PPAR ⁇ status is known. If the reference cell had been exposed to a PPAR ⁇ ligand a similar gene expression profile between the test cell population and the reference cell population indicates the agent is suitable for treating the pathophysiology in the subject. A difference in expression between sequences in the test cell population and those in the reference cell population indicates that the agent is not suitable for treating the PPAR ⁇ pathophysiology in the subject.
  • a similarity in gene expression patterns between the test cell population and the reference cell population indicates the agent is not suitable for treating the PPAR ⁇ pathophysiology in the subject, while a dissimilar gene expression patterns indicate the agent will be suitable for treating the subject.
  • a decrease in expression of one or more of the sequences ADIPOS: 1, 2, 3, 7, 16, 17, 20-22, 27, 29, 30, and 32-43 or an increase in expression of one or more of the sequences ADIPOS: 4, 5, 8 -10, 12, 13, 15, 18, 19, 23-26, 28 and 31 in a test cell population relative to a reference cell population is indicative that the agent is therapeutic.
  • test agent can be any compound or composition.
  • test agents are compounds and composition know to be PPAR ⁇ ligands, e.g. N-(2-benzoylphenyl)-L-tyrosine.
  • the invention further provides a method of diagnosing a pathophysiology associated with the PPAR ⁇ mediated pathway, e.g., non-insulin dependent diabetes millitus, in a subject.
  • a pathophysiology is diagnosed by examining the expression of one or more ADIPO nucleic acid sequences from a test population of cells from a subject suspected of have the pathophysiology.
  • ADIPO nucleic acid sequences e.g. ADIPO: 1-32 and optionally 33-43 is measured in the test cell and compared to the expression of the sequences in the reference cell population.
  • the reference cell population contains at least one cell whose PPAR ⁇ status, or disease status (i.e., the reference cell population is from a NIDDM subject) is known. If the reference cell population contains cells that have not been exposed to a PPAR ⁇ ligand, then a similarity in expression between ADIPO sequences in the test population and the reference cell population indicates the subject does not have a PPAR ⁇ mediated pathophysiology. A difference in expression between ADIPO sequences in the test population and the reference cell population indicates the reference cell population has a PPAR ⁇ mediated pathophysiology.
  • PPAR ⁇ ligand a similarity in expression pattern between the test cell population and the reference cell population indicates the test cell population has a PPAR ⁇ mediated pathophysiology.
  • a difference in expression between ADIPO sequences in the test population and the reference cell population indicates the subject does not have a PPAR ⁇ mediated pathophysiology.
  • Also included in the invention is a method of treating, i.e, preventing or delaying the onset of a pathophysiology associated with the PPAR ⁇ mediated pathway in a subject by administering to the subject an agent which modulates the expression or activity of one or more nucleic acids selected from the group consisting of ADIPO 1-32 and, optionally, ADIPO 33-43.
  • ""Modulates” is meant to include increase or decrease expression or activity of the ADIPO nucleic acids.
  • modulation results in alteration alter the expression or activity of the ADIPO genes or gene products in a subject to a level similar or identical to a subject not suffering from the pathophysiology.
  • the pathophysiologies can be any of the pathophysiologies described herein, e.g., NIDDM.
  • the subject can be, e.g., a human, a rodent such as a mouse or rat, or a dog or cat.
  • Therapeutics include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes.
  • the Therapeutics of the present invention may be administered by any convenient route, for example by infusion or bolus injection, by abso ⁇ tion through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically-active agents. Administration can be systemic or local.
  • Intraventricular injection may be facilitated by an intraventricular catheter attached to a reservoir (e.g., an Ommaya reservoir).
  • Pulmonary administration may also be employed by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
  • administration may be by direct injection at the site (or former site) of a malignant tumor or neoplastic or pre-neoplastic tissue.
  • a Therapeutic of the present invention including, e.g. : (i) encapsulation in liposomes, microparticles, microcapsules; (ii) recombinant cells capable of expressing the Therapeutic; (iii) receptor-mediated endocytosis (See, e.g., Wu and Wu, 1987. J Biol Chem 262:4429-4432); (iv) construction of a Therapeutic nucleic acid as part of a retroviral or other vector, and the like.
  • the Therapeutic may be delivered in a vesicle, in particular a liposome.
  • lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution.
  • amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution.
  • Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. Preparation of such liposomal formulations is within the level of skill in the art, as disclosed, for example, in U.S. Pat. No. 4,837,028; and U.S. Pat. No.
  • the Therapeutic can be delivered in a controlled release system including, e.g. : a delivery pump (See, e.g., Saudek, et al, 1989. New Engl J Med 321 :574 and a semi-permeable polymeric material (See, e.g., Howard, et al, 1989. J Neurosurg 71 :105).
  • a delivery pump See, e.g., Saudek, et al, 1989. New Engl J Med 321 :574
  • a semi-permeable polymeric material See, e.g., Howard, et al, 1989. J Neurosurg 71 :105.
  • the controlled release system can be placed in proximity of the therapeutic target (e.g., the brain), thus requiring only a fraction of the systemic dose. See, e.g., Goodson, In: Medical Applications of Controlled Release 1984. (CRC Press, Bocca Raton, FL).
  • the Therapeutic nucleic acid may be administered in vivo to promote expression of its encoded protein, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular (e.g., by use of a retroviral vector, by direct injection, by use of microparticle bombardment, by coating with lipids or cell-surface receptors or transfecting agents, or by administering it in linkage to a homeobox-like peptide which is known to enter the nucleus (See, e.g., Joliot, et al, 1991. Proc Natl Acad Sci USA 88:1864-1868), and the like.
  • a nucleic acid Therapeutic can be introduced intracellularly and inco ⁇ orated within host cell DNA for expression, by homologous recombination.
  • the term "therapeutically effective amount” means the total amount of each active component of the pharmaceutical composition or method that is sufficient to show a meaningful patient benefit, i.e., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions.
  • a meaningful patient benefit i.e., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions.
  • the term refers to that ingredient alone.
  • the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.
  • the amount of the Therapeutic of the invention which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and may be determined by standard clinical techniques by those of average skill within the art. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the overall seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Ultimately, the attending physician will decide the amount of protein of the present invention with which to treat each individual patient. Initially, the attending physician will administer low doses of protein of the present invention and observe the patient's response.
  • suitable dosage ranges for intravenous administration of the Therapeutics of the present invention are generally about 20-500 micrograms ( ⁇ g) of active compound per kilogram (Kg) body weight.
  • suitable dosage ranges for intranasal administration are generally about 0.01 pg/kg body weight to 1 mg/kg body weight.
  • Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • Suppositories generally contain active ingredient in the range of 0.5% to 10% by weight; oral formulations preferably contain 10% to 95% active ingredient.
  • the duration of intravenous therapy using the pharmaceutical composition of the present invention will vary, depending on the severity of the disease being treated and the condition and potential idiosyncratic response of each individual patient. It is contemplated that the duration of each application of the protein of the present invention will be in the range of 12 to 24 hours of continuous intravenous administration. Ultimately the attending physician will decide on the appropriate duration of intravenous therapy using the pharmaceutical composition of the present invention.
  • Polynucleotides of the present invention can also be used for gene therapy.
  • Gene therapy refers to therapy that is performed by the administration of a specific nucleic acid to a subject. Delivery of the Therapeutic nucleic acid into a mammalian subject may be either direct (i.e., the patient is directly exposed to the nucleic acid or nucleic acid-containing vector) or indirect (i.e., cells are first transformed with the nucleic acid in vitro, then transplanted into the patient). These two approaches are known, respectively, as in vivo or ex vivo gene therapy. Polynucleotides of the invention may also be administered by other known methods for introduction of nucleic acid into a cell or organism (including, without limitation, in the form of viral vectors or naked DNA). Any of the methodologies relating to gene therapy available within the art may be used in the practice of the present invention. See e.g., Goldspiel, et al, 1993. Clin Pharm 12:488-505.
  • Cells may also be cultured ex vivo in the presence of therapeutic agents or proteins of the present invention in order to proliferate or to produce a desired effect on or activity in such cells. Treated cells can then be introduced in vivo for therapeutic pu ⁇ oses.
  • the differentially expressed ADIPO sequences identified herein also allow for the course of treatment of a pathophysiology to be monitored.
  • a test cell population is provided from a subject undergoing treatment for pathophysiologies associated with the PPAR ⁇ mediated pathway. If desired, test cell populations can be taken from the subject at various time points before, during, or after treatment.
  • Expression of one or more of the ADIPO sequences, e.g., AD IPOs: 1-32 and, optionally, ADIPO 33-43, in the cell population is then measured and compared to a reference cell population which includes cells whose pathophysiologic state is known. Preferably, the reference cells not been exposed to the treatment.
  • the reference cell population contains no cells exposed to the treatment, a similarity in expression between ADIPO sequences in the test cell population and the reference cell population indicates that the treatment is efficacious. However, a difference in expression between ADIPO sequences in the test population and this reference cell population indicates the treatment is not efficacious.
  • efficacious is meant that the treatment leads to a decrease in the pathophysiology in a subject.
  • effcacious means that the treatment retards or prevents a pathophysiology.
  • PPAR ⁇ mediated pathophysiology is NIDDM
  • a "efficacious" treatment is one that increases insulin sensitivity in a subject.
  • Efficaciousness can be determined in association with any known method for treating the particular pathophysiology.
  • novel nucleic acids that include a nucleic acid sequence selected from the group consisting of ADIPOs:l-15, or its complement, as well as vectors and cells including these nucleic acids. Also provided are polypeptides encoded by ADIPO nucleic acid or biologically active portions thereof.
  • nucleic acid fragments sufficient for use as hybridization probes to identify ADIPO-encoding nucleic acids (e.g., ADIPO mRNA) and fragments for use as polymerase chain reaction (PCR) primers for the amplification or mutation of ADIPO nucleic acid molecules.
  • nucleic acid molecule is intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA generated using nucleotide analogs, and derivatives, fragments and homologs thereof.
  • the nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.
  • Probes refer to nucleic acid sequences of variable length, preferably between at least about 10 nucleotides (nt) or as many as about, e.g., 6,000 nt, depending on use. Probes are used in the detection of identical, similar, or complementary nucleic acid sequences. Longer length probes are usually obtained from a natural or recombinant source, are highly specific and much slower to hybridize than oligomers. Probes may be single- or double-stranded and designed to have specificity in PCR, membrane-based hybridization technologies, or ELISA-like technologies.
  • an "isolated" nucleic acid molecule is one that is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid.
  • isolated nucleic acid molecules include, but are not limited to, recombinant DNA molecules contained in a vector, recombinant DNA molecules maintained in a heterologous host cell, partially or substantially purified nucleic acid molecules, and synthetic DNA or RNA molecules.
  • an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.
  • the isolated ADIPO nucleic acid molecule can contain less than about 50 kb, 25 kb, 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived.
  • an "isolated" nucleic acid molecule such as a cDNA molecule, can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or of chemical precursors or other chemicals when chemically synthesized.
  • a nucleic acid molecule of the present invention e.g., a nucleic acid molecule having the nucleotide sequence of any of ADIPOS: 1-15, or a complement of any of these nucleotide sequences, can be isolated using standard molecular biology techniques and the sequence information provided herein.
  • ADIPO nucleic acid sequences can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook et al, eds., MOLECULAR CLONING: A LABORATORY MANUAL 2 nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausubel, et al, eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, 1993.)
  • a nucleic acid of the invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques.
  • the nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.
  • oligonucleotides corresponding to ADIPO nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
  • oligonucleotide refers to a series of linked nucleotide residues, which oligonucleotide has a sufficient number of nucleotide bases to be used in a PCR reaction.
  • a short oligonucleotide sequence may be based on, or designed from, a genomic or cDNA sequence and is used to amplify, confirm, or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue.
  • Oligonucleotides comprise portions of a nucleic acid sequence having at least about 10 nt and as many as 50 nt, preferably about 15 nt to 30 nt. They may be chemically synthesized and may be used as probes.
  • an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule that is a complement of the nucleotide sequence shown in ADIPOs: 1-15 .
  • an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule that is a complement of the nucleotide sequence shown in any of these sequences, or a portion of any of these nucleotide sequences.
  • a nucleic acid molecule that is complementary to the nucleotide sequence shown in ADIPOs: 1-15 is one that is sufficiently complementary to the nucleotide sequence shown, such that it can hydrogen bond with little or no mismatches to the nucleotide sequences shown, thereby forming a stable duplex.
  • binding means the physical or chemical interaction between two polypeptides or compounds or associated polypeptides or compounds or combinations thereof. Binding includes ionic, non-ionic, Non der Waals, hydrophobic interactions, etc.
  • a physical interaction can be either direct or indirect. Indirect interactions may be through or due to the effects of another polypeptide or compound. Direct binding refers to interactions that do not take place through, or due to, the effect of another polypeptide or compound, but instead are without other substantial chemical intermediates.
  • nucleic acid molecule of the invention can comprise only a portion of the nucleic acid sequence of AD IPOs: 1-15 e.g., a fragment that can be used as a probe or primer or a fragment encoding a biologically active portion of ADIPO.
  • Fragments provided herein are defined as sequences of at least 6 (contiguous) nucleic acids or at least 4 (contiguous) amino acids, a length sufficient to allow for specific hybridization in the case of nucleic acids or for specific recognition of an epitope in the case of amino acids, respectively, and are at most some portion less than a full length sequence. Fragments may be derived from any contiguous portion of a nucleic acid or amino acid sequence of choice.
  • Derivatives are nucleic acid sequences or ⁇ amino acid sequences formed from the native compounds either directly or by modification or partial substitution.
  • Analogs are nucleic acid sequences or amino acid sequences that have a structure similar to, but not identical to, the native compound but differs from it in respect to certain components or side chains. Analogs may be synthetic or from a different evolutionary origin and may have a similar or opposite metabolic activity compared to wild type.
  • Derivatives and analogs may be full length or other than full length, if the derivative or analog contains a modified nucleic acid or amino acid, as described below.
  • Derivatives or analogs of the nucleic acids or proteins of the invention include, but are not limited to, molecules comprising regions that are substantially homologous to the nucleic acids or proteins of the invention, in various embodiments, by at least about 45%, 50%, 70%, 80%, 95%, 98%, or even 99%o identity (with a preferred identity of 80-99%) over a nucleic acid or amino acid sequence of identical size or when compared to an aligned sequence in which the alignment is done by a computer homology program known in the art, or whose encoding nucleic acid is capable of hybridizing to the complement of a sequence encoding the aforementioned proteins under stringent, moderately stringent, or low stringent conditions.
  • a “homologous nucleic acid sequence” or “homologous amino acid sequence,” or variations thereof, refer to sequences characterized by a homology at the nucleotide level or amino acid level as discussed above.
  • Homologous nucleotide sequences encode those sequences coding for isoforms of a ADIPO polypeptide. Isoforms can be expressed in different tissues of the same organism as a result of, for example, alternative splicing of RNA. Alternatively, isoforms can be encoded by different genes.
  • homologous nucleotide sequences include nucleotide sequences encoding for a ADIPO polypeptide of species other than humans, including, but not limited to, mammals, and thus can include, e.g., mouse, rat, rabbit, dog, cat cow, horse, and other organisms.
  • homologous nucleotide sequences also include, but are not limited to, naturally occurring allelic variations and mutations of the nucleotide sequences set forth herein.
  • a homologous nucleotide sequence does not, however, include the nucleotide sequence encoding a human ADIPO protein.
  • Homologous nucleic acid sequences include those nucleic acid sequences that encode conservative amino acid substitutions (see below) in a
  • ADIPO polypeptide as well as a polypeptide having a ADIPO activity.
  • a homologous amino acid sequence does not encode the amino acid sequence of a human ADIPO polypeptide.
  • the nucleotide sequence determined from the cloning of human ADIPO genes allows for the generation of probes and primers designed for use in identifying and/or cloning ADIPO homologues in other cell types, e.g., from other tissues, as well as ADIPO homologues from other mammals.
  • the probe/primer typically comprises a substantially purified oligonucleotide.
  • the oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, 25, 50, 100, 150, 200, 250, 300, 350 or 400 consecutive sense strand nucleotide sequence of a nucleic acid comprising a ADIPO sequence, or an anti-sense strand nucleotide sequence of a nucleic acid comprising a ADIPO sequence, or of a naturally occurring mutant of these sequences.
  • Probes based on human ADIPO nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins.
  • the probe further comprises a label group attached thereto, e.g., the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.
  • Such probes can be used as a part of a diagnostic test kit for identifying cells or tissue which misexpress a ADIPO protein, such as by measuring a level of a ADIPO-encoding nucleic acid in a sample of cells from a subject e.g., detecting ADIPO mRNA levels or determining whether a genomic ADIPO gene has been mutated or deleted.
  • a polypeptide having a biologically active portion of ADIPO refers to polypeptides exhibiting activity similar, but not necessarily identical to, an activity of a polypeptide of the present invention, including mature forms, as measured in a particular biological assay, with or without dose dependency.
  • a nucleic acid fragment encoding a "biologically active portion of ADIPO” can be prepared by isolating a portion of ADIPOs: 1-15, that encodes a polypeptide having a ADIPO biological activity, expressing the encoded portion of ADIPO protein (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of ADIPO.
  • a nucleic acid fragment encoding a biologically active portion of a ADIPO polypeptide can optionally include an ATP -binding domain.
  • a nucleic acid fragment encoding a biologically active portion of ADIPO includes one or more regions.
  • the invention further encompasses nucleic acid molecules that differ from the disclosed or referenced ADIPO nucleotide sequences due to degeneracy of the genetic code. These nucleic acids thus encode the same ADIPO protein as that encoded by nucleotide sequence comprising a ADIPO nucleic acid as shown in, e.g. , ADIPO 1-15
  • ADIPO nucleotide sequence shown in ADIPOs:l-15
  • DNA sequence polymo ⁇ hisms that lead to changes in the amino acid sequences of a ADIPO polypeptide may exist within a population (e.g., the human population).
  • Such genetic polymo ⁇ hism in the ADIPO gene may exist among individuals within a population due to natural allelic variation.
  • the terms "gene” and "recombinant gene” refer to nucleic acid molecules comprising an open reading frame encoding a ADIPO protein, preferably a mammalian ADIPO protein.
  • Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence of the ADIPO gene. Any and all such nucleotide variations and resulting amino acid polymo ⁇ hisms in ADIPO that are the result of natural allelic variation and that do not alter the functional activity of ADIPO are intended to be within the scope of the invention.
  • nucleic acid molecules encoding ADIPO proteins from other species are intended to be within the scope of the invention.
  • Nucleic acid molecules corresponding to natural allelic variants and homologues of the ADIPO DNAs of the invention can be isolated based on their homology to the human ADIPO nucleic acids disclosed herein using the human cDNAs, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.
  • a soluble human ADIPO DNA can be isolated based on its homology to human membrane-bound ADIPO.
  • a membrane-bound human ADIPO DNA can be isolated based on its homology to soluble human ADIPO.
  • an isolated nucleic acid molecule of the invention is at least 6 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of AD IPOs: 1-15.
  • the nucleic acid is at least 10, 25, 50, 100, 250 or 500 nucleotides in length.
  • an isolated nucleic acid molecule of the invention hybridizes to the coding region.
  • hybridizes under stringent conditions is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60%> homologous to each other typically remain hybridized to each other.
  • Homologs i.e., nucleic acids encoding ADIPO proteins derived from species other than human
  • other related sequences e.g., paralogs
  • stringent hybridization conditions refers to conditions under which a probe, primer or oligonucleotide will hybridize to its target sequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures than shorter sequences. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50%> of the probes are occupied at equilibrium.
  • Tm 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 (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes, primers or oligonucleotides (e.g., 10 nt to 50 nt) and at least about 60°C for longer probes, primers and oligonucleotides.
  • Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.
  • the conditions are such that sequences at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99%) homologous to each other typically remain hybridized to each other.
  • a non-limiting example of stringent hybridization conditions is hybridization in a high salt buffer comprising 6X SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% FicoU, 0.02% BSA, and 500 mg/ml denatured salmon sperm D ⁇ A at 65 °C.
  • nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of ADIPOs: 1-15 corresponds to a naturally occurring nucleic acid molecule.
  • a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
  • a nucleic acid sequence that is hybridizable to the nucleic acid molecule comprising the nucleotide sequence of ADIPOs: 1-15 or fragments, analogs or derivatives thereof, under conditions of moderate stringency is provided.
  • moderate stringency hybridization conditions are hybridization in 6X SSC, 5X Denhardt's solution, 0.5% SDS and 100 mg/ml denatured salmon sperm DNA at 55°C, followed by one or more washes in IX SSC, 0.1% SDS at 37°C.
  • Other conditions of moderate stringency that may be used are well known in the art. See, e.g., Ausubel et al.
  • a nucleic acid that is hybridizable to the nucleic acid molecule comprising the nucleotide sequence of ADIPOs:l-15or fragments, analogs or derivatives thereof, under conditions of low stringency is provided.
  • a non-limiting example of low stringency hybridization conditions are hybridization in 35% formamide, 5X SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 mg/ml denatured salmon sperm DNA, 10%) (wt/vol) dextran sulfate at 40°C, followed by one or more washes in 2X SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS at 50°C.
  • ADIPO sequence In addition to naturally-occurring allelic variants of the ADIPO sequence that may exist in the population, the skilled artisan will further appreciate that changes can be introduced into an ADIPO nucleic acid or directly into an ADIPO polypeptide sequence without altering the functional ability of the ADIPO protein.
  • nucleotide sequence of ADIPOs: l-15 will be altered, thereby leading to changes in the amino acid sequence of the encoded ADIPO protein.
  • nucleotide substitutions that result in amino acid substitutions at various "non-essential" amino acid residues can be made in the sequence of ADIPOs: 1-15A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of ADIPO without altering the biological activity, whereas an "essential" amino acid residue is required for biological activity.
  • amino acid residues that are conserved among the ADIPO proteins of the present invention are predicted to be particularly unamenable to alteration.
  • amino acid residues that are conserved among family members of the ADIPO proteins of the present invention are also predicted to be particularly unamenable to alteration. As such, these conserved domains are not likely to be amenable to mutation. Other amino acid residues, however, (e.g., those that are not conserved or only semi-conserved among members of the ADIPO proteins) may not be essential for activity and thus are likely to be amenable to alteration.
  • nucleic acid molecules encoding ADIPO proteins that contain changes in amino acid residues that are not essential for activity. Such ADIPO proteins differ in amino acid sequence from the amino acid sequences of polypeptides encoded by nucleic acids containing ADIPOs: 1-15, yet retain biological activity.
  • the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 45% homologous, more preferably 60%, and still more preferably at least about 70%, 80%), 90%, 95%o, 98%, and most preferably at least about 99% homologous to the amino acid sequence of the amino acid sequences of polypeptides encoded by nucleic acids comprising ADIPOs: 1-15.
  • An isolated nucleic acid molecule encoding a ADIPO protein homologous to can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of a nucleic acid comprising ADIPOs: 1-15, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein.
  • Mutations can be introduced into a nucleic acid comprising ADIPOs: 1-15 by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues.
  • a "conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g.
  • a predicted nonessential amino acid residue in ADIPO is replaced with another amino acid residue from the same side chain family.
  • mutations can be introduced randomly along all or part of a ADIPO coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for ADIPO biological activity to identify mutants that retain activity.
  • the encoded protein can be expressed by any recombinant technology known in the art and the activity of the protein can be determined.
  • a mutant ADIPO protein can be assayed for (1) the ability to form protein: protein interactions with other ADIPO proteins, other cell-surface proteins, or biologically active portions thereof, (2) complex formation between a mutant ADIPO protein and a ADIPO ligand; (3) the ability of a mutant ADIPO protein to bind to an intracellular target protein or biologically active portion thereof; (e.g., avidin proteins); (4) the ability to bind ATP; or (5) the ability to specifically bind a ADIPO protein antibody.
  • ADIPO 1-15 wherein the fragment of the complementary polynucleotide sequence hybridizes to the first sequence.
  • the nucleic acid is RNA or DNA.
  • Another aspect of the invention pertains to isolated antisense nucleic acid molecules that are hybridizable to or complementary to the nucleic acid molecule comprising the nucleotide sequence of a ADIPO sequence or fragments, analogs or derivatives thereof.
  • An "antisense" nucleic acid comprises a nucleotide sequence that is complementary to a "sense" nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence.
  • antisense nucleic acid molecules are provided that comprise a sequence complementary to at least about 10, 25, 50, 100, 250 or 500 nucleotides or an entire ADIPO coding strand, or to only a portion thereof.
  • Nucleic acid molecules encoding fragments, homologs, derivatives and analogs of a ADIPO protein, or antisense nucleic acids complementary to a nucleic acid comprising a ADIPO nucleic acid sequence are additionally provided.
  • an antisense nucleic acid molecule is antisense to a "coding region" of the coding strand of a nucleotide sequence encoding ADIPO.
  • coding region refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues.
  • the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence encoding ADIPO.
  • noncoding region refers to 5' and 3' sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5' and 3' untranslated regions).
  • antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick or Hoogsteen base pairing.
  • the antisense nucleic acid molecule can be complementary to the entire coding region of ADIPO mRNA, but more preferably is an oligonucleotide that is antisense to only a portion of the coding or noncoding region of ADIPO mRNA.
  • the antisense oligonucleotide can be complementary to the region surrounding the translation start site of ADIPO mRNA.
  • An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.
  • an antisense nucleic acid of the invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known in the art.
  • an antisense nucleic acid e.g., an antisense oligonucleotide
  • an antisense nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used.
  • modified nucleotides that can be used to generate the antisense nucleic acid include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl- 2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-mefhylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'
  • the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
  • the antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a ADIPO protein to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation.
  • the hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the major groove of the double helix.
  • An example of a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site.
  • antisense nucleic acid molecules can be modified to target selected cells and then administered systemically.
  • antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens.
  • the antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.
  • the antisense nucleic acid molecule of the invention is an ⁇ -anomeric nucleic acid molecule.
  • An ⁇ -anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual ⁇ -units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids Res 15: 6625-6641).
  • the antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res 15: 6131-6148) or a chimeric RNA -DNA analogue (Inoue et al. (1987) EERS Lett 215: 327-330).
  • an antisense nucleic acid of the invention is a ribozyme.
  • Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region.
  • ribozymes e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave ADIPO mRNA transcripts to thereby inhibit translation of ADIPO mRNA.
  • a ribozyme having specificity for a ADIPO-encoding nucleic acid can be designed based upon the nucleotide sequence of a ADIPO DNA disclosed herein.
  • a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a ADIPO-encoding mRNA. See, e.g., Cech et al. U.S. Pat. No. 4,987,071 ; and Cech et al. U.S. Pat. No. 5,116,742.
  • ADIPO mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel et al, (1993) Science 261 :1411-1418.
  • ADIPO gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of a ADIPO nucleic acid (e.g., the ADIPO promoter and/or enhancers) to form triple helical structures that prevent transcription of the ADIPO gene in target cells.
  • a ADIPO nucleic acid e.g., the ADIPO promoter and/or enhancers
  • triple helical structures that prevent transcription of the ADIPO gene in target cells.
  • the nucleic acids of ADIPO can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule.
  • the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids (see Hyrup et al. (1996) Bioorg Med Chem 4: 5-23).
  • the terms "peptide nucleic acids” or "PNAs” refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained.
  • PNAs The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength.
  • the synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup et al. (1996) above; Perry-O'Keefe et al. (1996) PNAS 93: 14670-675.
  • PNAs of ADIPO can be used in therapeutic and diagnostic applications.
  • PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication.
  • PNAs of ADIPO can also be used, e.g., in the analysis of single base pair mutations in a gene by, e.g., PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., SI nucleases (Hyrup B. (1996) above); or as probes or primers for DNA sequence and hybridization (Hyrup et al. (1996), above; Perry-O'Keefe (1996), above).
  • PNAs of ADIPO can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art.
  • PNA-DNA chimeras of ADIPO can be generated that may combine the advantageous properties of PNA and DNA.
  • Such chimeras allow DNA recognition enzymes, e.g., RNase H and DNA polymerases, to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity.
  • PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup (1996) above).
  • the synthesis of PNA-DNA chimeras can be performed as described in Hyrup (1996) above and Finn et al. (1996) Nucl Acids Res 24: 3357-63.
  • a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry, and modified nucleoside analogs, e.g., 5'-(4-methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite, can be used between the PNA and the 5' end of DNA (Mag et al.
  • PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment (Finn et al. (1996) above).
  • chimeric molecules can be synthesized with a 5' DNA segment and a 3' PNA segment. See, Petersen et al. (1975) Bioorg Med Chem Lett 5: 1119-1 1124.
  • the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al, 1989, Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556; Lemaitre et al, 1987, Proc. Natl Acad. Sci. 84:648-652; PCT Publication No. W088/09810) or the blood-brain barrier (see, e.g., PCT Publication No. W089/10134).
  • peptides e.g., for targeting host cell receptors in vivo
  • agents facilitating transport across the cell membrane see, e.g., Letsinger et al, 1989, Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556; Lemaitre et al, 1987, Proc. Nat
  • oligonucleotides can be modified with hybridization triggered cleavage agents (See, e.g., Krol et al, 1988, BioTechniques 6:958-976) or intercalating agents. (See, e.g., Zon, 1988, Pharm. Res. 5: 539-549).
  • the oligonucleotide may be conjugated to another molecule, e.g., a peptide, a hybridization triggered cross-linking agent, a transport agent, a hybridization-triggered cleavage agent, etc.
  • ADIPO POLYPEPTIDES e.g., a peptide, a hybridization triggered cross-linking agent, a transport agent, a hybridization-triggered cleavage agent, etc.
  • ADIPO proteins and biologically active portions thereof, or derivatives, fragments, analogs or homologs thereof.
  • polypeptide fragments suitable for use as immunogens to raise anti-ADIPO antibodies are provided.
  • native ADIPO proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques.
  • ADIPO proteins are produced by recombinant DNA techniques.
  • a ADIPO protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques.
  • an “isolated” or “purified” protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the ADIPO protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized.
  • the language “substantially free of cellular material” includes preparations of ADIPO protein in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced. In one embodiment, the language
  • substantially free of cellular material includes preparations of ADIPO protein having less than about 30%) (by dry weight) of non-ADIPO protein (also referred to herein as a "contaminating protein"), more preferably less than about 20% of non-ADIPO protein, still more preferably less than about 10% of non-ADIPO protein, and most preferably less than about 5% non-ADIPO protein.
  • non-ADIPO protein also referred to herein as a "contaminating protein”
  • contaminating protein also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%), and most preferably less than about 5% of the volume of the protein preparation.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of ADIPO protein in which the protein is separated from chemical precursors or other chemicals that are involved in the synthesis of the protein.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of ADIPO protein having less than about 30% (by dry weight) of chemical precursors or non-ADIPO chemicals, more preferably less than about 20% chemical precursors or non-ADIPO chemicals, still more preferably less than about 10% chemical precursors or non-ADIPO chemicals, and most preferably less than about 5% chemical precursors or non-ADIPO chemicals.
  • Biologically active portions of a ADIPO protein include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequence of the ADIPO protein, e.g., the amino acid sequence encoded by a nucleic acid comprising ADIPO 1-20 that include fewer amino acids than the full length ADIPO proteins, and exhibit at least one activity of a ADIPO protein.
  • biologically active portions comprise a domain or motif with at least one activity of the ADIPO protein.
  • a biologically active portion of a ADIPO protein can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acids in length.
  • a biologically active portion of a ADIPO protein of the present invention may contain at least one of the above-identified domains conserved between the ADIPO proteins.
  • An alternative biologically active portion of a ADIPO protein may contain at least two of the above-identified domains.
  • Another biologically active portion of a ADIPO protein may contain at least three of the above-identified domains.
  • Yet another biologically active portion of a ADIPO protein of the present invention may contain at least four of the above-identified domains.
  • biologically active portions in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native ADIPO protein.
  • the ADIPO protein is substantially homologous to one of these ADIPO proteins and retains its the functional activity, yet differs in amino acid sequence due to natural allelic variation or mutagenesis, as described in detail below.
  • the invention includes an isolated polypeptide comprising an amino acid sequence that is 80%) or more identical to the sequence of a polypeptide whose expression is modulated in a mammal to which PPAR ⁇ ligand is administered. DETERMINING HOMOLOGY BETWEEN TWO OR MORE SEQUENCES
  • the sequences are aligned for optimal comparison pu ⁇ oses (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are homologous at that position (i.e., as used herein amino acid or nucleic acid "homology” is equivalent to amino acid or nucleic acid "identity").
  • the nucleic acid sequence homology may be determined as the degree of identity between two sequences.
  • the homology may be determined using computer programs known in the art, such as GAP software provided in the GCG program package. See Needleman and Wunsch 1970 J Mol Biol 48: 443-453.
  • GAP software provided in the GCG program package. See Needleman and Wunsch 1970 J Mol Biol 48: 443-453.
  • GAP creation penalty of 5.0 and GAP extension penalty of 0.3 the coding region of the analogous nucleic acid sequences referred to above exhibits a degree of identity preferably of at least 70%, 75%, 80%, 85%, 90%, 95%, 98%o, or 99%, with the CDS (encoding) part of a DNA sequence comprising ADIPOS: 1-15.
  • sequence identity refers to the degree to which two polynucleotide or polypeptide sequences are identical on a residue-by-residue basis over a particular region of comparison.
  • percentage of sequence identity is calculated by comparing two optimally aligned sequences over that region of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I, in the case of nucleic acids) 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 region of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • substantially identical denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 80 percent sequence identity, preferably at least 85 percent identity and often 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison region.
  • an ADIPO "chimeric protein” or “fusion protein” comprises an ADIPO polypeptide operatively linked to a non-ADIPO polypeptide.
  • a "ADIPO polypeptide” refers to a polypeptide having an amino acid sequence corresponding to ADIPO
  • a non-ADIPO polypeptide refers to a polypeptide having an amino acid sequence corresponding to a protein that is not substantially homologous to the ADIPO protein, e.g., a protein that is different from the ADIPO protein and that is derived from the same or a different organism.
  • ADIPO polypeptide can correspond to all or a portion of an ADIPO protein.
  • an ADIPO fusion protein comprises at least one biologically active portion of an ADIPO protein.
  • an ADIPO fusion protein comprises at least two biologically active portions of an ADIPO protein.
  • an ADIPO fusion protein comprises at least three biologically active portions of an ADIPO protein.
  • the term "operatively linked" is intended to indicate that the ADIPO polypeptide and the non-ADIPO polypeptide are fused in-frame to each other.
  • the non-ADIPO polypeptide can be fused to the N-terminus or C-terminus of the ADIPO polypeptide.
  • an ADIPO fusion protein comprises an ADIPO domain operably linked to the extracellular domain of a second protein.
  • Such fusion proteins can be further utilized in screening assays for compounds which modulate ADIPO activity (such assays are described in detail below).
  • the fusion protein is a GST- ADIPO fusion protein in which the ADIPO sequences are fused to the C-terminus of the GST (i.e., glutathione S-transferase) sequences.
  • Such fusion proteins can facilitate the purification of recombinant ADIPO.
  • the fusion protein is an ADIPO protein containing a heterologous signal sequence at its N-terminus.
  • a native ADIPO signal sequence can be removed and replaced with a signal sequence from another protein.
  • expression and/or secretion of ADIPO can be increased through use of a heterologous signal sequence.
  • the fusion protein is an ADIPO-immunoglobulin fusion protein in which the ADIPO sequences comprising one or more domains are fused to sequences derived from a member of the immunoglobulin protein family.
  • the ADIPO-immunoglobulin fusion proteins of the invention can be inco ⁇ orated into pharmaceutical compositions and administered to a subject to inhibit an interaction between a ADIPO ligand and a ADIPO protein on the surface of a cell, to thereby suppress ADIPO-mediated signal transduction in vivo.
  • the ADIPO-immunoglobulin fusion proteins can be used to affect the bioavailabihty of an ADIPO cognate ligand.
  • ADIPO-immunoglobulin fusion proteins of the invention can be used as immunogens to produce anti-ADIPO antibodies in a subject, to purify ADIPO ligands, and in screening assays to identify molecules that inhibit the interaction of ADIPO with a ADIPO ligand.
  • An ADIPO chimeric or fusion protein of the invention can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, e.g., by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Ausubel et al. (eds.) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, 1992).
  • anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence
  • many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide).
  • An ADIPO-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the ADIPO protein.
  • the present invention also pertains to variants of the ADIPO proteins that function as either ADIPO agonists (mimetics) or as ADIPO antagonists.
  • Variants of the ADIPO protein can be generated by mutagenesis, e.g., discrete point mutation or truncation of the ADIPO protein.
  • An agonist of the ADIPO protein can retain substantially the same, or a subset of, the biological activities of the naturally occurring form of the ADIPO protein.
  • An antagonist of the ADIPO protein can inhibit one or more of the activities of the naturally occurring form of the ADIPO protein by, for example, competitively binding to a downstream or upstream member of a cellular signaling cascade which includes the ADIPO protein.
  • specific biological effects can be elicited by treatment with a variant of limited function.
  • treatment of a subject with a variant having a subset of the biological activities of the naturally occurring form of the protein has fewer side effects in a subject relative to treatment with the naturally occurring form of the ADIPO proteins.
  • Variants of the ADIPO protein that function as either ADIPO agonists (mimetics) or as ADIPO antagonists can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of the ADIPO protein for ADIPO protein agonist or antagonist activity.
  • a variegated library of ADIPO variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library.
  • a variegated library of ADIPO variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential ADIPO sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of ADIPO sequences therein.
  • a degenerate set of potential ADIPO sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of ADIPO sequences therein.
  • methods which can be used to produce libraries of potential ADIPO variants from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector.
  • degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential ADIPO sequences.
  • Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu Rev Biochem 53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucl Acid Res 1 1 :477.
  • libraries of fragments of the ADIPO protein coding sequence can be used to generate a variegated population of ADIPO fragments for screening and subsequent selection of variants of an ADIPO protein.
  • a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of a ADIPO coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA that can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with SI nuclease, and ligating the resulting fragment library into an expression vector.
  • an expression library can be derived which encodes N-terminal and internal fragments of various sizes of the ADIPO protein.
  • Recursive ensemble mutagenesis (REM), a new technique that enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify ADIPO variants (Arkin and Yourvan (1992) PNAS 89:7811-7815; Delgrave et al. (1993) Protein Engineering 6:327-331).
  • REM Recursive ensemble mutagenesis
  • various suitable host animals e.g., rabbit, goat, mouse or other mammal
  • An appropriate immunogenic preparation can contain, for example, recombinantly expressed ADIPO protein or a chemically synthesized ADIPO polypeptide.
  • the preparation can further include an adjuvant.
  • adjuvants used to increase the immunological response include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.), human adjuvants such as Bacille Calmette-Guerin and Corynebacterium parvum, or similar immunostimulatory agents.
  • the antibody molecules directed against ADIPO can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as protein A chromatography to obtain the IgG fraction.
  • monoclonal antibody or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of ADIPO.
  • a monoclonal antibody composition thus typically displays a single binding affinity for a particular ADIPO protein with which it immunoreacts.
  • any technique that provides for the production of antibody molecules by continuous cell line culture may be utilized.
  • Such techniques include, but are not limited to, the hybridoma technique (see Kohler & Milstein, 1975 Nature 256: 495-497); the trioma technique; the human B-cell hybridoma technique (see Kozbor, et al, 1983 Immunol Today 4: 72) and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole, et al, 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).
  • Human monoclonal antibodies may be utilized in the practice of the present invention and may be produced by using human hybridomas (see Cote, et al, 1983.
  • techniques can be adapted for the production of single-chain antibodies specific to a ADIPO protein (see e.g., U.S. Patent No. 4,946,778).
  • methods can be adapted for the construction of F ab expression libraries (see e.g., Huse, et al, 1989 Science 246: 1275-1281) to allow rapid and effective identification of monoclonal F ab fragments with the desired specificity for a ADIPO protein or derivatives, fragments, analogs or homologs thereof.
  • Non-human antibodies can be "humanized" by techniques well known in the art. See e.g., U.S. Patent No. 5,225,539.
  • Antibody fragments that contain the idiotypes to a ADIPO protein may be produced by techniques known in the art including, but not limited to: (i) an F (ab , )2 fragment produced by pepsin digestion of an antibody molecule; (ii) an F ab fragment generated by reducing the disulfide bridges of an F (ab . )2 fragment; (iii) an F ab fragment generated by the treatment of the antibody molecule with papain and a reducing agent and (iv) F v fragments.
  • recombinant anti-ADIPO antibodies such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention.
  • Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in PCT International Application No. PCT/US86/02269; European Patent Application No. 184,187; European Patent Application No. 171,496; European Patent Application No. 173,494; PCT International Publication No. WO 86/01533; U.S. Pat. No. 4,816,567; European Patent Application No.
  • methods for the screening of antibodies that possess the desired specificity include, but are not limited to, enzyme-linked immunosorbent assay (ELISA) and other immunologically-mediated techniques known within the art.
  • ELISA enzyme-linked immunosorbent assay
  • other immunologically-mediated techniques known within the art.
  • Anti-ADIPO antibodies may be used in methods known within the art relating to the localization and/or quantitation of a ADIPO protein (e.g., for use in measuring levels of the ADIPO protein within appropriate physiological samples, for use in diagnostic methods, for use in imaging the protein, and the like).
  • antibodies for ADIPO proteins, or derivatives, fragments, analogs or homologs thereof, that contain the antibody derived binding domain are utilized as pharmacologically- active compounds [hereinafter "Therapeutics"].
  • an -ADIPO antibody e.g., monoclonal antibody
  • An anti-ADIPO antibody can facilitate the purification of natural ADIPO from cells and of recombinantly produced ADIPO expressed in host cells.
  • an anti-ADIPO antibody can be used to detect ADIPO protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the ADIPO protein.
  • Anti-ADIPO antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen.
  • Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance.
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, -galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include stf eptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fiuorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
  • an example of a luminescent material includes luminol;
  • bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include
  • vectors preferably expression vectors, containing a nucleic acid encoding ADIPO protein, or derivatives, fragments, analogs or homologs thereof.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a linear or circular double stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome.
  • vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • Other vectors e.g., non-episomal mammalian vectors
  • certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors”.
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and "vector” can be used interchangeably as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
  • viral vectors e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses
  • the recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, that is operatively linked to the nucleic acid sequence to be expressed.
  • "operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • regulatory sequence is intended to includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in
  • Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc.
  • the expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., ADIPO proteins, mutant forms of ADIPO, fusion proteins, etc.).
  • the recombinant expression vectors of the invention can be designed for expression of
  • ADIPO in prokaryotic or eukaryotic cells.
  • ADIPO can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).
  • the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
  • Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein.
  • Such fusion vectors typically serve three pu ⁇ oses: (1) to increase expression of recombinant protein; (2) to increase the solubility of the recombinant protein; and (3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification.
  • a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein.
  • enzymes, and their cognate recognition sequences include Factor Xa, thrombin and enterokinase.
  • Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and
  • GST glutathione S-transferase
  • E. coli expression vectors examples include pTrc (Amrann et al, (1988) Gene 69:301-315) and pET 1 Id (Studier et al, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89).
  • One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein. See, Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 119-128.
  • Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al, (1992) Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.
  • the ADIPO expression vector is a yeast expression vector.
  • yeast S. cerevisiae examples include pYepSecl (Baldari, et al, (1987) EMBO J 6:229-234), pMFa (Kuijan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al, (1987) Gene 54:113-123), pYES2 (Invitrogen Co ⁇ oration, San Diego, Calif), and picZ (InVitrogen Co ⁇ , San Diego, Calif).
  • ADIPO can be expressed in insect cells using baculovirus expression vectors.
  • Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series (Smith et al. (1983) Mol Cell Biol 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39).
  • a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector.
  • mammalian expression vectors include pCDM8 (Seed (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J 6: 187-195).
  • pCDM8 Seed (1987) Nature 329:840
  • pMT2PC Kaufman et al. (1987) EMBO J 6: 187-195.
  • promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.
  • suitable expression systems for both prokaryotic and eukaryotic cells See, e.g., Chapters 16 and 17 of Sambrook et al, MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.
  • the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid).
  • tissue-specific regulatory elements are known in the art.
  • suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al.
  • lymphoid-specific promoters Calame and Eaton (1988) Adv Immunol 43:235-275
  • promoters of T cell receptors Winoto and Baltimore (1989) EMBO J 8:729-733
  • immunoglobulins Bonerji et al. (1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748
  • neuron-specific promoters e.g., the neurofilament promoter; Byrne and Ruddle (1989) PNAS 86:5473-5477
  • pancreas-specific promoters Edlund et al.
  • mammary gland-specific promoters e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166.
  • Developmentally-regulated promoters are also encompassed, e.g., the murine hox promoters (Kessel and Grass (1990) Science 249:374-379) and the ⁇ -fetoprotein promoter (Campes and Tilghman (1989) Genes Dev 3:537-546).
  • the invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively linked to a regulatory sequence in a manner that allows for expression (by transcription of the DNA molecule) of an RNA molecule that is antisense to ADIPO mRNA. Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen that direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen that direct constitutive, tissue specific or cell type specific expression of antisense RNA.
  • the antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a
  • host cell and "recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • a host cell can be any prokaryotic or eukaryotic cell.
  • ADIPO protein can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells).
  • bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells).
  • mammalian cells such as Chinese hamster ovary cells (CHO) or COS cells.
  • Other suitable host cells are known to those skilled in the art.
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
  • transformation and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, D ⁇ A ⁇ -dextran-mediated transfection, lipofection, or electroporation.
  • Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NN., 1989), and other laboratory manuals.
  • Various selectable markers include those that confer resistance to drugs, such as G418, hygromycin and methotrexate.
  • Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding ADIPO or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drag selection (e.g., cells that have inco ⁇ orated the selectable marker gene will survive, while the other cells die).
  • a host cell of the invention such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) an ADIPO protein.
  • the invention further provides methods for producing ADIPO protein using the host cells of the invention.
  • the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding ADIPO has been introduced) in a suitable medium such that ADIPO protein is produced.
  • the method further comprises isolating ADIPO from the medium or the host cell.
  • the invention provides a kit useful for examining a pathophysiology associated with a PPAR ⁇ -mediated pathway.
  • the kit can include nucleic acids that detect two or more ADIPO sequences.
  • the kit includes reagents which detect 3, 4, 5, 6, 8, 10, 12, 15, 20, 25, 30, 35,40 or all of the ADIPO nucleic acid sequences.
  • the invention also includes an isolated plurality of sequences which can identify one or more ADIPO responsive nucleic acid sequences.
  • the kit or plurality may include, e.g., sequence homologous to ADIPO nucleic acid sequences, or sequences which can specifically identify one or more ADIPO nucleic acid sequences.
  • NUCLEOTIDE POLYMORPHISMS ASSOCIATED WITH ADIPO GENES The invention also includes nucleic acid sequences that include one or more polymo ⁇ hic
  • ADIPO sequences Also included are methods of identifying a base occupying a polymo ⁇ hic in
  • ADIPO sequence polymo ⁇ hisms an ADIPO sequence
  • the nucleotide polymo ⁇ hism can be a single nucleotide polymo ⁇ hism (SNP).
  • SNP occurs at a polymo ⁇ hic site occupied by a single nucleotide, which is the site of variation between allelic sequences. The site is usually preceded by and followed by highly conserved sequences of the allele (e.g., sequences that vary in less than 1/100 or 1/1000 members of the populations).
  • a single nucleotide polymo ⁇ hism usually arises due to substitution of one nucleotide for another at the polymo ⁇ hic site.
  • a transition is the replacement of one purine by another purine or one pyrimidine by another pyrimidine.
  • a transversion is the replacement of a purine by a pyrimidine or vice versa.
  • Single nucleotide polymo ⁇ hisms can also arise from a deletion of a nucleotide or an insertion of a nucleotide relative to a reference allele.
  • Polymo ⁇ hic sequences according to the present invention can include those shown in Table 2.
  • Table 2 describes nine ADIPO sequences for which polymo ⁇ hisms have been identified.
  • the first column of the table lists the names assigned to the sequences in which the polymo ⁇ hisms occur.
  • the second and third columns list the rat and human GenBank Accession numbers for the respective sequences.
  • the forth column lists the position in the sequence in which the polymo ⁇ hic site has been found.
  • the fifth column lists the base occupying the polymo ⁇ hic site in the sequence in the database, i.e., the wildtype.
  • the sixth column lists the alternative base at the polymo ⁇ hic site.
  • the seventh column lists any amino acid change that occurs due to the polymo ⁇ hism.
  • the polymo ⁇ hic sequence can include one or more of the following sequences: (1) a sequence having the nucleotide denoted in Table 2, column 5 at the polymo ⁇ hic site in the polymo ⁇ hic sequence, and (2) a sequence having a nucleotide other than the nucleotide denoted in Table 2, column 5.
  • An example of the latter sequence is a polymo ⁇ hic sequence having the nucleotide denoted in Table 2, column 6 at the polymo ⁇ hic site in the polymo ⁇ hic sequence.
  • a polymo ⁇ hism according to the invention includes a sequence polymo ⁇ hism in the ATP citrate lyase gene having the nucleotide sequence of GenBank Accession No. x64330, in which the cytosine at nucleotide 609 is replaced by adenosine.
  • the polymorphic sequence includes a nucleotide sequence of ATP citrate gene having the GenBank Accession No. x64330, wherein the nucleotide at 609 is any nucleotide other that cytosine.
  • the polymorphic sequence includes the full length of any one of the nine genes in Table2.
  • the polymo ⁇ hic sequence includes a polynucleotide that is between 10 and 100 nucleotides, 10 and 75 nucleotides, 10 and 50 nucleotides, or 10 and 25 nucleotides in length.
  • the invention also provides a method of identifying a base occupying a polymorphic site in a nucleic acid.
  • the method includes determining the nucleotide sequence of a nucleic acid that is obtained from a subject.
  • the nucleotide sequence is compared to a reference sequence. Difference in the nucleotide sequence in the test sequence relative to the reference sequence indicates a polymorphic site in the nucleic acid.
  • a target nucleic acid from an individual, e.g., a mammal, human or rodent (such as mouse or rat) being analyzed.
  • a mammal e.g., human or rodent (such as mouse or rat) being analyzed.
  • rodent such as mouse or rat
  • genomic DNA virtually any biological sample (other than pure red blood cells) is suitable.
  • tissue samples include whole blood, semen, saliva, tears, urine, fecal material, sweat, buccal, skin and hair.
  • the tissue sample must be obtained from an organ in which the target nucleic acid is expressed.
  • polymo ⁇ hisms in specific DNA sequences can be accomplished by a variety of methods including, e.g., restriction-fragment-length-polymo ⁇ hism detection based on allele-specific restriction-endonuclease cleavage (Kan and Dozy Lancet ii:910-912 (1978)), hybridization with allele-specific oligonucleotide probes (Wallace et al. Nucl. Acids Res.
  • DGGE denaturing- gradient gel electrophoresis
  • Single-strand-conformation-polymo ⁇ hism detection Orita et al. Genomics 5:874-879 (1983)
  • RNAase cleavage at mismatched base-pairs Myers et al. Science 230:1242 (1985)
  • chemical Cotton et al. Proc. Natl. w Sci.
  • Specific hybridization or “selective hybridization” refers to the binding, or duplexing, of a nucleic acid molecule only to a second particular nucleotide sequence to which the nucleic acid is complementary, under suitably stringent conditions when that sequence is present in a complex mixture (e.g., total cellular DNA or RNA).
  • stringent conditions are conditions under which a probe will hybridize to its target subsequence, but to no other sequences. Stringent conditions are sequence-dependent and are different in different circumstances. Longer sequences hybridize specifically at higher temperatures than shorter ones.
  • stringent conditions are selected such that the temperature is about 5°C lower than the thermal melting point (Tm) for the specific sequence to which hybridization is intended to occur at a defined ionic strength and pH.
  • Tm is the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the target sequence hybridizes to the complementary probe at equilibrium.
  • stringent conditions include a salt concentration of at least about 0.01 to about 1.0 M Na ion concentration (or other salts), at pH 7.0 to 8.3.
  • the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides) .
  • Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide. For example, conditions of 5X SSPE (750 mM NaCl, 50 mM NaPhosphate, 5 mM EDTA, pH 7.4) and a temperature of 25-30°C are suitable for allele- specific probe hybridizations.
  • “Complementary” or “target” nucleic acid sequences refer to those nucleic acid sequences which selectively hybridize to a nucleic acid probe. Proper annealing conditions depend, for example, upon a probe's length, base composition, and the number of mismatches and their position on the probe, and must often be determined empirically. For discussions of nucleic acid probe design and annealing conditions, see, for example, Sambrook et al., or Current Protocols in Molecular Biology, F. Ausubel et al., ed., Greene Publishing and Wiley-Interscience, New York (1987).
  • PCR PCR Technology: Principles and Applications for DNA Amplification (ed. H. A. Erlich, Freeman Press, N.Y., N.Y., 1992); PCR Protocols: A Guide to Methods and Applications (eds. Innis, et al, Academic Press, San Diego,
  • LCR ligase chain reaction
  • NASBA nucleic acid based sequence amplification
  • the latter two amplification methods involve isothermal reactions based on isothermal transcription, which produce both single stranded RNA (ssRNA) and double stranded DNA (dsDNA) as the amplification products in a ratio of about 30 or 100 to 1, respectively.
  • ssRNA single stranded RNA
  • dsDNA double stranded DNA
  • the invention also provides a method of selecting an individualized therapeutic agent for treating a PPAR ⁇ associated pathology, e.g., NIDDM, in a subject using ADIPO polymo ⁇ hisms.
  • the therapeutic agent can be identified by providing a nucleic acid sample from the subject, determining the nucleotide sequence of at least a portion of one or more of the ADIPO 1-32 and, optionally, ADIPO 33-43 sequences, and comparing the ADIPO nucleotide sequence in the subject to the corresponding ADIPO nucleic acid sequence in a reference nucleic acid sample.
  • the reference nucleic acid sample is obtained from a reference individual (who is preferably as similar to the test subject as possible), whose responsiveness to the agent for treating the PPAR ⁇ associated pathology is known.
  • the presence of the same sequence in the test and reference nucleic acid sample indicates the subject will demonstrate the same responsiveness to the agent as the reference individual, while the presence of a different sequence indicates the subject will have a different response to the therapeutic agent.
  • the ADIPO-associated sequence polymo ⁇ hisms can be used to predict the outcome of treatment for a PPAR ⁇ associated pathology, e.g., NIDDM, in a subject.
  • a PPAR ⁇ associated pathology e.g., NIDDM
  • a region of an ADIPO nucleic acid sequence from the subject is compared to the corresponding ADIPO sequence in a reference individual whose outcome in response to the treatment for the PPAR ⁇ associated pathology is known.
  • a similarity in the ADIPO sequence in the test subject as compared to the sequence in the reference individual suggests the outcome in the subject will be
  • ADIPO sequence in the test and reference individual indicates the outcome of treatment will differ in the subject and reference individuals.
  • test cell population comprising a cell capable of expressing one or more nucleic acid sequences selected from the group consisting of ADIPO: 1-5; 7-13; 15-31 and 32;
  • test cell population (b) contacting the test cell population with a test agent; (c) measuring expression of one or more of the nucleic acid sequences in the test cell population;

Abstract

Disclosed are methods of identifying ligands for the peroxisome proliferator activated receptor gamma (PPARη) using differential gene expression. Also disclosed are novel nucleic acid sequences whose expression is differentially regulated by PPARη ligands.

Description

METHOD OF IDENTIFYING LIGANDS FOR THE PEROXISOME
PROLIFERATOR ACTIVATED RECEPTOR GAMMA USING
DIFFERENTIAL GENE EXPRESSION
RELATED U.S. APPLICATIONS This application claims priority to USSN 60/130,821 filed April 23, 1999, which is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
The invention relates generally to nucleic acids and polypeptides and in particular to the identification of ligands for the Peroxisome Proliferator Activated Receptor Gamma (PPARγ) using differential gene expression.
BACKGROUND OF THE INVENTION
Diabetes is known to affect approximately eight million people in the United States. Over 90% of diagnosed diabetics suffer from noninsulin-dependent diabetes mellitus (NIDDM), which is also known as type II diabetes. An additional eight million people may have undiagnosed NIDDM. Obesity, advancing age, a family history of NIDDM, a sedentary lifestyle, a history of gestational diabetes, and the presence of co-morbid conditions such as hypertension or hyperlipidemia are risk factors for this disorder.
NIDDM is associated with functional and biochemical abnormalities in the pancreas, liver and peripheral insulin-sensitive tissues such as skeletal muscle and adipose tissue. The abnormalities can include, e.g. relative, but not absolute deficiency of pancreatic insulin secretion, an increased rate of hepatic glucose production and extreme insulin resistance in peripheral tissues such as adipose and skeletal muscle.
One hypothesis for the pathogenesis of NIDDM suggests that the initial event is not pancreatic failure but the development of peripheral tissue insulin resistance. During this "pre- diabetic state" candidate NIDDM patients actually demonstrate hyperinsulinemia, which is an increase level of insulin in the plasma due to an increase in secretion of insulin by the beta cells of the pancreatic islets. The observed pancreatic insulin deficiency that follows is most likely related to pancreatic burnout from maintaining the hyperinsulinemic state. Persistent, untreated hyperglycemia can result in, e.g., increased risk of urinary tract infections and dehydration related to polyuria. However, often the most important sequellae of diabetes are its long term complications. Following 15-20 years of poorly managed diabetes, patients are at risk for peripheral vascular disease with risk of limb-amputating gangrene, blindness, myocardial infarction and renal failure.
One class of therapeutics used to control of NIDDM is the thiazolidenedione compounds. These compounds have been classified as synthetic ligands for the Peroxisome Proliferator Activated Receptor γ (PPARγ). PPARγ is a nuclear hormone receptor and it has been shown to have metabolic activity primarily in the peripheral tissues where insulin resistance takes place.
PPARγ receptors exist in three forms, which have been named γl and γ2 and γ3. PPARγ 1 has a ubiquitous tissue distribution with increased expression levels in the heart, liver and kidney. Its function in these tissues is largely uncharacterized. PPARγ2 expression however, is reported to be almost exclusively expressed within the white adipocyte. Activation of this receptor in adipose tissue has been associated with adipocyte differentiation and improved glycemic control. A natural ligand for PPARγ is 15-deoxy-Δ-J2 prostaglandin.
SUMMARY OF THE INVENTION
The invention is based in part on the discovery that certain nucleic acids are differentially expressed in adipose tissue of animals treated with N-(2-benzoylphenyl)-L-tyrosine, a synthetic Peroxisome Proliferator Activated Receptor Gamma ligand (PPARγL). These differentially expressed nucleic acids include previously undescribed sequences and nucleic acids sequences that, while previously described, have not heretofore been identified as PPARγ responsive.
In various aspects, the invention includes methods of identifying PPARγ ligands, methods of diagnosing PPARγ pathophysiologies, and methods of treating those pathophysiologies. For example, in one aspect, the invention provides a method of identifying a PPARγ ligand by providing a test cell population that includes one or more cells capable of expressing one or more nucleic acids sequences responsive to PPARγ ligands and contacting the test cell population with the test agent. Levels of expression of one or more sequences, termed ADIPO sequences, are then compared to the levels of expression of the corresponding nucleic acids in a reference cell population. The reference cell population contains cells whose PPARγ ligand exposure status is known, i.e., the reference cells are known to have been exposed to a PPARγ ligand, or are known not to have been exposed to the ligand. When the former type of reference cell population is used, an similar expression profiles of the ADIPO nucleic acids sequences in the test cell population and the reference cell population indicates that the test agent is a ligand for PPARγ.
The invention in a further aspect includes a method of selecting an individualized therapeutic agent appropriate for a particular subject. The method includes providing from the subject a test cell population comprising a cell capable of expressing one or more nucleic acids sequences responsive to PPARγ ligands, contacting the test cell population with the therapeutic agent, and comparing the expression of the nucleic acids sequences in the test cell population to the expression of the nucleic acids sequences in a reference cell population.
In a further aspect, the invention provides a method of diagnosing or determining susceptibility to a PPARγ mediated pathophysiology, e.g., noninsulin-dependent diabetes mellitus, or adipocyte differentiation. The method includes providing from the subject a cell population comprising a cell capable of expressing one or more PPARγ-responsive genes, and comparing the expression of the nucleic acids sequences to the expression of the nucleic acids sequences in a reference cell population that includes cells from a subject not suffering from a PPARγ mediated pathophysiology.
Also provided are novel nucleic acids whose expression is responsive to the effects of N-(2-benzoylphenyl)-L-tyrosine, as well as single nucleotide polymorphisms in ADIPO sequences, as well as methods of using the ADIPO single nucleotide polymorphisms.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description and claims.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is based in part on the discovery of changes in expression patterns of multiple nucleic acid sequences in rodent adipose cells following exposure to a ligand for the Peroxisome Proliferator Activated Receptor Gamma (PPARγ). The differences in gene expression were identified following administration of a PPARγ-specific ligand, N- (2-benzoylphenyl)-L-tyrosine, referred to herein as PPARγL. This compound is described in Henke et al., J. Med. Chem.41 :5020-5036, 1998, whose contents are incoφorated herein in their entirety.
The differentially expressed nucleic acids were identified by administering PPARγL to male 10-14 week old Sprague Dawley rats at 5.0 mg/kg/day b.i.d. dosing for 3 days. Control animals received N-methylglucamine. The animals were sacrificed 24 hours following the last dose. White adipose tissue was dissected from the animals, and total RNA was recovered from the dissected tissue and cDNA prepared. Genes whose transcript levels varied between the two tissue types were identified using GENECALLING™ differential expression analysis as described in U. S. Patent No. 5,871,697 and in Shimkets et al., Nature Biotechnology 17:798- 803 (1999). The contents of these patents and publications are incoφorated herein by reference in their entirety.
106 gene fragments were initially found to be differentially expressed in rat white adipose tissue in response PPARγL. Genes fragments whose expression levels appeared to increase or decrease more than 4-fold compared to control tissues were selected for further analysis. An unlabeled oligonucleotide competition assay as described in Shimkets et al.,
Nature Biotechnology 17:198-803 was used to verify the identity of differentially expressed sequences.
43 single copy nucleic acid sequences whose expression levels differed in PPARγL- treated tissue and tissue treated with vehcicle alone were chosen for further characterization. These sequences are referred to herein as ADIPO 1-43. A summary of the ADIPO sequences analyzed is presented in Table 1.
Ten sequences (ADIPO: 1-10) represent novel rat genes for which the sequence identity to previously described sequences is either high (i.e., > 90%, observed for 5 fragments), moderate (i.e., between about 70% and about 90%, observed for 4 genes) or low ( i.e., < 70%, observed for 1 fragment) suggesting a putative homology. For five of the nucleic acids (ADIPO: 1 1-15), no homology was found to known nucleic acid sequences.
The 28 other sequenced identified have been previously described. 17 of the sequences (ADIPO 16-32) are newly shown to be PPARγ responsive. 1 1 sequences have been previously recognized as being differentially expressed as part of the PPARγ response (ADIPO: 33-43). Six of these 11 sequences have been shown to be differentially expressed as part of the PPARγ response specifically in adipose tissue (ADIPO: 38-43), while the other 5 have been shown to be differentially expressed as part of the PPARγ response in liver tissue (ADIPO: 33-37). For some of the novel sequences (i.e., ADIPO1-15), a cloned sequence is provided along with one or more additional sequence fragments (e.g., ESTs or contigs) which contain sequences identical to, or substantially identical to, the cloned sequence. Also provided is a consensus sequences which includes a composite sequence assembled from the cloned and additional fragments. For a given ADIPO sequence, its expression can be measured using any of the associated nucleic acid sequence in the methods described herein. For previously described sequences (ADIPO: 16-43), database accession numbers are provided. This information allows for one of ordinary skill in the art to deduce information necessary for detecting and measuring expression of the ADIPO nucleic acid sequences.
The PPARγ -responsive nucleic acids discussed herein include the following:
Table 1
Figure imgf000008_0001
Figure imgf000009_0001
Figure imgf000010_0001
Below follows additional discussion of nucleic acid sequences whose expression is differentially regulated in the presence of PPARγL.
ADIPO 1
ADIPO 1 is a novel 744 bp gene fragment. The nucleic acid has the following sequence:
1 GGGTTTTTTTCTGGTTTATTAAAACAAAACTCCATTTCCATGCTTCTCAGGTTGTACCATCATTGGGCATAAAAATCAGT
81 CAACATTTGCAAGGCAGTGTCACTCACAGAATCACCCAGTGCTATTTTACAGTGGTCATTCTCTTTACAACAATCAGGCA
161 TTGCTTTGTACAATGGGCAAACGCTATAACAAACGCCTTCCATAAACAGTTAGCCCTTTAGCTATTGGGAAGGTCAAGAG 241 TCGCTTGAAGCAGTCACTGGTCGCACTAAACAAAGTACCAGGTGGCAGCTCTCAAGCCCCTAAAAAGTGTTTTTAAAAAA
321 CAGATTTACATCCCATAGTAAGTCTTAGAAACTTCAAAGATAGGGCCTATCATTTCAAAACACTACGGTTATCTACGGAT 401 AAAAATCTGCATCCAACTGTGCAGTGTTTTCTTCTAGATCTGGATTAAAACCTTTATTATTTACCCCAATTAGTTCCTCT 481 ATTAATTCTGCTACTCTCGGTTTTAAAAAGGCCAGGGTTGCCAGTTGATCTTCCACAGAGGAAAACACTGCAGCCCGTTC 561 CCTGTGCTTACTCTCCTGTGAGGAAAGACAGTCAGAGCACGCGGTGCATCGAGAGTGATACAGAAAGCATGCAAGAGTCT 641 GCTTGTCACCGTGGTGACATTTGGTGACATTTGCTTGTCACCGTGGTGACATTTGGTCTGNACTAAAGAGCGATTCGTGT
721 AGCTTCTGAAAACGTTTTCCATGG (SEQ ID NO : 1 )
Its expression is increased 20-fold in PPARγL treated rats.
ADIPO2
ADIPO2 is a novel 434 bp gene fragment. The nucleic acid was initially identified in a cloned fragment having the following sequence:
1 gccggcatcn ttnatagtga aaggtgttca caggtgccat tgtggaatan cacggacctn
61 ttgtcgactc catntcctgt acnggcatgg caacaatttg caanatggtt gcagaaattn
121 ggnccattan atcagttttc ccctanaacc acattatgna naagtacctg agcaagaanc
181 ccaggccgan cagacattgc caacctagca taagaattc (SEQ ID NO: 2)
The cloned sequence was assembled into a contig that includes: gaatt ct ctgct aggttggcaatgtctgctcggcctgtcttgctcaggt act ttt teat cctgtggttgtaggggaacactgatgtagtggccccaatttctgcacccatgttgcaaat tgttgccatgccagtgcaggagatggagtcgacaccaggtccatggtattccacaatggc acctgtgcacctttcactgtgaggatgccggc, (SEQ ID NO : 3 )
gaattcttctgctaggttggcaatgtctgcgcggcctgtcttgctcaggtactttttcat cctgtggttgtaggggaacactgatgtagtggccccaatttctgcacccatgttgcaaat tgttgccatgccagtgcaggagatggagtcgacaccaggtccatggtat ccacaatggc acctgtgcacctttcactgtgaggatgccggc, (SEQ ID NO : 4 )
gaattcttctgctaggttggcaatgtctgcncggcctgtcttgctcaggtactttttcat cctgtggttgtaggggaacactgatgtagtggccccaatttctgcacccatgttgcaaat tgttgccatgccagtgcaggagatggngtcgacaccaggtccatgg attccacaatggc acctgtgcacctttcactgtgaggatgccggccactttcaggatcacatctttgggtgag gtccaaccagagagggtgcccgtcagettcacgccaatcaccttgggacact cagetec caggggatcc, (SEQ ID NO : 5 )
tctagaaaactatgcataccctggagttcttctga tggcactgactcccacacccccaa tgggtggncctgggagg atctgcattggagtagggggtgctgatgctgtggatgtcatg gctgggacccctgggagctgaagtgtcccaaggtgattggcgtgaagctgacgggcaccc tctctggttggacctcacccaaagatgtgatcntgaaagtggccggcatcctcacagtga aaggtggcacaggtgccattgtggaataccatgg, (SEQ ID NO : 6 ) and gaattcttctgctaggttggcaatgtctgctcgncctgtcttgctcaggtactttttcat cctgtggttgtaggggaacactgatgtagtggccccaatttctgcacccatgttgcaaat tgttgccatgccagtgcaggagatggagtcgac (SEQ ID NO : 7 )
The resulting consensus sequence is:
1 GAATTCTTCTGCTAGGTTGGCAATGTCTGCTCGGCCTGTCTTGCTCAGGTACTTTTTCATCCTGTGGTTGTAGGGGAACA
81 CTGATGTAGTGGCCCCAATTTCTGCACCCATGTTGCAAATTGTTGCCATGCCAGTGCAGGAGATGGAGTCGACACCAGGT
161 CCATGGTATTCCACAATGGCACCTGTGCACCTTTCACTGTGAGGATGCCGGCCACTTTCAGGATCACATCTTTGGGTGAG 241 GTCCAACCAGAGAGGGTGCCCGTCAGCTTCACGCCAATCACCTTGGGACACTTCAGCTCCCAGGGGATCCCAGCCATGAC 321 ATCCACAGCATCAGCACCCCCTACTCCAATGCAGATACCTCCCAGGNCCACCCATTGGGGGTGTGGGAGTCAGTGCCAAT 401 CAGAAGAACTCCAGGGTATGCATAGTTTTCTAGA (SEQ ID NO : 8 )
Its expression is increased 2-fold in PPARγL -treated rats.
ADIPO3 ADIPO3 is a novel 181 bp gene fragment. The cloned sequence is:
1 agatctgccc cttcgagggc acagtggctt acaagaaact gtgtccccac ggccgaggat
61 tcatgaccaa cggagcagat antgatgagt gcaaggtaat ccacgatgtt tnccgaaacg
121 gggagtgtgt caatgacaga ggatcatatc actgcatctg taaaactggc tacactccgg
181 a (SEQ ID NO : 9 ) Its expression is decreased 70-fold in PPARγL treated rats.
ADIPO4
ADIPO4 is a novel 63 bp gene fragment. The cloned sequence is:
1 tgtacaggct agtgcaattc ttgaactgta ggtggatnga tttcagggac ttcttggtcc 61 gga (SEQ ID NO: 10) Its expression is decreased 100-fold in PPARγL -treated rats.
ADIPO5
ADIPO5 is a novel 317 bp gene fragment. The nucleic acid has the following sequence:
1 TCCGGAATCAAAAAGACCCTCGAGCCAATCCTAGTGCCTTTCTATGAGATTTGATCTCCCAGACAGGATCTTTGCAGCCT 81 CGGCTGGTTTCTACCCAGCTCTAGCCATGGAGGGCCTTGAACTCCGCATTCTCCTGCCTCAGCCTCCTGCCAGTATTGCA
2 1 TAGGATGGCCACCCCCCAACAGGGGTTTTTGTTTTAGAGAGAAAAAGGTCCCTTACCTCACTCCTAAGAACTGATCA (SEQ ID NO: 11)
Its expression is decreased 10-fold in PPARγL -treated rats. ADIPO6
ADIPO6 is a novel 106 bp gene fragment. The cloned sequence is:
1 tgatcagtct tccagcttcc tcatcctgag tcggacgtct gcagtgctgt tgagatggtg 61 tnagctcctt ttctctttga gcccattaaa cagaattcng agtccg (SEQ ID NO: 12) Its expression is modulated 1-fold in PPARγL-treated rats.
ADIPO7
ADIPO7 is a novel 258 bp gene fragment. The cloned sequence is:
1 tgatcattct cctccatgta tttcttggct gcctcactgg cactttctgc ctgcttttta
61 aaggcttcat tggaggccag cagtgtggcc tgctgggaga tgagagtcac caggcgtcta 121 agcaggaagg acagcagcaa ggaaaagcca gcaatataga gattcctctg agcacggaaa
181 agcttcatgt ggaagtgctc catggcacct ggattgttct ggaggttcac cttttctgtt
241 ncatcatcat atttccgg (SEQ ID NO: 13)
Its expression is increased 10-fold in PPARγL-treated rats.
ADIPO8 ADIPO8 is a novel 531 bp gene fragment. The nucleic acid was initially identified in a cloned fragment having the following sequence:
1 gaattctgtg gaacgcaaag gcctccagac cttgacacca gcagcaacgc cgtggatctg
61 ctgttcttca cggatgagtc cggggacagc cgaggctgga agctt (SEQ ID NO: 14)
The cloned sequence was assembled into a contig that includes: ctcgagtacccgaagccctatccacccgatctgcgctgtaactacagcatccgggtagag aggggcctcactgtgcatctcaagttcctggatcc tttgaaattgatgaccaccagcaa gtacactgcccctatgaccagctccagatctacgctaatgggaagaacttgggtgaattc tgtggaacgcaaaggcctccagaccttgacaccagcagcaacgccgtggatctgctgttc ttcacggatgagtccggggacagccgaggctggaagct , (SEQ ID NO: 15)
ggatccttttaaattgatgaccaccagcaagtacactgcccctatnatcagctccagatc tacgctaatgggaagnncttgggtgaattctgtggaacgcaaaggcctccagaccttgac accagcagcaacgccgtggatctgctgttcttcacggatgagtccggggacagccgaggc tggaagctt, (SEQ ID NO: 16)
tccggactcatccgtgaagaagagcanatccanggcgttnctnctggtgtcaaganctgg aggcctttgcgttccacagaattcacccaagttcttcccattat gtagatctggagctg gtcataggggcagtgtacttnctgggtggtcatcaatttnaaaaggatccaggaacttga gggtncacagngagggcccctctctacccgggatgctggtagttacagcgcagatcgggt ggatagggctttcgggttactcgaggntggnagatgtagcccgagggctccgtgtaagan cccactgctncactcngcccttggcaggattgcctnntctttcnggantctgn, (SEQ ID NO: 17)
nacttccagcctcgnctgtgcccggactcatccgtgaagaacagcagatccacggcgttg ctgctggtgtcaaggtctggaggcctttgcgttccacagaattcacccaagttcttccca ttagcgtagatct, (SEQ ID NO: 18)
ctcgagtacccgangccctatccacccgatctgngctgtaactacagcntccgggtagag aggggcctcactgtgcacctcaagttcctggatccttttgaaattgatgaccaccagcaa gtacactgcccctatgaccagctccagatctacgctaatgggaagaacttgggtgaattc, (SEQ ID NO: 19)
ngaccatcatgttctacaagggcttcctggcctactaccaggctgtgtaccttgatgaat gtacgtcccagcccaactcagtggaggagggtttgcagccccgatgccaacacctgtgcc acaactatgttggagnttacttctgttcctgccgtcctggctacgagcttcagaaagacg ggcaatcctgccaggccgagtgcagcagtgggctctacacggagccctcgggctacatct ccagcctcgagtact, (SEQ ID NO: 20)
ctcgagtacccgangccctatccacccgatctgcgctgtaactacagcanccgggtagag aggggcctcactgtgcatctcaagttcctggatccttttgaaattgatgaccaccagcaa gtacactgcccctatgaccagctccagatct (SEQ ID NO: 21) and gaattcacccaagttcttcccattagcgtanatctgganctggtcat ggggcagtgtac ttgctggtggtcatcaatttcaaaaggatccaggaacttgaggtgcn (SEQ ID NO: 22)
The resulting consensus sequence is:
1 NAAGCTTCCAGCCTCGGCTGTCCCCGGACTCATCCGTGAAGAACAGCAGATCCACGGCGTTGCTGCTGGTGTCAAGGTCT
81 GGAGGCCTTTGCGTTCCACAGAATTCACCCAAGTTCTTCCCATTAGCGTAGATCTGGAGCTGGTCATAGGGGCAGTGTAC
161 TTGCTGGTGGTCATCAATTTCAAAAGGATCCAGGAACTTGAGGTGCACAGTGAGGCCCCTCTCTACCCGGATGCTGTAGT
241 TACAGCGCAGATCGGGTGGATAGGGCTTCGGGTACTCGAGGCTGGNAGATGTAGCCCGAGGGCTCCGTGTAAGAGCCCAC 321 TGCTGCACTCGGCCCTTGGCAGGATTGCCTCGTCTTTCTGGAAGCTCGNTAGCCAGGACGGCAGGAACAGAAGTAANCTC
401 CAACATAGTTGTGGCACAGGTGTTGGCATCGGGGCTGCAAACCCTCCTCCACTGAGTTGGGCTGGGACGTACATTCATCA
481 AGGTACACAGCCTGGTAGTAGGCCAGGAAGCCCTTGTAGAACATGATGGTCN (SEQ ID NO: 23)
Its expression is decreased 30-fold in PPARγL -treated rats.
ADIPO9 ADIPO9 is a novel 316 bp gene fragment. The nucleic acid has the following sequence:
1 tgatcagata catctacaac cgngaggagt tacctgcgct acgacagcga cgtgggcnag
61 taccgnncgg tnaccgagct ggggcggccc tcanccgagt actttaacaa ncagtacctg
121 gancggacgg tgtcntagct gnaacacggt ctncagcaca caactacnag taagtncagn
181 aaangggttc cccacctccc tggcggcggc ttnagcagcc caatgtggcc atctccctgt 241 ccaggacaga ggccctcaac caccacaact tgctggtctg ctcagtgacn ctatttgccc
301 cnaagcccag atcaaa (SEQ ID NO: 24)
Its expression is decreased 7-fold in PPARγL-treated rats.
ADIPOIO
ADIPOIO is a novel 713 bp gene fragment. The nucleic acid has the following sequence: 1 TCTGAACGTTCTACAGCAGAAGCAGAAGGCTCTCAATGCAGGTTACATCCTAAACGGTCTGACCGTGTCCATCCCTGGAC
81 TGGAGAAAGCCCTACAGCAGTATACACTAGAACCATCAGAAAAGCCATTTGACCTCAAGTCTGTGCCTCTGGCCACCACA
161 CCTATGACAGAGCAGAGACCACGAAAGCACCTCCACTGCAGCAGTCAAACAGCCGGAGAAGGTGGCAGCCACACGGCAGG
241 AGATATTCCAGGAGCAGTTAGCAGCGGTGCCAGAGTTCCAGGGGCTAGGGCCCCTCTTCAAGTCCTCTCCTGAGCCGGTG 321 GCCCTCACCGAGTCAGAGACCGAGTATGTCATCCGTTGCACCAAACACACCTTCTCTGACCACTTGGTGTTCCAGTTTGA 401 CTGCACAAACACCCTCAATGACCAGACTCTGGAGAATGTCACAGTGCAAATGGAACCCACTGAGGCATATGAAGTGCTCT 481 CTTATGTGCCTGTGCGAAGCTTGCCCTACAACCAGCCTGGGACCTGCTACACACTAGTGCTCTGCCCAAGGAAGACCCCA 561 CAGCTGTGGCATGCACATTCAGCTGTGTGATGAAGTTCACTGTTAAGGACTGTGACCCCAACACTGGAGAAATCGATGAA 641 GAAGGCTATGAGGATGAGTATGTGCCGGAGGATCTAGAAGTTACTGTAGCTGATCACATCCAGAAAGTCATGA (SEQ ID NO:25)
Its expression is decreased 5-fold in PPARγL -treated rats.
ADIPO11
ADIPO 1 1 is a novel 407 bp gene fragment. The cloned sequence is:
1 gctagcattt aaaaatgtag actttcgtaa gtgtataggc cagcttgtgg ggaagtgacc
61 aagatacata aggtaggaga ttcaagtgac cgcctggagt ttctatactt cattctgctt
121 acagaacatt tattaaggtt gtttaatatg tgacttttca tttgacattg tggaagtcct
181 ttattgtaag gctaatcctt ctgcctggtc acaaggcagc agccacaaag angatcactc
241 tggacccnnn cgctggataa atcccatttc ctcctgagan cataggnttc ctcctgtatt 301 tctgaggtaa tgattttatt aantnngcag tgncggtggg ccccgatggc atnagtaaat
361 ttgggganaa atttccttag aatgcngggg aattaccgag gagtgng (SEQ ID NO:26)
Its expression is increased 35 -fold in PPARγL-treated rats.
ADIPO12
ADIPO 12 is a novel 120 bp gene fragment. The cloned sequence is: 1 actagtgaaa aggcctttgt cctcaggtct tcgctccctg gtataggtgg ggtgcagggc
61 tggttcgtga ctgccccttg caggagtgga agtttcctga tgttctgact ttccaaatty (SEQ ID NO:27)
Its expression is decreased 100-fold in PPARγL-treated rats.
ADIPO13 ADIPO 13 is a novel 231 bp gene fragment. The nucleic acid has the following sequence:
1 NTAGNCTAACTNTTAAAAA AAA NAATTTTCAATCCCATTANTNNTGTGTGCTNAAGAGACAGCAGTCANAGCTCGCCT 81 AATGGCCTGACTTTAATTCTGGCTTCTTGGAAACTAGTATCACTCTGCCACCTCACACACGAGAGAGAAGCTTGATGCTT 161 AATAATCTGCTGCTNCNGCTGTAACTATATTTAATATATAATTTNCAGTATNAAACCTNTTAACAAATCN ( SEQ ID NO : 28 )
Its expression is decreased 100-fold in PPARγL-treated rats.
ADIPO14
ADIPO14 is a novel 315 bp gene fragment. The nucleic acid has the following sequence:
1 acggggtcac aggcaaatgg ctttattggc ttatnattta aaatgggctt gtnatcacaa
61 ggcttgggaa gttcagacac ttccaagaga gatgttcaaa caaacccctt tctaaccagt
121 caaggggaaa gancagtagg cccgagattg actgcagcgt gagtncagcc tactcagtat
181 aaccaggcat gccaggccta ctgcaagcca catgtgcttt tactacctaa gagttctaga
241 gtctggattc agaactgtta ctagcagacc tgagctcata ccctcggtga ctctgccaag 301 gnaggagggg gaggg (SEQ ID NO: 29)
Its expression is modulated 1-fold in N-(2-benzoylphenyl)-L-tyrosine -treated rats.
ADIPO15
ADIPO 15 is a novel 178 bp gene fragment. The cloned sequence is:
1 tctagagtct ggattcagaa ctgttactag cagacctgat ctcataccct cngtgactcn 61 gccaanggat nagggggang ggaacacana ttaganaagg tgncccgana cccaccttct 121 aggcctttgt tagnggtcca agtgtggctc tctgggccta ctgtgggaac aaaccatg (SEQ ID NO: 30)
Its expression is decreased 20-fold in PPARγL-treated rats.
ADIPO16
ADIPO 16 corresponds to the gene encoding tricarboxylate transport protein (rat; L12016, human; U25147). Its transcription is increased 4-fold in PPARγL treated rats.
Tricarboxylate transport protein functions in fatty acid synthesis. The protein transports citrate synthesized in the mitochondria from oxaloacetate and acetyl-CoA across the mitochondrial membrane to the cytosol where the citrate is reconverted to oxaloacetate and acetyl-CoA. ADIPO17
ADIPO 17 corresponds to the gene encoding ATP-citrate lyase (rat; J05210, human; X64330). Its transcription is increased 90-fold in PPARγL treated rats.
Gene encoding ATP-citrate lyase cleaves off acetyl-CoA from cytosolic citrate to allow acetyl-CoA to enter the fatty acid synthesis cycle.
ADIPO 18
ADIPO 18 corresponds to the gene encoding 12-lipoxygenase (rat; L06040, human; M58704). Its transcription is decreased 5-fold in PPARγL treated rats.
The major pathway of arachidonic acid metabolism in human platelets proceeds via a 12- lipoxygenase enzyme. The enzyme introduces molecular oxygen into arachidonic acid in the C- 12 position to create 12(S)-hydroperoxy-5,8,10,14-eicosatetraenoic acid (12-S HETE).
ADIPO19
ADIPO19 corresponds to a gene encoding rhodanese (rat; X56228, human; D87292). Its transcription is decreased 2.0-fold in PPARγL treated rats. It is also known as nuclear-encoded mitochondrial thiosulfate sulfotransferase. Rhodanese is involved in forming iron-sulfur compounds,cyanide detoxification, and modification of sulfur containing enzymes. Rhodanese converts cyanide to thiocyanate.
ADIPO20
ADIPO20 corresponds to a gene encoding NADH-ubiquinone oxidoreductase chain 4 (rat ; P05508, C06662, human ; P03905, V00662, D381120). Its expression is increased 80-fold in PPARγL treated rats.
The NADH-ubiquinone oxidoreductase chain 4 protein is one of the proteins involed in the one of the first steps of the electron transport chain.
ADIPO21 ADIPO21 corresponds to cytochrome C oxidase polypeptide I (rat; S79304, human;
AF035429). Its expression is increased 100-fold in PPARγL treated reats. Cytochrome C oxidase polypeptide I is one of 3 cytochrome C oxidase subunits encoded in the mitochondrial genome. Cytochrome C oxidase is the last step in the electron transport chain and is responsible for collecting electrons from reduced cytochrome C and then transfering the electrons to oxygen to generate water.
ADIPO22
ADIPO22 corresponds to adenine nucleotide translocator (rat; D12771, human; J02683). Its expression is increased 2.0-fold PPARγL treated rats.
Adenine nucleotide translocator is the most abundant mitochondrial protein. The protein exists as a homodimer of two 30 kDa subunits. The dimer forms a gated pore that shuttles ADP across the mitochondrial membrane. The pore controls the rate of ADP/ATP flux between the mitochondrion and the cytosol.
ADIPO23
ADIPO23 corresponds to Δ4-3-ketosteroid 5β reductase (rat; D 17309, human; Z28339). Its expresssion is decreased 50 fold in PPARγL treated rats.
Δ4-3-Ketosteroid 5β reductase is a key enzyme in bile acid synthesis. It catalyzes the reduction of Δ-4-3-oxosteroids to give the cis conformation and is required for synthesis of chenodeoxycholic acid and cholic acid.
ADIPO24
ADIPO24 corresponds to major α-globin (rat; Ml 7083, human; N00493). Its expression is decreased 10-fold in PPARγL treated rats.
Major α-globin is one of the two protein constituents of adult major hemoglobin, fetal hemoglobin and A2 hemoglobin. Hemoglobin forms a tetramer of two alpha-class chains and two beta-class chains and serves as the major carrier of molecular oxygen in the blood to peripheral tissues. Hemoglobin has also been shown to have minor transport functions for some essential cofactors, including folate. ADIPO25
ADIPO25 corresponds to β-globin (rat; X05080, human; N00497). Its expresssion is decreased 7.0-fold in PPARγL treated rats.
β globin forms the β-class component of adult major hemoglobin, where two beta chains heterodimerize with two alpha chains (α-1 chains for major hemoglobin and α2 chains for minor hemoglobin).
ADIPO26
ADIPO26 corresponds to keratin 19 (rat; X81449, human; Y00503). Its expression is decreased 15.0-fold in PPARγL treated rats. There are two classes of keratin proteins : (i) acidic (or type I) and (ii) basic (or type II). Generally, the proteins are coordinately synthesized in pairs so that at least 1 member of each class is expressed in each epithelial cell. This is known as the keratin pair rule. The most striking exception to the keratin-pair rule is the smallest known (40- kD) acidic keratin, KRT19. This keratin is found in the periderm, which is the transient superficial layer that envelopes the developing epidermis. It is expressed often in epithelial cells in culture and in some carcinomas. Its expression is increased by vitamin A treatment of cultured human keratinocytes.
ADIPO27
ADIPO27 corresponds to brain S-100 β subunit (rat; X01090, human; M59488). Its expression is increased 90-fold in Ν-(2-benzoylphenyl)-L-tyrosine teated rats.
Brain S-100 β subunit is abundantly expressed in glial cells and other tissues. The protein contains two calcium-binding domains. It is thought to be involved in signaling to cause axonal growth in nervous tissue and mediate neuron/glial communication, S-100 proteins bind glial fibrillary acidic proteins and desmin intermediate filaments in the N-terminal head domains. This binding both causes disassembly of existing polymers and inhibits reassemblies of these intermediate filaments, thereby antagonizing the effects of annexin-II2 in micro filament assembly. ADIPO28
ADIPO28 corresponds to SPI-3 serine protease inhbitor (rat; XI 6539, human; KOI 500). Its expression is decreased 5-fold in PPARγL-treated rats.
SPI-3 serine protease inhbitor is a liver acidic glycoprotein. Under normal conditions, expression of the protein in rats is minimal. Its transcription is regulated by IL-6 and glucocorticoids. Thus, expression levels are correlated with inflammation.
ADIPO29
ADIPO29 corresponds to WAP four disulfide core domain protein (rat; AF037272, human; ESTAA 005075). Its expression is increased 1.5-fold in PPARγL-treated rats.
WAP four disulfide core domain protein member of the class of WAP serine protease inhibitors. The gene was initially isolated from a urogenital sinusmesenchymal cell line and is shown to have growth-inhibitory properties.
ADIPO30
ADIPO30 corresponds to β3 adrenergic receptor (rat; M74716, human; X70811). Its expresssion increases 2-fold in PPARγL treated rats.
β3 adrenergic receptor is a G-protein that is involved in thermoregulation and in lipolysis. Evolutionarily, the protein is linked to brown fat and is used in uncoupled Ox-Phos to generate heat in hibernating animals. There is also evidence that the responsiveness of the receptor decreases with age. This relative lack of responsiveness may be related to a decrease in B-3- ADR which can be restored by exposing older animals to the cold. Additionally, it may be involved in regulating intestinal absorption of foodstuffs. Correlation of a mutation of this gene with an increased incidence of NIDDM onset has been demonstrated in some populations.
ADIPO31
ADIPO31 corresponds to GTP binding protein (rat; Ml 7528, human; X04828). Its expresssion decreases 1.5-fold in PPARγL treated rats. GTP binding protein was originally cloned out of olfactory epithelium. The protein consists of three subunits; alpha, beta and gamma. The alpha subunit tends to be the effector subunit. When no ligand is bound the associated receptor, the alpha subunit stays bound to the beta/gamma complex and is complexed with GDP. The receptor, when bound to its ligand then associates with the beta/gamma complex and dissociates from the alpha subunit. The free alpha subunit exchanges GDP for GTP and goes to adenylate cyclase.
ADIPO32
ADIPO32 corresponds to insulin induced growth response protein (rat; L13619, human; U96876). The protein is also known as insulin-induced-gene- 1 (INSIG1). Its expresssion increases 30-fold in PPARγL treated rats.
This gene was discovered following treating liver cells with insulin and then collecting differentially expressed proteins. Insulin induced growth response protein is the most abundantly induced insulin-responsive gene and is highly hydrophobic In addition to hepatic expression, it has been induced in a murine model of adipocyte differentiation suggesting that it may play a role in regulating growth/differentiation of tissues involved in metabolic control.
ADIPO33
ADIPO33 corresponds to malic enzyme (rat; M26581, M30596, M26594, M26585, human; X77244), whose expression has previously been reported to be differentially regulated by PPARγ in liver tissue. Its expression increased 100-fold in PPARγL treated rats. ADIPO34
ADIPO34 corresponds to long chain 3-ketoacyl-CoA thiolase (rat; D 16479, human; D 16481), whose expression has been previously reported to be differentially regulated by PPARγ in liver tissue. In the present studies, its expresssion increased 15-fold in PPARγL treated rats.
ADIPO35 ADIPO35 corresponds to catalase (rat; Ml 6670, human; X04076), whose expression has been previously reported to be differentially regulated by PPARγ in liver tissue. In the present studies, its expression increased 8-fold in PPARγL treated rats.
ADIPO36
ADIPO36 correponds to carnitine/acylcarnitine carrier protein (rat; X97831, human; Y 10319), whose expression has been previously reported to be differentially regulated by PPARγ in liver tissue. In the present studies, its expression increased 1.5-fold in PPARγL treated rats.
ADIPO37
ADIPO37 corresponds to fatty acid synthase (rat; M76767, human; U26644), whose expression has previously been reported to be differentially regulated by PPARγ in liver tissue. In the present studies, its expression increased 40fold in PPARγL treated rats. ADIPO38
ADIPO38 corresponds to stearyl-CoA desaturase (rat; J02585, human; Y13647), whose expression has been previously reported to be differentially regulated by PPARγ in adipose tissue. In the present studies, its expresssion increased 20-fold in PPARγL treated rats.
ADIPO39 ADIPO39 corresponds to phosphoenolpyruvate carboxykinase (rat; K03243, human;
LI 2760), whose expression has been previously reported to be differentially regulated by PPARγ in adipose tissue. In the present studies, its expression increased 2-fold in PPARγL treated rats.
ADIPO40
ADIPO40 correponds to glycogen synthase (rat; J05446, human; D29685), whose expression has been previously reported to be differentially regulated by PPARγ in adipose tissue. In the present studies, its expression increased 100-fold in PPARγL treated rats.
ADIPO41
ADIPO41 corresponds to uncoupling protein (rat; AB010743, human; U76367), whose expression has been previously reported to be differentially regulated by PPARγ in adipose tissue. In the present studies, its expresssion increased 1.5-fold in PPARγL treated rats. ADIPO42
ADIPO42 corresponds to fatty acid transport protein (rat; U89529, human; AF055899), whose expression has been previously reported to be differentially regulated by PPARγ in adipose tissue. In the present studies, its expression increased 1.5-fold in PPARγL treated rats.
ADIPO43
ADIPO43 correponds to adipocyte fatty acid binding protein (rat; U75581, human; X56549), whose expression has been previously reported to be differentially regulated by PPARγ in adipose tissue.
In the present studies, its expression increased 5-fold in PPARγL treated rats.
GENERAL SCREEENING AND DIAGNOSTIC METHODS USING ADIPO SEQUENCES
Several of the herein disclosed methods relate to comparing the levels of expression of one or more ADIPO nucleic acids in a test and reference cell populations. The sequence information disclosed herein, coupled with nucleic acid detection methods known in the art, allow for detection and comparison of the various ADIPO transcripts. In some embodiments, the ADIPO nucleic acids and polypeptide correspond to nucleic acids or polypeptides which include the various sequences (referenced by SEQ ID NOs) disclosed for each ADIPO nucleic acid sequence.
In its various aspects and embodiments, the invention includes providing a test cell population which includes at least one cell that is capable of expressing one or more of the sequences ADIPO 1-32, or any combination of ADIPO sequences thereof. By "capable of expressing" is meant that the gene is present in an intact form in the cell and can be expressed. Expression of one, some, or all of the ADIPO sequences is then detected, if present, and, preferably, measured. Using sequence information provided by the database entries for the known sequences, or the sequence information for the newly described sequences, expression of the ADIPO sequences can be detected (if expressed) and measured using techniques well known to one of ordinary skill in the art. For example, sequences within the sequence database entries corresponding to ADIPO sequences, or within the sequences disclosed herein, can be used to construct probes for detecting ADIPO RNA sequences in, e.g., northern blot hybridization analyses or methods which specifically, and, preferably, quantitatively amplify specific nucleic acid sequences. As another example, the sequences can be used to construct primers for specifically amplifying the ADIPO sequences in, e.g., amplification-based detection methods such as reverse-transcription based polymerase chain reaction. When alterations in gene expression are associated with gene amplification or deletion, sequence comparisons in test and reference populations can be made by comparing relative amounts of the examined DNA sequences in the test and reference cell populations.
For ADIPO sequences whose polypeptide product is known, expression can be also measured at the protein level, i.e., by measuring the levels of polypeptides encoded by the gene products described herein. Such methods are well known in the art and include, e.g., immunoassays based on antibodies to proteins encoded by the genes.
Expression level of one or more of the ADIPO sequences in the test cell population is then compared to expression levels of the sequences in one or more cells from a reference cell population. Expression of sequences in test and control populations of cells can be compared using any art-recognized method for comparing expression of nucleic acid sequences. For example, expression can be compared using GENECALLING® methods as described in US Patent No. 5,871,697 and in Shimkets et al., Nat. Biotechnol. 17:798-803.
In various embodiments, the expression of 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, 25, 28, 30, 35, 40, or all of the sequences represented by ADIPO 1-32 are measured. If desired, expression of these sequences can be measured along with other sequences whose expression is known to be altered according to one of the herein described parameters or conditions. For example, expression of one or more of sequences represented by ADIPO 1-32 can be compared with sequences from ADIPO33-43, which have been previously shown to be response to PPARγ ligands. The latter genes correspond to adipocyte fatty acid binding protein, phosphoenolpyruvate carboxykinase, stearyl-CoA desaturase, glycogen sythatase, uncoupling protein-2, fatty acid transport protein, long chain acyl-CoA sythatase and leptin. The reference cell population includes cells one or more cells capable of expressing the measured ADIPO sequences and for which the compared parameter is known, e.g., PPARγ expression status. By "PPARγ expression status" is meant that is known whether the reference cell has been exposed to a PPARγ ligand, e.g. N-(2-benzoylphenyl)-L-tyrosine. Whether or not comparison of the gene expression profile in the test cell population to the reference cell population reveals the presence, or degree, of the measured parameter depends on the composition of the reference cell population. For example, if the reference cell population is composed of cells that have not been treated with a known PPARγ ligand, a similar gene expression level in the test cell population and a reference cell population indicates the test agent is not a PPARγ ligand. Conversely, if the reference cell population is made up of cells that have been treated with a known PPAR γ ligand , a similar gene expression profile between the test cell population and the reference cell population indicates the test agent is a PPARγ ligand.
In various embodiments, a ADIPO sequence in a test cell population is considered comparable in expression level to the expression level of the ADIPO sequence in the reference cell population if its expression level varies within a factor of 2.0, 1.5, or 1.0 fold to the level of the ADIPO transcript in the reference cell population. In various embodiments, a ADIPO sequence in a test cell population can be considered altered in levels of expression if its expression level varies from the reference cell population by more than 1.0, 1.5, 2.0 or more fold from the expression level of the corresponding ADIPO sequence in the reference cell population. In some embodiments, the variation in expression of a particular ADIPO sequence corresponds to the change in expression level observed for the ADIPO sequence in the presence and absence of the PPARγ ligand as shown in Table 1.
If desired, comparison of differentially expressed sequences between a test cell population and a reference cell population can be done with respect to a control nucleic acid whose expression is independent of the parameter or condition being measured. Expression levels of the control nucleic acid in the test and reference nucleic acid can be used to normalize signal levels in the compared populations. Suitable control nucleic acids can readily be determined by one of ordinary skill in the art. In some embodiments, the test cell population is compared to multiple reference cell populations. Each of the multiple reference populations may differ in the known parameter. Thus, a test cell population may be compared to a first reference cell population known to have been exposed to a PPARγ ligand, as well as a second reference population known have not been exposed to a PPARy ligand.
The test cell population that is exposed to, i.e., contacted with, the test PPARγ ligand can be any number of cells, i.e., one or more cells, and can be provided in vitro, in vivo, or ex vivo.
In other embodiments, the test cell population can be divided into two or more subpopulations. The subpopulations can be created by dividing the first population of cells to create as identical a subpopulation as possible. This will be suitable, in, for example, in vitro or ex vivo screening methods. In some embodiments, various sub populations can be exposed to a control agent, and/or a test agent, multiple test agents, or, e.g., varying dosages of one or multiple test agents administered together, or in various combinations.
Preferably, cells in the reference cell population are derived from a tissue type as similar as possible to test cell, e.g., adipose tissue. In some embodiments, the control cell is derived from the same subject as the test cell, e.g., from a region proximal to the region of origin of the test cell. In other embodiments, the reference cell population is derived from a plurality of cells. For example, the reference cell population can be a database of expression patterns from previously tested cells for which one of the herein-described parameters or conditions (e.g., PPARγ status, screening, diagnostic, or therapeutic claims) is known.
The subject is preferably a mammal. The mammal can be, e.g., a human, non-human primate, mouse, rat, dog, cat, horse, or cow.
SCREENING FOR PPARγ LIGANDS
In one aspect, the invention provides a method of identifying PPARγ ligands. The PPARγ ligand can be identified by providing a cell population that includes cells capable of expressing one or more nucleic acid sequences homologous to those listed in Table 1 as ADIPO 1-32 and optionally 33-43. The sequences need not be identical to sequences including ADIPO 1-32 and optionally 33-43, as long as the sequence is sufficiently similar that specific hybridization can be detected. Preferably, the cell includes sequences that are identical, or nearly identical to those identifying the ADIPO nucleic acids shown in Table 1.
Expression of the nucleic acid sequences in the test cell population is then compared to the expression of the nucleic acid sequences in a reference cell population, which is a cell population that has not been exposed to the test agent, or, in some embodiments, a cell population exposed the test agent. Comparison can be performed on test and reference samples measured concurrently or at temporally distinct times. An example of the latter is the use of compiled expression information, e.g., a sequence database, which assembles information about expression levels of known sequences following administration of various agents. For example, alteration of expression levels following administration of test agent can be compared to the expression changes observed in the nucleic acid sequences following administration of a control agent, such as N-(2-benzoylphenyl)-L-tyrosine.
An alteration in expression of the nucleic acid sequence in the test cell population compared to the expression of the nucleic acid sequence in the reference cell population that has not been exposed to the test agent indicates the test agent is a PPARγ ligand.
The invention also includes a PPARγ ligand identified according to this screening method, and a pharmaceutical composition which includes the PPARγ ligands.
SCREENING ASSAYS FOR IDENTIFYING A CANDIDATE THERAPEUTIC AGENT FOR TREATING OR PREVENTING A PATHOPHYSIOLOGIES ASSOCIATED WITH THE PPARγ MEDIATED PATHWAY
The differentially expressed sequences disclosed herein can also be used to identify candidate therapeutic agents pathophysiologies associated with the PPARγ mediated pathway. The method is based on screening a candidate therapeutic agent to determine if it induces an expression profile of one or more ADIPO 1-32 sequences, and, optionally, ADIPO33-43 sequences in a test cell population that is characteristic of a PPARXresponse. In the method, a test cell population is exposed to a test agent or a combination of test agents (sequentially or consequentially), and the expression of one or more of the ADIPO sequences is measured. The expression of the ADIPO sequences in the test population is compared to expression level of the ADIPO sequences in a reference cell population whose PPARγ status is known. If the reference cell population contains cells that have not been exposed to a PPARγ ligand, alteration of expression of the nucleic acids in the test cell population as compared to the reference cell population indicates that the test agent is a candidate therapeutic agent.
In some embodiments, the reference cell population includes cells that have been exposed to a test agent. When this cell population is used, an alteration in expression of the nucleic acid sequences in the presence of the agent from the expression profile of the cell population in the absence of the agent indicates the agent is a candidate therapeutic agent. In other embodiments, the test cell population includes cells that have not been exposed to a PPARγ ligand. For this cell population, a similarity in expression of the ADIPO sequences in the test and control cell populations indicates the test agent is not a candidate therapeutic agent, while a difference suggests it is a candidate.
The test agent can be a compound not previously described or can be a previously known compound but which is not known to be a PPARγ ligand
An agent effective in stimulating expression of underexpressed genes, or in suppressing expression of overexpressed genes can be further tested for its ability to prevent the PPARγ mediated pathophysiology, e.g. NIDDM, and as a potential therapeutic useful for the treatment of such pathophysiology. Further evaluation of the clinical usefulness of such a compound can be performed using standard methods of evaluating toxicity and clinical effectiveness of anti-diabetic agents. SELECTING A THERAPEUTIC AGENT FOR TREATING A PATHOPHYSIOLOGY ASSOCIATED WITH THE PPARγ MEDIATED PATHWAY THAT IS APPROPRIATE FOR A PARTICULAR INDIVIDUAL
Differences in the genetic makeup of individuals can result in differences in their relative abilities to metabolize various drugs. An agent that is metabolized in a subject to act as a PPARγ ligand can manifest itself by inducing a change in gene expression pattern from that characteristic of a pathophysiologic state to a gene expression pattern characteristic of a non-pathophysiologic state. Accordingly, the differentially expressed ADIPO sequences disclosed herein allow for a putative therapeutic or prophylactic agent to be tested in a test cell population from a selected subject in order to determine if the agent is a suitable PPARγ ligand in the subject.
To identify a PPARγ ligand, that is appropriate for a specific subject, a test cell population from the subject is exposed to a therapeutic agent, and the expression of one or more of ADIPO 1-32 and, optionally, ADIPO33-43 sequences is measured.
In some embodiments, the test cell population contains an adipocyte. In other embodiments, the agent is first mixed with a cell extract, e.g., an adipose cell extract, which contains enzymes that metabolize drugs into an active form. The activated form of the therapeutic agent can then be mixed with the test cell population and gene expression measured. Preferably, the cell population is contacted ex vivo with the agent or activated form of the agent.
Expression of the nucleic acid sequences in the test cell population is then compared to the expression of the nucleic acid sequences a reference cell population. The reference cell population includes at least one cell whose PPARγ status is known. If the reference cell had been exposed to a PPARγ ligand a similar gene expression profile between the test cell population and the reference cell population indicates the agent is suitable for treating the pathophysiology in the subject. A difference in expression between sequences in the test cell population and those in the reference cell population indicates that the agent is not suitable for treating the PPARγ pathophysiology in the subject.
If the reference cell has not been exposed to a PPARγ ligand, a similarity in gene expression patterns between the test cell population and the reference cell population indicates the agent is not suitable for treating the PPARγ pathophysiology in the subject, while a dissimilar gene expression patterns indicate the agent will be suitable for treating the subject.
In some embodiments, a decrease in expression of one or more of the sequences ADIPOS: 1, 2, 3, 7, 16, 17, 20-22, 27, 29, 30, and 32-43 or an increase in expression of one or more of the sequences ADIPOS: 4, 5, 8 -10, 12, 13, 15, 18, 19, 23-26, 28 and 31 in a test cell population relative to a reference cell population is indicative that the agent is therapeutic.
The test agent can be any compound or composition. In some embodiments the test agents are compounds and composition know to be PPARγ ligands, e.g. N-(2-benzoylphenyl)-L-tyrosine.
METHODS OF DIAGNOSING PATHOPHYSIOLOGIES ASSOCIATED WITH THE PPARγ MEDIATED
PATHWAY
The invention further provides a method of diagnosing a pathophysiology associated with the PPARγ mediated pathway, e.g., non-insulin dependent diabetes millitus, in a subject. A pathophysiology is diagnosed by examining the expression of one or more ADIPO nucleic acid sequences from a test population of cells from a subject suspected of have the pathophysiology.
Expression of one or more of the ADIPO nucleic acid sequences, e.g. ADIPO: 1-32 and optionally 33-43 is measured in the test cell and compared to the expression of the sequences in the reference cell population. The reference cell population contains at least one cell whose PPAR γ status, or disease status (i.e., the reference cell population is from a NIDDM subject) is known. If the reference cell population contains cells that have not been exposed to a PPARγ ligand, then a similarity in expression between ADIPO sequences in the test population and the reference cell population indicates the subject does not have a PPARγ mediated pathophysiology. A difference in expression between ADIPO sequences in the test population and the reference cell population indicates the reference cell population has a PPAR γ mediated pathophysiology.
Conversely, when the reference cell population contains cells that have been exposed to a
PPARγ ligand, a similarity in expression pattern between the test cell population and the reference cell population indicates the test cell population has a PPARγ mediated pathophysiology. A difference in expression between ADIPO sequences in the test population and the reference cell population indicates the subject does not have a PPARγ mediated pathophysiology.
METHODS OF TREATING PATHOPHYSIOLOGIES ASSOCIATED WITH THE PPARγ MEDIATED PATHWAY IN A SUBJECT
Also included in the invention is a method of treating, i.e, preventing or delaying the onset of a pathophysiology associated with the PPARγ mediated pathway in a subject by administering to the subject an agent which modulates the expression or activity of one or more nucleic acids selected from the group consisting of ADIPO 1-32 and, optionally, ADIPO 33-43. ""Modulates" is meant to include increase or decrease expression or activity of the ADIPO nucleic acids. Preferably, modulation results in alteration alter the expression or activity of the ADIPO genes or gene products in a subject to a level similar or identical to a subject not suffering from the pathophysiology.
The pathophysiologies can be any of the pathophysiologies described herein, e.g., NIDDM. The subject can be, e.g., a human, a rodent such as a mouse or rat, or a dog or cat.
The herein described ADIPO nucleic acids, polypeptides, antibodies, agonists, and antagonists when used therapeutically are referred to herein as "Therapeutics". Methods of administration of Therapeutics include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The Therapeutics of the present invention may be administered by any convenient route, for example by infusion or bolus injection, by absoφtion through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically-active agents. Administration can be systemic or local. In addition, it may be advantageous to administer the Therapeutic into the central nervous system by any suitable route, including intraventricular and intrathecal injection. Intraventricular injection may be facilitated by an intraventricular catheter attached to a reservoir (e.g., an Ommaya reservoir). Pulmonary administration may also be employed by use of an inhaler or nebulizer, and formulation with an aerosolizing agent. It may also be desirable to administer the Therapeutic locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, by injection, by means of a catheter, by means of a suppository, or by means of an implant. In a specific embodiment, administration may be by direct injection at the site (or former site) of a malignant tumor or neoplastic or pre-neoplastic tissue.
Various delivery systems are known and can be used to administer a Therapeutic of the present invention including, e.g. : (i) encapsulation in liposomes, microparticles, microcapsules; (ii) recombinant cells capable of expressing the Therapeutic; (iii) receptor-mediated endocytosis (See, e.g., Wu and Wu, 1987. J Biol Chem 262:4429-4432); (iv) construction of a Therapeutic nucleic acid as part of a retroviral or other vector, and the like. In one embodiment of the present invention, the Therapeutic may be delivered in a vesicle, in particular a liposome. In a liposome, the protein of the present invention is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution. Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. Preparation of such liposomal formulations is within the level of skill in the art, as disclosed, for example, in U.S. Pat. No. 4,837,028; and U.S. Pat. No. 4,737,323, all of which are incoφorated herein by reference. In yet another embodiment, the Therapeutic can be delivered in a controlled release system including, e.g. : a delivery pump (See, e.g., Saudek, et al, 1989. New Engl J Med 321 :574 and a semi-permeable polymeric material (See, e.g., Howard, et al, 1989. J Neurosurg 71 :105). Additionally, the controlled release system can be placed in proximity of the therapeutic target (e.g., the brain), thus requiring only a fraction of the systemic dose. See, e.g., Goodson, In: Medical Applications of Controlled Release 1984. (CRC Press, Bocca Raton, FL).
In a specific embodiment of the present invention, where the Therapeutic is a nucleic acid encoding a protein, the Therapeutic nucleic acid may be administered in vivo to promote expression of its encoded protein, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular (e.g., by use of a retroviral vector, by direct injection, by use of microparticle bombardment, by coating with lipids or cell-surface receptors or transfecting agents, or by administering it in linkage to a homeobox-like peptide which is known to enter the nucleus (See, e.g., Joliot, et al, 1991. Proc Natl Acad Sci USA 88:1864-1868), and the like. Alternatively, a nucleic acid Therapeutic can be introduced intracellularly and incoφorated within host cell DNA for expression, by homologous recombination.
As used herein, the term "therapeutically effective amount" means the total amount of each active component of the pharmaceutical composition or method that is sufficient to show a meaningful patient benefit, i.e., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions. When applied to an individual active ingredient, administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.
The amount of the Therapeutic of the invention which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and may be determined by standard clinical techniques by those of average skill within the art. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the overall seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Ultimately, the attending physician will decide the amount of protein of the present invention with which to treat each individual patient. Initially, the attending physician will administer low doses of protein of the present invention and observe the patient's response. Larger doses of protein of the present invention may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further. However, suitable dosage ranges for intravenous administration of the Therapeutics of the present invention are generally about 20-500 micrograms (μg) of active compound per kilogram (Kg) body weight. Suitable dosage ranges for intranasal administration are generally about 0.01 pg/kg body weight to 1 mg/kg body weight. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. Suppositories generally contain active ingredient in the range of 0.5% to 10% by weight; oral formulations preferably contain 10% to 95% active ingredient.
The duration of intravenous therapy using the pharmaceutical composition of the present invention will vary, depending on the severity of the disease being treated and the condition and potential idiosyncratic response of each individual patient. It is contemplated that the duration of each application of the protein of the present invention will be in the range of 12 to 24 hours of continuous intravenous administration. Ultimately the attending physician will decide on the appropriate duration of intravenous therapy using the pharmaceutical composition of the present invention.
Polynucleotides of the present invention can also be used for gene therapy. Gene therapy refers to therapy that is performed by the administration of a specific nucleic acid to a subject. Delivery of the Therapeutic nucleic acid into a mammalian subject may be either direct (i.e., the patient is directly exposed to the nucleic acid or nucleic acid-containing vector) or indirect (i.e., cells are first transformed with the nucleic acid in vitro, then transplanted into the patient). These two approaches are known, respectively, as in vivo or ex vivo gene therapy. Polynucleotides of the invention may also be administered by other known methods for introduction of nucleic acid into a cell or organism (including, without limitation, in the form of viral vectors or naked DNA). Any of the methodologies relating to gene therapy available within the art may be used in the practice of the present invention. See e.g., Goldspiel, et al, 1993. Clin Pharm 12:488-505.
Cells may also be cultured ex vivo in the presence of therapeutic agents or proteins of the present invention in order to proliferate or to produce a desired effect on or activity in such cells. Treated cells can then be introduced in vivo for therapeutic puφoses.
ASSESSING EFFICACY OF TREATMENT OF A PATHOPHYSIOLOGIES ASSOCIATED WITH THE PPARγ MEDIATED PATHWAY IN A SUBJECT
The differentially expressed ADIPO sequences identified herein also allow for the course of treatment of a pathophysiology to be monitored. In this method, a test cell population is provided from a subject undergoing treatment for pathophysiologies associated with the PPARγ mediated pathway. If desired, test cell populations can be taken from the subject at various time points before, during, or after treatment. Expression of one or more of the ADIPO sequences, e.g., AD IPOs: 1-32 and, optionally, ADIPO 33-43, in the cell population is then measured and compared to a reference cell population which includes cells whose pathophysiologic state is known. Preferably, the reference cells not been exposed to the treatment.
If the reference cell population contains no cells exposed to the treatment, a similarity in expression between ADIPO sequences in the test cell population and the reference cell population indicates that the treatment is efficacious. However, a difference in expression between ADIPO sequences in the test population and this reference cell population indicates the treatment is not efficacious.
By "efficacious" is meant that the treatment leads to a decrease in the pathophysiology in a subject. When treatment is applied prophylactically, "efficacious" means that the treatment retards or prevents a pathophysiology. For example, if the PPARγ mediated pathophysiology is NIDDM, a "efficacious" treatment is one that increases insulin sensitivity in a subject.
Efficaciousness can be determined in association with any known method for treating the particular pathophysiology.
ADIPO NUCLEIC ACIDS
Also provided in the invention are novel nucleic acids that include a nucleic acid sequence selected from the group consisting of ADIPOs:l-15, or its complement, as well as vectors and cells including these nucleic acids. Also provided are polypeptides encoded by ADIPO nucleic acid or biologically active portions thereof.
Also included in the invention are nucleic acid fragments sufficient for use as hybridization probes to identify ADIPO-encoding nucleic acids (e.g., ADIPO mRNA) and fragments for use as polymerase chain reaction (PCR) primers for the amplification or mutation of ADIPO nucleic acid molecules. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA generated using nucleotide analogs, and derivatives, fragments and homologs thereof. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.
"Probes" refer to nucleic acid sequences of variable length, preferably between at least about 10 nucleotides (nt) or as many as about, e.g., 6,000 nt, depending on use. Probes are used in the detection of identical, similar, or complementary nucleic acid sequences. Longer length probes are usually obtained from a natural or recombinant source, are highly specific and much slower to hybridize than oligomers. Probes may be single- or double-stranded and designed to have specificity in PCR, membrane-based hybridization technologies, or ELISA-like technologies.
An "isolated" nucleic acid molecule is one that is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid. Examples of isolated nucleic acid molecules include, but are not limited to, recombinant DNA molecules contained in a vector, recombinant DNA molecules maintained in a heterologous host cell, partially or substantially purified nucleic acid molecules, and synthetic DNA or RNA molecules. Preferably, an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated ADIPO nucleic acid molecule can contain less than about 50 kb, 25 kb, 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or of chemical precursors or other chemicals when chemically synthesized.
A nucleic acid molecule of the present invention, e.g., a nucleic acid molecule having the nucleotide sequence of any of ADIPOS: 1-15, or a complement of any of these nucleotide sequences, can be isolated using standard molecular biology techniques and the sequence information provided herein. Using all or a portion of these nucleic acid sequences as a hybridization probe, ADIPO nucleic acid sequences can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook et al, eds., MOLECULAR CLONING: A LABORATORY MANUAL 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausubel, et al, eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, 1993.)
A nucleic acid of the invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to ADIPO nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
As used herein, the term "oligonucleotide" refers to a series of linked nucleotide residues, which oligonucleotide has a sufficient number of nucleotide bases to be used in a PCR reaction. A short oligonucleotide sequence may be based on, or designed from, a genomic or cDNA sequence and is used to amplify, confirm, or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue. Oligonucleotides comprise portions of a nucleic acid sequence having at least about 10 nt and as many as 50 nt, preferably about 15 nt to 30 nt. They may be chemically synthesized and may be used as probes.
In another embodiment, an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule that is a complement of the nucleotide sequence shown in ADIPOs: 1-15 . In another embodiment, an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule that is a complement of the nucleotide sequence shown in any of these sequences, or a portion of any of these nucleotide sequences. A nucleic acid molecule that is complementary to the nucleotide sequence shown in ADIPOs: 1-15 is one that is sufficiently complementary to the nucleotide sequence shown, such that it can hydrogen bond with little or no mismatches to the nucleotide sequences shown, thereby forming a stable duplex. As used herein, the term "complementary" refers to Watson-Crick or Hoogsteen base pairing between nucleotides units of a nucleic acid molecule, and the term "binding" means the physical or chemical interaction between two polypeptides or compounds or associated polypeptides or compounds or combinations thereof. Binding includes ionic, non-ionic, Non der Waals, hydrophobic interactions, etc. A physical interaction can be either direct or indirect. Indirect interactions may be through or due to the effects of another polypeptide or compound. Direct binding refers to interactions that do not take place through, or due to, the effect of another polypeptide or compound, but instead are without other substantial chemical intermediates.
Moreover, the nucleic acid molecule of the invention can comprise only a portion of the nucleic acid sequence of AD IPOs: 1-15 e.g., a fragment that can be used as a probe or primer or a fragment encoding a biologically active portion of ADIPO. Fragments provided herein are defined as sequences of at least 6 (contiguous) nucleic acids or at least 4 (contiguous) amino acids, a length sufficient to allow for specific hybridization in the case of nucleic acids or for specific recognition of an epitope in the case of amino acids, respectively, and are at most some portion less than a full length sequence. Fragments may be derived from any contiguous portion of a nucleic acid or amino acid sequence of choice. Derivatives are nucleic acid sequences or ■ amino acid sequences formed from the native compounds either directly or by modification or partial substitution. Analogs are nucleic acid sequences or amino acid sequences that have a structure similar to, but not identical to, the native compound but differs from it in respect to certain components or side chains. Analogs may be synthetic or from a different evolutionary origin and may have a similar or opposite metabolic activity compared to wild type.
Derivatives and analogs may be full length or other than full length, if the derivative or analog contains a modified nucleic acid or amino acid, as described below. Derivatives or analogs of the nucleic acids or proteins of the invention include, but are not limited to, molecules comprising regions that are substantially homologous to the nucleic acids or proteins of the invention, in various embodiments, by at least about 45%, 50%, 70%, 80%, 95%, 98%, or even 99%o identity (with a preferred identity of 80-99%) over a nucleic acid or amino acid sequence of identical size or when compared to an aligned sequence in which the alignment is done by a computer homology program known in the art, or whose encoding nucleic acid is capable of hybridizing to the complement of a sequence encoding the aforementioned proteins under stringent, moderately stringent, or low stringent conditions. See e.g. Ausubel, et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, 1993, and below. An exemplary program is the Gap program (Wisconsin Sequence Analysis Package, Version 8 for UNIX, Genetics Computer Group, University Research Park, Madison, WI) using the default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2: 482-489, which in incoφorated herein by reference in its entirety).
A "homologous nucleic acid sequence" or "homologous amino acid sequence," or variations thereof, refer to sequences characterized by a homology at the nucleotide level or amino acid level as discussed above. Homologous nucleotide sequences encode those sequences coding for isoforms of a ADIPO polypeptide. Isoforms can be expressed in different tissues of the same organism as a result of, for example, alternative splicing of RNA. Alternatively, isoforms can be encoded by different genes. In the present invention, homologous nucleotide sequences include nucleotide sequences encoding for a ADIPO polypeptide of species other than humans, including, but not limited to, mammals, and thus can include, e.g., mouse, rat, rabbit, dog, cat cow, horse, and other organisms. Homologous nucleotide sequences also include, but are not limited to, naturally occurring allelic variations and mutations of the nucleotide sequences set forth herein. A homologous nucleotide sequence does not, however, include the nucleotide sequence encoding a human ADIPO protein. Homologous nucleic acid sequences include those nucleic acid sequences that encode conservative amino acid substitutions (see below) in a
ADIPO polypeptide, as well as a polypeptide having a ADIPO activity. A homologous amino acid sequence does not encode the amino acid sequence of a human ADIPO polypeptide.
The nucleotide sequence determined from the cloning of human ADIPO genes allows for the generation of probes and primers designed for use in identifying and/or cloning ADIPO homologues in other cell types, e.g., from other tissues, as well as ADIPO homologues from other mammals. The probe/primer typically comprises a substantially purified oligonucleotide. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, 25, 50, 100, 150, 200, 250, 300, 350 or 400 consecutive sense strand nucleotide sequence of a nucleic acid comprising a ADIPO sequence, or an anti-sense strand nucleotide sequence of a nucleic acid comprising a ADIPO sequence, or of a naturally occurring mutant of these sequences.
Probes based on human ADIPO nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins. In various embodiments, the probe further comprises a label group attached thereto, e.g., the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used as a part of a diagnostic test kit for identifying cells or tissue which misexpress a ADIPO protein, such as by measuring a level of a ADIPO-encoding nucleic acid in a sample of cells from a subject e.g., detecting ADIPO mRNA levels or determining whether a genomic ADIPO gene has been mutated or deleted.
"A polypeptide having a biologically active portion of ADIPO" refers to polypeptides exhibiting activity similar, but not necessarily identical to, an activity of a polypeptide of the present invention, including mature forms, as measured in a particular biological assay, with or without dose dependency. A nucleic acid fragment encoding a "biologically active portion of ADIPO" can be prepared by isolating a portion of ADIPOs: 1-15, that encodes a polypeptide having a ADIPO biological activity, expressing the encoded portion of ADIPO protein (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of ADIPO. For example, a nucleic acid fragment encoding a biologically active portion of a ADIPO polypeptide can optionally include an ATP -binding domain. In another embodiment, a nucleic acid fragment encoding a biologically active portion of ADIPO includes one or more regions.
ADIPO VARIANTS
The invention further encompasses nucleic acid molecules that differ from the disclosed or referenced ADIPO nucleotide sequences due to degeneracy of the genetic code. These nucleic acids thus encode the same ADIPO protein as that encoded by nucleotide sequence comprising a ADIPO nucleic acid as shown in, e.g. , ADIPO 1-15
In addition to the rat ADIPO nucleotide sequence shown in ADIPOs:l-15, it will be appreciated by those skilled in the art that DNA sequence polymoφhisms that lead to changes in the amino acid sequences of a ADIPO polypeptide may exist within a population (e.g., the human population). Such genetic polymoφhism in the ADIPO gene may exist among individuals within a population due to natural allelic variation. As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules comprising an open reading frame encoding a ADIPO protein, preferably a mammalian ADIPO protein. Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence of the ADIPO gene. Any and all such nucleotide variations and resulting amino acid polymoφhisms in ADIPO that are the result of natural allelic variation and that do not alter the functional activity of ADIPO are intended to be within the scope of the invention.
Moreover, nucleic acid molecules encoding ADIPO proteins from other species, and thus that have a nucleotide sequence that differs from the human sequence of ADIPOl-15, are intended to be within the scope of the invention. Nucleic acid molecules corresponding to natural allelic variants and homologues of the ADIPO DNAs of the invention can be isolated based on their homology to the human ADIPO nucleic acids disclosed herein using the human cDNAs, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions. For example, a soluble human ADIPO DNA can be isolated based on its homology to human membrane-bound ADIPO. Likewise, a membrane-bound human ADIPO DNA can be isolated based on its homology to soluble human ADIPO.
Accordingly, in another embodiment, an isolated nucleic acid molecule of the invention is at least 6 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of AD IPOs: 1-15. In another embodiment, the nucleic acid is at least 10, 25, 50, 100, 250 or 500 nucleotides in length. In another embodiment, an isolated nucleic acid molecule of the invention hybridizes to the coding region. As used herein, the term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60%> homologous to each other typically remain hybridized to each other. Homologs (i.e., nucleic acids encoding ADIPO proteins derived from species other than human) or other related sequences (e.g., paralogs) can be obtained by low, moderate or high stringency hybridization with all or a portion of the particular human sequence as a probe using methods well known in the art for nucleic acid hybridization and cloning.
As used herein, the phrase "stringent hybridization conditions" refers to conditions under which a probe, primer or oligonucleotide will hybridize to its target sequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures than shorter sequences. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50%> of the probes are occupied at equilibrium. Typically, 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 (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes, primers or oligonucleotides (e.g., 10 nt to 50 nt) and at least about 60°C for longer probes, primers and oligonucleotides. Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.
Stringent conditions are known to those skilled in the art and can be found in CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NN. (1989), 6.3.1-6.3.6. Preferably, the conditions are such that sequences at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99%) homologous to each other typically remain hybridized to each other. A non-limiting example of stringent hybridization conditions is hybridization in a high salt buffer comprising 6X SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% FicoU, 0.02% BSA, and 500 mg/ml denatured salmon sperm DΝA at 65 °C. This hybridization is followed by one or more washes in 0.2X SSC, 0.01% BSA at 50°C. An isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of ADIPOs: 1-15 corresponds to a naturally occurring nucleic acid molecule. As used herein, a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
In a second embodiment, a nucleic acid sequence that is hybridizable to the nucleic acid molecule comprising the nucleotide sequence of ADIPOs: 1-15 or fragments, analogs or derivatives thereof, under conditions of moderate stringency is provided. A non-limiting example of moderate stringency hybridization conditions are hybridization in 6X SSC, 5X Denhardt's solution, 0.5% SDS and 100 mg/ml denatured salmon sperm DNA at 55°C, followed by one or more washes in IX SSC, 0.1% SDS at 37°C. Other conditions of moderate stringency that may be used are well known in the art. See, e.g., Ausubel et al. (eds.), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY, and Kriegler, 1990, GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY.
In a third embodiment, a nucleic acid that is hybridizable to the nucleic acid molecule comprising the nucleotide sequence of ADIPOs:l-15or fragments, analogs or derivatives thereof, under conditions of low stringency, is provided. A non-limiting example of low stringency hybridization conditions are hybridization in 35% formamide, 5X SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 mg/ml denatured salmon sperm DNA, 10%) (wt/vol) dextran sulfate at 40°C, followed by one or more washes in 2X SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS at 50°C. Other conditions of low stringency that may be used are well known in the art (e.g., as employed for cross-species hybridizations). See, e.g., Ausubel et al. feds.), 1993, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY, and Kriegler, 1990, GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY; Shilo et al., 1981, Proc Natl Acad Sci USA 78: 6789-6792.
CONSERVATIVE MUTATIONS
In addition to naturally-occurring allelic variants of the ADIPO sequence that may exist in the population, the skilled artisan will further appreciate that changes can be introduced into an ADIPO nucleic acid or directly into an ADIPO polypeptide sequence without altering the functional ability of the ADIPO protein. In some embodiments, the nucleotide sequence of ADIPOs: l-15will be altered, thereby leading to changes in the amino acid sequence of the encoded ADIPO protein. For example, nucleotide substitutions that result in amino acid substitutions at various "non-essential" amino acid residues can be made in the sequence of ADIPOs: 1-15A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of ADIPO without altering the biological activity, whereas an "essential" amino acid residue is required for biological activity. For example, amino acid residues that are conserved among the ADIPO proteins of the present invention, are predicted to be particularly unamenable to alteration.
In addition, amino acid residues that are conserved among family members of the ADIPO proteins of the present invention, are also predicted to be particularly unamenable to alteration. As such, these conserved domains are not likely to be amenable to mutation. Other amino acid residues, however, (e.g., those that are not conserved or only semi-conserved among members of the ADIPO proteins) may not be essential for activity and thus are likely to be amenable to alteration.
Another aspect of the invention pertains to nucleic acid molecules encoding ADIPO proteins that contain changes in amino acid residues that are not essential for activity. Such ADIPO proteins differ in amino acid sequence from the amino acid sequences of polypeptides encoded by nucleic acids containing ADIPOs: 1-15, yet retain biological activity. In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 45% homologous, more preferably 60%, and still more preferably at least about 70%, 80%), 90%, 95%o, 98%, and most preferably at least about 99% homologous to the amino acid sequence of the amino acid sequences of polypeptides encoded by nucleic acids comprising ADIPOs: 1-15.
An isolated nucleic acid molecule encoding a ADIPO protein homologous to can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of a nucleic acid comprising ADIPOs: 1-15, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced into a nucleic acid comprising ADIPOs: 1-15 by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in ADIPO is replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a ADIPO coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for ADIPO biological activity to identify mutants that retain activity. Following mutagenesis of the nucleic acid,, the encoded protein can be expressed by any recombinant technology known in the art and the activity of the protein can be determined.
In one embodiment, a mutant ADIPO protein can be assayed for (1) the ability to form protein: protein interactions with other ADIPO proteins, other cell-surface proteins, or biologically active portions thereof, (2) complex formation between a mutant ADIPO protein and a ADIPO ligand; (3) the ability of a mutant ADIPO protein to bind to an intracellular target protein or biologically active portion thereof; (e.g., avidin proteins); (4) the ability to bind ATP; or (5) the ability to specifically bind a ADIPO protein antibody. In other embodiment, the fragment of the complementary polynucleotide sequence of
ADIPO 1-15 wherein the fragment of the complementary polynucleotide sequence hybridizes to the first sequence.
In other specific embodiments, the nucleic acid is RNA or DNA. The fragment or the fragment of the complementary polynucleotide sequence of ADIPO 1-15, wherein the fragment is between about 10 and about 100 nucleotides in length, e.g., between about 10 and about 90 nucleotides in length, or about 10 and about 75 nucleotides in length, about 10 and about 50 bases in length, about 10 and about 40 bases in length, or about 15 and about 30 bases in length.
ANTISENSE
Another aspect of the invention pertains to isolated antisense nucleic acid molecules that are hybridizable to or complementary to the nucleic acid molecule comprising the nucleotide sequence of a ADIPO sequence or fragments, analogs or derivatives thereof. An "antisense" nucleic acid comprises a nucleotide sequence that is complementary to a "sense" nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. In specific aspects, antisense nucleic acid molecules are provided that comprise a sequence complementary to at least about 10, 25, 50, 100, 250 or 500 nucleotides or an entire ADIPO coding strand, or to only a portion thereof. Nucleic acid molecules encoding fragments, homologs, derivatives and analogs of a ADIPO protein, or antisense nucleic acids complementary to a nucleic acid comprising a ADIPO nucleic acid sequence are additionally provided.
In one embodiment, an antisense nucleic acid molecule is antisense to a "coding region" of the coding strand of a nucleotide sequence encoding ADIPO. The term "coding region" refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues. In another embodiment, the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence encoding ADIPO. The term "noncoding region" refers to 5' and 3' sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5' and 3' untranslated regions).
Given the coding strand sequences encoding ADIPO disclosed herein, antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick or Hoogsteen base pairing. The antisense nucleic acid molecule can be complementary to the entire coding region of ADIPO mRNA, but more preferably is an oligonucleotide that is antisense to only a portion of the coding or noncoding region of ADIPO mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of ADIPO mRNA. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid of the invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used.
Examples of modified nucleotides that can be used to generate the antisense nucleic acid include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl- 2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-mefhylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
The antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a ADIPO protein to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation. The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the major groove of the double helix. An example of a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens. The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.
In yet another embodiment, the antisense nucleic acid molecule of the invention is an α-anomeric nucleic acid molecule. An α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β-units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids Res 15: 6625-6641). The antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res 15: 6131-6148) or a chimeric RNA -DNA analogue (Inoue et al. (1987) EERS Lett 215: 327-330).
RlBOZYMΕS AND PNA MOIETIES
In still another embodiment, an antisense nucleic acid of the invention is a ribozyme. Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave ADIPO mRNA transcripts to thereby inhibit translation of ADIPO mRNA. A ribozyme having specificity for a ADIPO-encoding nucleic acid can be designed based upon the nucleotide sequence of a ADIPO DNA disclosed herein. For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a ADIPO-encoding mRNA. See, e.g., Cech et al. U.S. Pat. No. 4,987,071 ; and Cech et al. U.S. Pat. No. 5,116,742. Alternatively, ADIPO mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel et al, (1993) Science 261 :1411-1418.
Alternatively, ADIPO gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of a ADIPO nucleic acid (e.g., the ADIPO promoter and/or enhancers) to form triple helical structures that prevent transcription of the ADIPO gene in target cells. See generally, Helene. (1991) Anticancer Drug Des. 6: 569-84; Helene. et al. (1992) Ann. N Y. Acad. Sci. 660:27-36; and Maher (1992) Bioassays 14: 807-15.
In various embodiments, the nucleic acids of ADIPO can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule. For example, the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids (see Hyrup et al. (1996) Bioorg Med Chem 4: 5-23). As used herein, the terms "peptide nucleic acids" or "PNAs" refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup et al. (1996) above; Perry-O'Keefe et al. (1996) PNAS 93: 14670-675.
PNAs of ADIPO can be used in therapeutic and diagnostic applications. For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication. PNAs of ADIPO can also be used, e.g., in the analysis of single base pair mutations in a gene by, e.g., PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., SI nucleases (Hyrup B. (1996) above); or as probes or primers for DNA sequence and hybridization (Hyrup et al. (1996), above; Perry-O'Keefe (1996), above). In another embodiment, PNAs of ADIPO can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art. For example, PNA-DNA chimeras of ADIPO can be generated that may combine the advantageous properties of PNA and DNA. Such chimeras allow DNA recognition enzymes, e.g., RNase H and DNA polymerases, to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity. PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup (1996) above). The synthesis of PNA-DNA chimeras can be performed as described in Hyrup (1996) above and Finn et al. (1996) Nucl Acids Res 24: 3357-63. For example, a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry, and modified nucleoside analogs, e.g., 5'-(4-methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite, can be used between the PNA and the 5' end of DNA (Mag et al. (1989) Nucl Acid Res 17: 5973-88). PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment (Finn et al. (1996) above). Alternatively, chimeric molecules can be synthesized with a 5' DNA segment and a 3' PNA segment. See, Petersen et al. (1975) Bioorg Med Chem Lett 5: 1119-1 1124.
In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al, 1989, Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556; Lemaitre et al, 1987, Proc. Natl Acad. Sci. 84:648-652; PCT Publication No. W088/09810) or the blood-brain barrier (see, e.g., PCT Publication No. W089/10134). In addition, oligonucleotides can be modified with hybridization triggered cleavage agents (See, e.g., Krol et al, 1988, BioTechniques 6:958-976) or intercalating agents. (See, e.g., Zon, 1988, Pharm. Res. 5: 539-549). To this end, the oligonucleotide may be conjugated to another molecule, e.g., a peptide, a hybridization triggered cross-linking agent, a transport agent, a hybridization-triggered cleavage agent, etc. ADIPO POLYPEPTIDES
One aspect of the invention pertains to isolated ADIPO proteins, and biologically active portions thereof, or derivatives, fragments, analogs or homologs thereof. Also provided are polypeptide fragments suitable for use as immunogens to raise anti-ADIPO antibodies. In one embodiment, native ADIPO proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques. In another embodiment, ADIPO proteins are produced by recombinant DNA techniques. Alternative to recombinant expression, a ADIPO protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques.
An "isolated" or "purified" protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the ADIPO protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. The language "substantially free of cellular material" includes preparations of ADIPO protein in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced. In one embodiment, the language
"substantially free of cellular material" includes preparations of ADIPO protein having less than about 30%) (by dry weight) of non-ADIPO protein (also referred to herein as a "contaminating protein"), more preferably less than about 20% of non-ADIPO protein, still more preferably less than about 10% of non-ADIPO protein, and most preferably less than about 5% non-ADIPO protein. When the ADIPO protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%), and most preferably less than about 5% of the volume of the protein preparation.
The language "substantially free of chemical precursors or other chemicals" includes preparations of ADIPO protein in which the protein is separated from chemical precursors or other chemicals that are involved in the synthesis of the protein. In one embodiment, the language "substantially free of chemical precursors or other chemicals" includes preparations of ADIPO protein having less than about 30% (by dry weight) of chemical precursors or non-ADIPO chemicals, more preferably less than about 20% chemical precursors or non-ADIPO chemicals, still more preferably less than about 10% chemical precursors or non-ADIPO chemicals, and most preferably less than about 5% chemical precursors or non-ADIPO chemicals.
Biologically active portions of a ADIPO protein include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequence of the ADIPO protein, e.g., the amino acid sequence encoded by a nucleic acid comprising ADIPO 1-20 that include fewer amino acids than the full length ADIPO proteins, and exhibit at least one activity of a ADIPO protein. Typically, biologically active portions comprise a domain or motif with at least one activity of the ADIPO protein. A biologically active portion of a ADIPO protein can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acids in length.
A biologically active portion of a ADIPO protein of the present invention may contain at least one of the above-identified domains conserved between the ADIPO proteins. An alternative biologically active portion of a ADIPO protein may contain at least two of the above-identified domains. Another biologically active portion of a ADIPO protein may contain at least three of the above-identified domains. Yet another biologically active portion of a ADIPO protein of the present invention may contain at least four of the above-identified domains.
Moreover, other biologically active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native ADIPO protein.
In some embodiments, the ADIPO protein is substantially homologous to one of these ADIPO proteins and retains its the functional activity, yet differs in amino acid sequence due to natural allelic variation or mutagenesis, as described in detail below. In specific embodiments, the invention includes an isolated polypeptide comprising an amino acid sequence that is 80%) or more identical to the sequence of a polypeptide whose expression is modulated in a mammal to which PPARγ ligand is administered. DETERMINING HOMOLOGY BETWEEN TWO OR MORE SEQUENCES
To determine the percent homology of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison puφoses (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are homologous at that position (i.e., as used herein amino acid or nucleic acid "homology" is equivalent to amino acid or nucleic acid "identity").
The nucleic acid sequence homology may be determined as the degree of identity between two sequences. The homology may be determined using computer programs known in the art, such as GAP software provided in the GCG program package. See Needleman and Wunsch 1970 J Mol Biol 48: 443-453. Using GCG GAP software with the following settings for nucleic acid sequence comparison: GAP creation penalty of 5.0 and GAP extension penalty of 0.3, the coding region of the analogous nucleic acid sequences referred to above exhibits a degree of identity preferably of at least 70%, 75%, 80%, 85%, 90%, 95%, 98%o, or 99%, with the CDS (encoding) part of a DNA sequence comprising ADIPOS: 1-15.
The term "sequence identity" refers to the degree to which two polynucleotide or polypeptide sequences are identical on a residue-by-residue basis over a particular region of comparison. The term "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over that region of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I, in the case of nucleic acids) 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 region of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. The term "substantial identity" as used herein denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 80 percent sequence identity, preferably at least 85 percent identity and often 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison region.
CHIMERIC AND FUSION PROTEINS
The invention also provides ADIPO chimeric or fusion proteins. As used herein, an ADIPO "chimeric protein" or "fusion protein" comprises an ADIPO polypeptide operatively linked to a non-ADIPO polypeptide. A "ADIPO polypeptide" refers to a polypeptide having an amino acid sequence corresponding to ADIPO, whereas a "non-ADIPO polypeptide" refers to a polypeptide having an amino acid sequence corresponding to a protein that is not substantially homologous to the ADIPO protein, e.g., a protein that is different from the ADIPO protein and that is derived from the same or a different organism. Within an ADIPO fusion protein the
ADIPO polypeptide can correspond to all or a portion of an ADIPO protein. In one embodiment, an ADIPO fusion protein comprises at least one biologically active portion of an ADIPO protein. In another embodiment, an ADIPO fusion protein comprises at least two biologically active portions of an ADIPO protein. In yet another embodiment, an ADIPO fusion protein comprises at least three biologically active portions of an ADIPO protein. Within the fusion protein, the term "operatively linked" is intended to indicate that the ADIPO polypeptide and the non-ADIPO polypeptide are fused in-frame to each other. The non-ADIPO polypeptide can be fused to the N-terminus or C-terminus of the ADIPO polypeptide.
For example, in one embodiment an ADIPO fusion protein comprises an ADIPO domain operably linked to the extracellular domain of a second protein. Such fusion proteins can be further utilized in screening assays for compounds which modulate ADIPO activity (such assays are described in detail below).
In yet another embodiment, the fusion protein is a GST- ADIPO fusion protein in which the ADIPO sequences are fused to the C-terminus of the GST (i.e., glutathione S-transferase) sequences. Such fusion proteins can facilitate the purification of recombinant ADIPO.
In another embodiment, the fusion protein is an ADIPO protein containing a heterologous signal sequence at its N-terminus. For example, a native ADIPO signal sequence can be removed and replaced with a signal sequence from another protein. In certain host cells (e.g., mammalian host cells), expression and/or secretion of ADIPO can be increased through use of a heterologous signal sequence.
In yet another embodiment, the fusion protein is an ADIPO-immunoglobulin fusion protein in which the ADIPO sequences comprising one or more domains are fused to sequences derived from a member of the immunoglobulin protein family. The ADIPO-immunoglobulin fusion proteins of the invention can be incoφorated into pharmaceutical compositions and administered to a subject to inhibit an interaction between a ADIPO ligand and a ADIPO protein on the surface of a cell, to thereby suppress ADIPO-mediated signal transduction in vivo. The ADIPO-immunoglobulin fusion proteins can be used to affect the bioavailabihty of an ADIPO cognate ligand. Inhibition of the ADIPO ligand/ ADIPO interaction may be useful therapeutically for both the treatments of proliferative and differentiative disorders, as well as modulating (e.g. promoting or inhibiting) cell survival. Moreover, the ADIPO-immunoglobulin fusion proteins of the invention can be used as immunogens to produce anti-ADIPO antibodies in a subject, to purify ADIPO ligands, and in screening assays to identify molecules that inhibit the interaction of ADIPO with a ADIPO ligand.
An ADIPO chimeric or fusion protein of the invention can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, e.g., by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Ausubel et al. (eds.) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). An ADIPO-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the ADIPO protein.
ADIPO AGONISTS AND ANTAGONISTS
The present invention also pertains to variants of the ADIPO proteins that function as either ADIPO agonists (mimetics) or as ADIPO antagonists. Variants of the ADIPO protein can be generated by mutagenesis, e.g., discrete point mutation or truncation of the ADIPO protein. An agonist of the ADIPO protein can retain substantially the same, or a subset of, the biological activities of the naturally occurring form of the ADIPO protein. An antagonist of the ADIPO protein can inhibit one or more of the activities of the naturally occurring form of the ADIPO protein by, for example, competitively binding to a downstream or upstream member of a cellular signaling cascade which includes the ADIPO protein. Thus, specific biological effects can be elicited by treatment with a variant of limited function. In one embodiment, treatment of a subject with a variant having a subset of the biological activities of the naturally occurring form of the protein has fewer side effects in a subject relative to treatment with the naturally occurring form of the ADIPO proteins.
Variants of the ADIPO protein that function as either ADIPO agonists (mimetics) or as ADIPO antagonists can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of the ADIPO protein for ADIPO protein agonist or antagonist activity. In one embodiment, a variegated library of ADIPO variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A variegated library of ADIPO variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential ADIPO sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of ADIPO sequences therein. There are a variety of methods which can be used to produce libraries of potential ADIPO variants from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector. Use of a degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential ADIPO sequences. Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu Rev Biochem 53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucl Acid Res 1 1 :477.
POLYPEPTIDE LIBRARIES
In addition, libraries of fragments of the ADIPO protein coding sequence can be used to generate a variegated population of ADIPO fragments for screening and subsequent selection of variants of an ADIPO protein. In one embodiment, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of a ADIPO coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA that can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with SI nuclease, and ligating the resulting fragment library into an expression vector. By this method, an expression library can be derived which encodes N-terminal and internal fragments of various sizes of the ADIPO protein.
Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property. Such techniques are adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of ADIPO proteins. The most widely used techniques, which are amenable to high throughput analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected. Recursive ensemble mutagenesis (REM), a new technique that enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify ADIPO variants (Arkin and Yourvan (1992) PNAS 89:7811-7815; Delgrave et al. (1993) Protein Engineering 6:327-331). For the production of polyclonal antibodies, various suitable host animals (e.g., rabbit, goat, mouse or other mammal) may be immunized by injection with the native protein, or a synthetic variant thereof, or a derivative of the foregoing. An appropriate immunogenic preparation can contain, for example, recombinantly expressed ADIPO protein or a chemically synthesized ADIPO polypeptide. The preparation can further include an adjuvant. Various adjuvants used to increase the immunological response include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.), human adjuvants such as Bacille Calmette-Guerin and Corynebacterium parvum, or similar immunostimulatory agents. If desired, the antibody molecules directed against ADIPO can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as protein A chromatography to obtain the IgG fraction.
The term "monoclonal antibody" or "monoclonal antibody composition", as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of ADIPO. A monoclonal antibody composition thus typically displays a single binding affinity for a particular ADIPO protein with which it immunoreacts. For preparation of monoclonal antibodies directed towards a particular ADIPO protein, or derivatives, fragments, analogs or homologs thereof, any technique that provides for the production of antibody molecules by continuous cell line culture may be utilized. Such techniques include, but are not limited to, the hybridoma technique (see Kohler & Milstein, 1975 Nature 256: 495-497); the trioma technique; the human B-cell hybridoma technique (see Kozbor, et al, 1983 Immunol Today 4: 72) and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole, et al, 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies may be utilized in the practice of the present invention and may be produced by using human hybridomas (see Cote, et al, 1983. Proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et al, 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).
59 According to the invention, techniques can be adapted for the production of single-chain antibodies specific to a ADIPO protein (see e.g., U.S. Patent No. 4,946,778). In addition, methods can be adapted for the construction of Fab expression libraries (see e.g., Huse, et al, 1989 Science 246: 1275-1281) to allow rapid and effective identification of monoclonal Fab fragments with the desired specificity for a ADIPO protein or derivatives, fragments, analogs or homologs thereof. Non-human antibodies can be "humanized" by techniques well known in the art. See e.g., U.S. Patent No. 5,225,539. Antibody fragments that contain the idiotypes to a ADIPO protein may be produced by techniques known in the art including, but not limited to: (i) an F(ab,)2 fragment produced by pepsin digestion of an antibody molecule; (ii) an Fab fragment generated by reducing the disulfide bridges of an F(ab.)2 fragment; (iii) an Fab fragment generated by the treatment of the antibody molecule with papain and a reducing agent and (iv) Fv fragments.
Additionally, recombinant anti-ADIPO antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in PCT International Application No. PCT/US86/02269; European Patent Application No. 184,187; European Patent Application No. 171,496; European Patent Application No. 173,494; PCT International Publication No. WO 86/01533; U.S. Pat. No. 4,816,567; European Patent Application No.
125,023; Better et /.(1988) Science 240: 1041-1043; Liu et al. (1987) PNAS 84:3439-3443; Liu et al. (1987) J Immunol. 139:3521-3526; Sun et al. (1987) PNAS 84:214-218; Nishimura et al. (1987) Cancer Res 47:999-1005; Wood et al. (1985) Nature 314:446-449; Shaw et al. (1988) J Natl Cancer Inst. 80:1553-1559); Morrison( 1985) Science 229:1202-1207; Oi et al. (1986) BioTechniques 4:214; U.S. Pat. No. 5,225,539; Jones et al. (1986) Nature 321 :552-525;
Verhoeyan et al. (1988) Science 239: 1534; and Beidler et al. (1988) J Immunol 141 :4053-4060.
In one embodiment, methods for the screening of antibodies that possess the desired specificity include, but are not limited to, enzyme-linked immunosorbent assay (ELISA) and other immunologically-mediated techniques known within the art. In a specific embodiment,
- 60 - selection of antibodies that are specific to a particular domain of a ADIPO protein is facilitated by generation of hybridomas that bind to the fragment of a ADIPO protein possessing such a domain. Antibodies that are specific for one or more domains within a ADIPO protein, e.g., domains spanning the above-identified conserved regions of ADIPO family proteins, or derivatives, fragments, analogs or homologs thereof, are also provided herein.
Anti-ADIPO antibodies may be used in methods known within the art relating to the localization and/or quantitation of a ADIPO protein (e.g., for use in measuring levels of the ADIPO protein within appropriate physiological samples, for use in diagnostic methods, for use in imaging the protein, and the like). In a given embodiment, antibodies for ADIPO proteins, or derivatives, fragments, analogs or homologs thereof, that contain the antibody derived binding domain, are utilized as pharmacologically- active compounds [hereinafter "Therapeutics"].
An an -ADIPO antibody (e.g., monoclonal antibody) can be used to isolate ADIPO by standard techniques, such as affinity chromatography or immunoprecipitation. An anti-ADIPO antibody can facilitate the purification of natural ADIPO from cells and of recombinantly produced ADIPO expressed in host cells. Moreover, an anti-ADIPO antibody can be used to detect ADIPO protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the ADIPO protein. Anti-ADIPO antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, -galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include stf eptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fiuorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125I, 13'I, 35S or 3H.
- 61 - ADIPO RECOMBINANT EXPRESSION VECTORS AND HOST CELLS
Another aspect of the invention pertains to vectors, preferably expression vectors, containing a nucleic acid encoding ADIPO protein, or derivatives, fragments, analogs or homologs thereof. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a linear or circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors". In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, that is operatively linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). The term "regulatory sequence" is intended to includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in
- 62 - Goeddel; GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., ADIPO proteins, mutant forms of ADIPO, fusion proteins, etc.).
The recombinant expression vectors of the invention can be designed for expression of
ADIPO in prokaryotic or eukaryotic cells. For example, ADIPO can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
Expression of proteins in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non- fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three puφoses: (1) to increase expression of recombinant protein; (2) to increase the solubility of the recombinant protein; and (3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and
63 pRIT5 (Pharmacia, Piscataway, NJ.) that fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.
Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amrann et al, (1988) Gene 69:301-315) and pET 1 Id (Studier et al, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89).
One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein. See, Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 119-128. Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al, (1992) Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.
In another embodiment, the ADIPO expression vector is a yeast expression vector.
Examples of vectors for expression in yeast S. cerevisiae include pYepSecl (Baldari, et al, (1987) EMBO J 6:229-234), pMFa (Kuijan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al, (1987) Gene 54:113-123), pYES2 (Invitrogen Coφoration, San Diego, Calif), and picZ (InVitrogen Coφ, San Diego, Calif).
Alternatively, ADIPO can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith et al. (1983) Mol Cell Biol 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39).
In yet another embodiment, a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J 6: 187-195). When used in mammalian cells, the expression vector's control functions are often
64 provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells. See, e.g., Chapters 16 and 17 of Sambrook et al, MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.
In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev 1 :268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv Immunol 43:235-275), in particular promoters of T cell receptors (Winoto and Baltimore (1989) EMBO J 8:729-733) and immunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle (1989) PNAS 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, e.g., the murine hox promoters (Kessel and Grass (1990) Science 249:374-379) and the α-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev 3:537-546).
The invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively linked to a regulatory sequence in a manner that allows for expression (by transcription of the DNA molecule) of an RNA molecule that is antisense to ADIPO mRNA. Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen that direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen that direct constitutive, tissue specific or cell type specific expression of antisense RNA. The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a
- 65 - high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced. For a discussion of the regulation of gene expression using antisense genes see Weintraub et al, "Antisense RNA as a molecular tool for genetic analysis," Reviews— Trends in Genetics, Vol. 1(1) 1986.
Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
A host cell can be any prokaryotic or eukaryotic cell. For example, ADIPO protein can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.
Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DΕAΕ-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NN., 1989), and other laboratory manuals.
For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DΝA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host
- 66 - cells along with the gene of interest. Various selectable markers include those that confer resistance to drugs, such as G418, hygromycin and methotrexate. Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding ADIPO or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drag selection (e.g., cells that have incoφorated the selectable marker gene will survive, while the other cells die).
A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) an ADIPO protein. Accordingly, the invention further provides methods for producing ADIPO protein using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding ADIPO has been introduced) in a suitable medium such that ADIPO protein is produced. In another embodiment, the method further comprises isolating ADIPO from the medium or the host cell.
KITS AND NUCLEIC ACID COLLECTIONS FOR IDENTIFYING ADIPO NUCLEIC ACIDS
In another aspect, the invention provides a kit useful for examining a pathophysiology associated with a PPARγ-mediated pathway. The kit can include nucleic acids that detect two or more ADIPO sequences. In prefened embodiments, the kit includes reagents which detect 3, 4, 5, 6, 8, 10, 12, 15, 20, 25, 30, 35,40 or all of the ADIPO nucleic acid sequences.
The invention also includes an isolated plurality of sequences which can identify one or more ADIPO responsive nucleic acid sequences.
The kit or plurality may include, e.g., sequence homologous to ADIPO nucleic acid sequences, or sequences which can specifically identify one or more ADIPO nucleic acid sequences.
NUCLEOTIDE POLYMORPHISMS ASSOCIATED WITH ADIPO GENES The invention also includes nucleic acid sequences that include one or more polymoφhic
ADIPO sequences. Also included are methods of identifying a base occupying a polymoφhic in
- 67 - an ADIPO sequence, as well as methods of identifying an individualized therapeutic agent for treating PPARγ associated pathologies based on ADIPO sequence polymoφhisms.
The nucleotide polymoφhism can be a single nucleotide polymoφhism (SNP). A SNP occurs at a polymoφhic site occupied by a single nucleotide, which is the site of variation between allelic sequences. The site is usually preceded by and followed by highly conserved sequences of the allele (e.g., sequences that vary in less than 1/100 or 1/1000 members of the populations). A single nucleotide polymoφhism usually arises due to substitution of one nucleotide for another at the polymoφhic site. A transition is the replacement of one purine by another purine or one pyrimidine by another pyrimidine. A transversion is the replacement of a purine by a pyrimidine or vice versa. Single nucleotide polymoφhisms can also arise from a deletion of a nucleotide or an insertion of a nucleotide relative to a reference allele.
Polymoφhic sequences according to the present invention can include those shown in Table 2. Table 2 describes nine ADIPO sequences for which polymoφhisms have been identified. The first column of the table lists the names assigned to the sequences in which the polymoφhisms occur. The second and third columns list the rat and human GenBank Accession numbers for the respective sequences. The forth column lists the position in the sequence in which the polymoφhic site has been found. The fifth column lists the base occupying the polymoφhic site in the sequence in the database, i.e., the wildtype. The sixth column lists the alternative base at the polymoφhic site. The seventh column lists any amino acid change that occurs due to the polymoφhism.
The polymoφhic sequence can include one or more of the following sequences: (1) a sequence having the nucleotide denoted in Table 2, column 5 at the polymoφhic site in the polymoφhic sequence, and (2) a sequence having a nucleotide other than the nucleotide denoted in Table 2, column 5. An example of the latter sequence is a polymoφhic sequence having the nucleotide denoted in Table 2, column 6 at the polymoφhic site in the polymoφhic sequence.
For example, a polymoφhism according to the invention includes a sequence polymoφhism in the ATP citrate lyase gene having the nucleotide sequence of GenBank Accession No. x64330, in which the cytosine at nucleotide 609 is replaced by adenosine. In
68 some embodiments the polymorphic sequence includes a nucleotide sequence of ATP citrate gene having the GenBank Accession No. x64330, wherein the nucleotide at 609 is any nucleotide other that cytosine.
In some embodiments, the polymorphic sequence includes the full length of any one of the nine genes in Table2. In other embodiments, the polymoφhic sequence includes a polynucleotide that is between 10 and 100 nucleotides, 10 and 75 nucleotides, 10 and 50 nucleotides, or 10 and 25 nucleotides in length.
Table 2
Figure imgf000070_0001
The invention also provides a method of identifying a base occupying a polymorphic site in a nucleic acid. The method includes determining the nucleotide sequence of a nucleic acid that is obtained from a subject. The nucleotide sequence is compared to a reference sequence. Difference in the nucleotide sequence in the test sequence relative to the reference sequence indicates a polymorphic site in the nucleic acid.
- 69 - Polymoφhisms are detected in a target nucleic acid from an individual, e.g., a mammal, human or rodent (such as mouse or rat) being analyzed. For assay of genomic DNA, virtually any biological sample (other than pure red blood cells) is suitable. For example, convenient tissue samples include whole blood, semen, saliva, tears, urine, fecal material, sweat, buccal, skin and hair. For assay of cDNA or mRNA, the tissue sample must be obtained from an organ in which the target nucleic acid is expressed.
The detection of polymoφhisms in specific DNA sequences, can be accomplished by a variety of methods including, e.g., restriction-fragment-length-polymoφhism detection based on allele-specific restriction-endonuclease cleavage (Kan and Dozy Lancet ii:910-912 (1978)), hybridization with allele-specific oligonucleotide probes (Wallace et al. Nucl. Acids Res.
6:3543-3557 (1978)), including immobilized oligonucleotides (Saiki et al. Proc. Natl. Acad. SCI. USA. 86:6230-6234 (1969)) or oligonucleotide arrays (Maskos and Southern Nucl. Acids Res 21 :2269-2270 (1993)), allele-specific PCR (Newton et al. Nucl Acids Res 17:2503-.2516 (1989)), mismatch-repair detection (MRD) (Faham and Cox Genome Res 5:474-482 (1995)), binding of MutS protein (Wagner et al. Nucl Acids Res 23:3944-3948 (1995), denaturing- gradient gel electrophoresis (DGGE) (Fisher and Lerman et al. Proc. Natl. Acad. Sci. U.S.A. 80:1579-1 583 (1983)), single-strand-conformation-polymoφhism detection (Orita et al. Genomics 5:874-879 (1983)), RNAase cleavage at mismatched base-pairs (Myers et al. Science 230:1242 (1985)), chemical (Cotton et al. Proc. Natl. w Sci. U.S.A, 8Z4397-4401 (1988)) or enzymatic (Youil et al. Proc. Natl. Acad. Sci. U.S.A. 92:87-91 (1995)) cleavage of heteroduplex DNA, methods based on allele specific primer_extension (Syvanen et al. Genomics 8:684-692 (1990)), genetic bit analysis (GBA) (Nikiforov et al. &&I Acids 22:4167-4175 (1994)), the oligonucleotide-ligation assay (OLA) (Landegren et al. Science_241:1077 (1988)), the allele-specific ligation chain reaction (LCR) (Barrany Proc. Natl. Acad. Sci. U.S.A. 88:189-1 93 (1991)), gap-LCR (Abravaya et al. Nucl Acids Res 23:675-682 (1995)), radioactive and/or fluorescent DNA sequencing using standard procedures well known in the art, and peptide nucleic acid (PNA) assays (Orum et al., Nucl. Acids Res, 21 :5332-5356 (1993); Thiede et al., Nucl. Acids Res. 24:983-984 (1996)).
70 For the puφoses of identifying single nucleotide polymoφhisms, "Specific hybridization" or "selective hybridization" refers to the binding, or duplexing, of a nucleic acid molecule only to a second particular nucleotide sequence to which the nucleic acid is complementary, under suitably stringent conditions when that sequence is present in a complex mixture (e.g., total cellular DNA or RNA). "Stringent conditions" are conditions under which a probe will hybridize to its target subsequence, but to no other sequences. Stringent conditions are sequence-dependent and are different in different circumstances. Longer sequences hybridize specifically at higher temperatures than shorter ones. Generally, stringent conditions are selected such that the temperature is about 5°C lower than the thermal melting point (Tm) for the specific sequence to which hybridization is intended to occur at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the target sequence hybridizes to the complementary probe at equilibrium. Typically, stringent conditions include a salt concentration of at least about 0.01 to about 1.0 M Na ion concentration (or other salts), at pH 7.0 to 8.3. The temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides) . Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide. For example, conditions of 5X SSPE (750 mM NaCl, 50 mM NaPhosphate, 5 mM EDTA, pH 7.4) and a temperature of 25-30°C are suitable for allele- specific probe hybridizations.
"Complementary" or "target" nucleic acid sequences refer to those nucleic acid sequences which selectively hybridize to a nucleic acid probe. Proper annealing conditions depend, for example, upon a probe's length, base composition, and the number of mismatches and their position on the probe, and must often be determined empirically. For discussions of nucleic acid probe design and annealing conditions, see, for example, Sambrook et al., or Current Protocols in Molecular Biology, F. Ausubel et al., ed., Greene Publishing and Wiley-Interscience, New York (1987).
Many of the methods described above require amplification of DNA from target samples. This can be accomplished by e.g., PCR. See generally, PCR Technology: Principles and Applications for DNA Amplification (ed. H. A. Erlich, Freeman Press, N.Y., N.Y., 1992); PCR Protocols: A Guide to Methods and Applications (eds. Innis, et al, Academic Press, San Diego,
- 71 - Calif, 1990); Mattila et al, Nucleic Acids Res. 19, 4967 (1991); Eckert et al, PCR Methods and Applications 1, 17 (1991); PCR (eds. McPherson et al, IRL Press, Oxford); and U.S. Pat. No. 4,683,202 (each of which is incoφorated by reference for all puφoses).
Other suitable amplification methods include the ligase chain reaction (LCR), (See Wu and Wallace, Genomics 4, 560 (1989), Landegren et al., Science 241, 1077 (1988)), transcription amplification (Kwoh et al, Proc. Natl. Acad. Sci. USA 86, 1173 (1989)), and self- sustained sequence replication (Guatelli et al, Proc. Nat. Acad. Sci. USA, 87, 1874 (1990)) and nucleic acid based sequence amplification (NASBA). The latter two amplification methods involve isothermal reactions based on isothermal transcription, which produce both single stranded RNA (ssRNA) and double stranded DNA (dsDNA) as the amplification products in a ratio of about 30 or 100 to 1, respectively.
The invention also provides a method of selecting an individualized therapeutic agent for treating a PPARγ associated pathology, e.g., NIDDM, in a subject using ADIPO polymoφhisms. The therapeutic agent can be identified by providing a nucleic acid sample from the subject, determining the nucleotide sequence of at least a portion of one or more of the ADIPO 1-32 and, optionally, ADIPO 33-43 sequences, and comparing the ADIPO nucleotide sequence in the subject to the corresponding ADIPO nucleic acid sequence in a reference nucleic acid sample. The reference nucleic acid sample is obtained from a reference individual (who is preferably as similar to the test subject as possible), whose responsiveness to the agent for treating the PPARγ associated pathology is known. The presence of the same sequence in the test and reference nucleic acid sample indicates the subject will demonstrate the same responsiveness to the agent as the reference individual, while the presence of a different sequence indicates the subject will have a different response to the therapeutic agent.
Similarly, the ADIPO-associated sequence polymoφhisms can be used to predict the outcome of treatment for a PPARγ associated pathology, e.g., NIDDM, in a subject. A region of an ADIPO nucleic acid sequence from the subject is compared to the corresponding ADIPO sequence in a reference individual whose outcome in response to the treatment for the PPARγ associated pathology is known. A similarity in the ADIPO sequence in the test subject as compared to the sequence in the reference individual suggests the outcome in the subject will be
- 72 - the same as that of the reference individual. An altered ADIPO sequence in the test and reference individual indicates the outcome of treatment will differ in the subject and reference individuals.
OTHER EMBODIMENTS
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
73 CLAIMS
What is claimed is:
1. A method of identifying a ligand for peroxisome proliferator activated receptor gamma (PPARγ), the method comprising;
(a) providing a test cell population comprising a cell capable of expressing one or more nucleic acid sequences selected from the group consisting of ADIPO: 1-5; 7-13; 15-31 and 32;
(b) contacting the test cell population with a test agent; (c) measuring expression of one or more of the nucleic acid sequences in the test cell population;
(d) comparing the expression of the nucleic acid sequences in the test cell population to the expression of the nucleic acid sequences in a reference cell population comprising at least one cell whose PPARγ expression status is known; and
(e) identifying a difference in expression levels of the ADIPO sequence, if present, in the test cell population and reference cell population, thereby identifying a ligand for PPARγ.
2. The method of claim 1 , wherein the method comprises comparing the expression of five or more of the nucleic acid sequences.
3 The method of claim 1 , wherein the method comprises comparing the expression of 20 or more of the nucleic acid sequences.
74

Claims

4. The method of claim 1, wherein the method comprises comparing the expression of 25 or more of the nucleic acid sequences.
5. The method of claim 1, wherein the method further comprises comparing the expression of at least one nucleic acid sequences selected from the group consisting of ADIPO 38-43.
6. The method of claim 1, wherein the expression of the nucleic acid sequences in the test cell population is decreased as compared to the reference cell population.
7. The method of claim 1, wherein the expression of the nucleic acid sequences in the test cell population is increased as compared to the reference cell population.
8. The method of claim 1, wherein the test cell population is provided in vitro.
9. The method of claim 1 , wherein the test cell population is provided ex vivo from a mammalian subject.
10. The method of claim 1, wherein the test cell is provided in vivo in a mammalian subject.
11. The method of claim 1 , wherein the test cell population is derived from a human or rodent subject.
12. The method of claim 1, wherein the test cell includes an adipocyte.
75
13. A PPARγ ligand identified according to the method of claim 1.
14. A pharmaceutical composition comprising the PPARγ ligand of claim 13.
15. A method of identifying a candidate therapeutic agent for a pathophysiology associated with a PPARγ mediated metabolic pathway, the method comprising;
(a) providing a test cell population comprising a cell capable of expressing one or more nucleic acid sequences selected from the group consisting of ADIPO: 1-5; 7-13; 15-31 and 32; (b) contacting the test cell population with a test agent;
(c) measuring expression of one or more of the nucleic acid sequences in the test cell population;
(d) comparing the expression of the gene in the test cell population to the expression of the nucleic acid sequences in a reference cell population comprising at least one cell whose PPARγ expression status is known; and
(e) identifying a difference in expression levels of the nucleic acid sequences, if present, in the test cell population and reference cell population, thereby identifying a therapeutic agent for a pathophysiology associated with the PPARγ mediated metabolic pathway.
16. The method of claim 15, wherein the pathophysiology is noninsulin-dependent diabetes mellitus (NIDMM).
17. The method of claim 15, wherein the therapeutic agent is a ligand for the PPARγ.
- 76 -
18. A method of identifying an individualized therapeutic agent suitable for treating a pathophysiology associated with a PPARγ mediated pathway appropriate in a selected subject, the method comprising:
(a) providing from the subject a test cell population comprising cells capable of expressing one or more nucleic acid sequences selected from the group consisting of ADIPO: 1-5; 7-13; 15-31 and 32;
(b) contacting the test cell population with the therapeutic agent ;
(c) measuring expression of one or more of the nucleic acid sequences in the test cell population; (d) comparing the expression of the nucleic acid sequences in the test cell population to the expression of the nucleic acid sequences in a reference cell population comprising at least one cell whose PPARγ expression status is known; and
(e) identifying a difference in expression levels of the nucleic acid sequences, if present, in the test cell population and reference cell population, thereby identifying a therapeutic agent appropriate for the subject.
19. The method of claim 18, wherein the subj ect is a human or rodent.
20. A method of diagnosing or determining the susceptibility to a pathophysiology associated with a PPARγ mediated pathway in a subject, the method comprising:
(a) providing from the subject a test cell population comprising cells capable of expressing one or more nucleic acid sequences selected from the group consisting of ADIPO: 1-5; 7-13; 15-31 and 32; (b) measuring expression of one or more of the nucleic acid sequences in the test cell population; and
- 77 - (c) comparing the expression of the nucleic acid sequences in the test cell population to the expression of the nucleic acid sequences in a reference cell population comprising at least one cell from a subject not suffering from pathophysiology associated with the for PPARγ mediated pathway; and (d) identifying a difference in expression levels of the nucleic acid sequences, if present, in the test cell population and reference cell population, thereby diagnosing or determining the susceptibility to a pathophysiology associated with the PPARγ mediated pathway in the subject.
21. A method of treating a pathophysiology associated with the PPARγ mediated pathway in a subject, the method comprising administering to the subject an agent that modulates the expression or the activity of one or more nucleic acids selected from the group consisting of ADIPO: 1-5; 7-13; 15-31 and 32.
22. A method of assessing the efficacy of a treatment of pathophysiology associated with the PPARγ mediated pathway in a subject, the method comprising:
(a) providing from the subject a test cell population comprising cells capable of expressing one or more nucleic acid sequences selected from the group consisting of ADIPO: 1-5; 7-13; 15-31 and 32; (b) detecting expression of one or more of the nucleic acid sequences in the test cell population;
(c) comparing the expression of the nucleic acid sequences in the test cell population to the expression of the nucleic acid sequences in a reference cell population comprising at least one cell from a subject not suffering from the pathophysiology associated with the PPARγ mediated pathway; and
78 (e) identifying a difference in expression levels of the nucleic acid sequences, if present, in the test cell population and reference cell population, thereby assessing the efficacy of treatment of the pathophysiology in the subject.
23. An isolated nucleic acid comprising a nucleic acid sequence selected from the group consisting of a ADIPO 1-15 gene, or its complement.
24. A vector comprising the nucleic acid of claim 23.
25. A cell comprising the vector of claim 24.
26. A pharmaceutical composition comprising the nucleic acid of claim 23.
27. A polypeptide encoded by the nucleic acid of claim 23.
28. An antibody which specifically binds to the polypeptide of claim 27
29. A kit which detects two or more of the nucleic acid sequences selected from the group consisting of ADIPOs: 1-43.
30. An anay which detects one or more of the nucleic acid selected from the group consisting of ADIPOs: 1-43.
79
31. A plurality of nucleic acid comprising one or more of the nucleic acid selected from the group consisting of ADIPOs: 1-43.
32. A method of identifying a base occupying a polymoφhic site in a nucleic acid, the method comprising:
(a) obtaining a nucleic acid from a subject;
(b) determining at least one portion of a region of nucleotide sequence corresponding to a contiguous region of any one ADIPO nucleotide sequence listed in Table 1 ; (c) comparing the determined nucleotide sequence to a reference sequence of the nucleic acid; and
(d) identifying a difference in the determined nucleic acid sequence relative to the reference sequence, wherein a difference in the determined nucleic acid sequence indicates a polymoφhic site in the nucleic acid.
33. The method of claim 32, wherein the subject suffers from or is at risk for, a pathophysiology associated with the PPARγ mediated pathway.
34. The method of claim 32, wherein the presence of the polymoφhic site is conelated with the presence of the pathophysiology associated with the PPARγ mediated pathway.
35. The method of claim 32, wherein the nucleic acid is genomic DNA.
36. The method of claim 32, wherein the nucleic acid is cDNA.
- 80 -
37. A nucleic acid sequence 20-100 nucleotides in length comprising the polymoφhic site identified in the method of claim 32.
38. The method of claim 32, wherein the nucleic acid is obtained from a plurality of subjects, and a base occupying one of the polymoφhic sites is determined in each of the subjects.
39. The method of claim 32, wherein the subject is a human or rodent.
40. A method of identifying an individualized therapeutic agent suitable for treating a PPARγ associated pathology in a subject, the method comprising;
(a) providing a nucleic acid sample from the subject;
(b) determining the nucleotide sequence in said subject nucleic acid sample of at least a portion of one or more nucleic acid sequences selected from the group consisting of ADIPO: 1-32;
(c) comparing the nucleic acid sequence in said subject nucleic acid sample to the corresponding nucleic acid sequence from a reference nucleic acid from a reference individual whose reactivity to said agent is known; and
(d) identifying a difference in the nucleic acid sequence, if present, between the subject sample and the reference nucleic acid sample, thereby identifying a ligand suitable for the subject.
41. The method of claim 40, wherein the nucleic acid sequence is selected from the group consisting of any one nucleotide sequence listed in Table 2.
- 81 -
42. The method of claim 40, wherein the subject is a human or rodent.
43. A method of determining the efficacy of treatment of a PPAR associated pathology in a subject, the method comprising: (a) providing a nucleic acid sample from the subject;
(b) determining the nucleotide sequence of at least a portion of one or more nucleic acid sequences selected from the group consisting of ADIPO: 1-32;
(c) comparing the sequence to the conesponding nucleic acid sequence from a cell population whose responsiveness to said treatment for the PPARγ associated pathology agent is known,
thereby determining the efficacy of treatment of a PPARγ associated pathology in said subject.
44. An isolated polynucleotide selected from the group consisting of: (a) a nucleotide sequence comprising one or more polymoφhic sequences of Table 2;
(b) a fragment of the nucleotide sequence including a polymoφhic site in the polymoφhic sequence;
(c) a complementary nucleotide sequence comprising a sequence complementary to one or more of the polymoφhic sequence of Table 2; and (d) a fragment of the complementary nucleotide sequence including a polymoφhic site in the polymoφhic sequence.
45. The polynucleotide of claim 44, wherein the polynucleotide is DNA.
46. The polynucleotide of claim 44, wherein the polynucleotide is RNA.
- 82 -
47. The polynucleotide of claim 44, wherein the polynucleotide is between about 10 and about 100 nucleotides in length.
48. The polynucleotide of claim 44, wherein the polynucleotide is between about 10 and about 75 nucleotides in length.
49. The polynucleotide of claim 44, wherein the polynucleotide is between about 10 and about 50 nucleotides in length.
50. The polynucleotide of claim 44, wherein the polynucleotide is between about 10 and about 25 nucleotides in length.
51. The polynucleotide of claim 44, wherein the polynucleotide is a nucleic acid encoding a polypeptide selected from the group consisting of ATP citrate lyase, long chain
3-ketoacyl-CoA, 12-lipoxygenase, catalase, rhodanese, adipocyte fatty acid binding protein, β-3 adrenergic receptor, complement component Clr, and ADIPO 14.
52. The polynucleotide of claim 44, wherein the polymoφhic site includes a nucleotide other than the nucleotide listed in Table 2, column 5 for the polymoφhic sequence.
53. The polynucleotide of claim 44, wherein the complement of the polymoφhic site includes a nucleotide other than the complement of the nucleotide listed in Table 2, column 5 for the complement of the polymoφhic sequence.
- 83 -
54. The polynucleotide of claim 44, wherein the polymoφhic site includes the nucleotide listed in Table 2, column 6 for the polymoφhic sequence.
55. An isolated allele-specific oligonucleotide that hybridizes to a first polynucleotide at a polymoφhic site encompassed therein, wherein the first polynucleotide is chosen from the group consisting of:
(a) a nucleotide sequence comprising one or more polymoφhic sequences provided that the polymoφhic sequence includes a nucleotide other than the nucleotide recited in Table 2, column 5 for said polymoφhic sequence;
(b) a nucleotide sequence that is a fragment of said polymoφhic sequence, provided that the fragment includes a polymoφhic site in said polymoφhic sequence;
(c) a complementary nucleotide sequence comprising a sequence complementary to one or more polymoφhic sequences ,provided that the complementary nucleotide sequence includes a nucleotide other than the complement of the nucleotide recited in Table 2, column 5; and
(d) a nucleotide sequence that is a fragment of said complementary sequence, provided that the fragment includes a polymoφhic site in said polymoφhic sequence.
- 84 -
PCT/US2000/010757 1999-04-23 2000-04-21 Method of identifying ligands for the peroxisome proliferator activated receptor gamma using differential gene expression WO2000065091A2 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU46526/00A AU4652600A (en) 1999-04-23 2000-04-21 Method of identifying ligands for the peroxisome proliferator activated receptorgamma using differential gene expression

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US13082199P 1999-04-23 1999-04-23
US60/130,821 1999-04-23
US54603800A 2000-04-10 2000-04-10
US09/546,038 2000-04-10

Publications (2)

Publication Number Publication Date
WO2000065091A2 true WO2000065091A2 (en) 2000-11-02
WO2000065091A3 WO2000065091A3 (en) 2002-09-12

Family

ID=26828851

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2000/010757 WO2000065091A2 (en) 1999-04-23 2000-04-21 Method of identifying ligands for the peroxisome proliferator activated receptor gamma using differential gene expression

Country Status (2)

Country Link
AU (1) AU4652600A (en)
WO (1) WO2000065091A2 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002086122A2 (en) * 2001-03-14 2002-10-31 Hybrigenics Protein-protein interactions in adipocytes

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995021944A1 (en) * 1994-02-14 1995-08-17 Smithkline Beecham Corporation Differentially expressed genes in healthy and diseased subjects
WO1997010813A1 (en) * 1995-09-18 1997-03-27 Ligand Pharmaceuticals Incorporated Ppar gamma antagonists for treating obesity
WO1998005331A2 (en) * 1996-08-02 1998-02-12 Ligand Pharmaceuticals Incorporated Prevention or treatment of type 2 diabetes or cardiovascular disease with ppar modulators
WO2000063435A2 (en) * 1999-04-15 2000-10-26 Curagen Corporation Method of identifying toxic agents using differential gene expression

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995021944A1 (en) * 1994-02-14 1995-08-17 Smithkline Beecham Corporation Differentially expressed genes in healthy and diseased subjects
WO1997010813A1 (en) * 1995-09-18 1997-03-27 Ligand Pharmaceuticals Incorporated Ppar gamma antagonists for treating obesity
WO1998005331A2 (en) * 1996-08-02 1998-02-12 Ligand Pharmaceuticals Incorporated Prevention or treatment of type 2 diabetes or cardiovascular disease with ppar modulators
WO2000063435A2 (en) * 1999-04-15 2000-10-26 Curagen Corporation Method of identifying toxic agents using differential gene expression

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DATABASE EMBL [Online] Acc. No./Seq. ID No. AA900711, 8 April 1998 (1998-04-08) "UI-R-E0-by-g-09-0-UI.s4 UI-R-E0 Rattus norvegicus cDNA clone" XP002180486 & BONALDO FATIMA DE M; LENNON G; SOARES M B: "NORMALIZATION AND SUBTRACTION: TWO APPROACHES TO FACILITATE GENE DISCOVERY" GENOME RESEARCH, vol. 6, no. 9, 1 September 2001 (2001-09-01), pages 791-806, XP002039972 GENOME RESEARCH, COLD SPRING HARBOR LABORATORY PRESS, US *
MARTIN ET AL: "Coordinate regulation of the expression of the fatty acid transport protein and acyl-CoA synthase genes by PPAR-alpha and PPAR-gamma activators" JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS, BALTIMORE, MD, US, vol. 272, no. 45, 7 November 1997 (1997-11-07), pages 28210-28217, XP002081439 ISSN: 0021-9258 *
SCHOONJANS K ET AL: "PPARALPHA AND PPARGAMMA ACTIVATORS DIRECT A DISTINCT TISSUE-SPECIFIC TRANSCRIPTIONAL RESPONSE VIA A PPRE IN THE LIPOPROTEIN LIPASE GENE" EMBO JOURNAL, OXFORD UNIVERSITY PRESS, SURREY, GB, vol. 15, no. 19, 1 October 1996 (1996-10-01), pages 5336-5348, XP000615472 ISSN: 0261-4189 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002086122A2 (en) * 2001-03-14 2002-10-31 Hybrigenics Protein-protein interactions in adipocytes
WO2002086122A3 (en) * 2001-03-14 2003-10-30 Hybrigenics Sa Protein-protein interactions in adipocytes

Also Published As

Publication number Publication date
WO2000065091A3 (en) 2002-09-12
AU4652600A (en) 2000-11-10

Similar Documents

Publication Publication Date Title
US20020144298A1 (en) Novel human genes and gene expression products
JP2008154589A (en) Method of detecting and treating tuberous sclerosis complex associated disorders
US20020142284A1 (en) Methods of identifying renal protective factors
EP1169481A1 (en) Method of identifying toxic agents using differential gene expression
US7258973B2 (en) Method for detecting a differentially expressed sequence
JP2003518365A (en) Methods for identifying virulence factors using differential gene expression
US6852845B1 (en) Method of identifying toxic agents using NSAID-induced differential gene expression in liver
WO1999026976A1 (en) Tnf ligand family gene
JP2004500068A (en) Polynucleotides and polypeptides encoded thereby
WO2000065091A2 (en) Method of identifying ligands for the peroxisome proliferator activated receptor gamma using differential gene expression
WO2001044512A2 (en) Method of identifying ligands for the peroxisome proliferator activated receptor gamma using differential gene expression
JP2002533099A (en) Methods for identifying psychotropic drugs using differential gene expression
WO2001063279A2 (en) Method of identifying toxic agents using differential gene expression
US6355430B1 (en) Diagnostic and screening methods employing KIAA0101
US20030091973A1 (en) Method of identifying osteoregenerative agents using differential gene expression
EP1494031A2 (en) Novel human genes and gene expression products
AU783994B2 (en) Novel hematopoietic regulatory factors and methods of use thereof
WO2001074860A2 (en) Methods of identifying integrin ligands using differential gene expression
EP1593687A2 (en) Human genes differentially expressed in colon cancer
AU4350200A (en) Method of identifying toxic agents using differential gene expression

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DE DK DM DZ EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

AK Designated states

Kind code of ref document: A3

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DE DK DM DZ EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A3

Designated state(s): GH GM KE LS MW SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase in:

Ref country code: JP