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WO2003033513A1 - Genes exprimes de maniere differentielle, associes a l'obesite et au diabete de type 2 - Google Patents

Genes exprimes de maniere differentielle, associes a l'obesite et au diabete de type 2 Download PDF

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Publication number
WO2003033513A1
WO2003033513A1 PCT/AU2002/001405 AU0201405W WO03033513A1 WO 2003033513 A1 WO2003033513 A1 WO 2003033513A1 AU 0201405 W AU0201405 W AU 0201405W WO 03033513 A1 WO03033513 A1 WO 03033513A1
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agt
seq
nucleotide sequence
nucleic acid
acid molecule
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PCT/AU2002/001405
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English (en)
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Greg Collier
Paul Zev Zimmet
Ken Walder
Janine Mcmillan
Andrea Michelle De Silva
Kelly Windmill
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Autogen Research Pty Ltd
International Diabetes Institute
Deakin University
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Priority to CA002463578A priority Critical patent/CA2463578A1/fr
Priority to MXPA04003635A priority patent/MXPA04003635A/es
Priority to EP02801234A priority patent/EP1446414A4/fr
Priority to KR10-2004-7005672A priority patent/KR20040088019A/ko
Priority to IL16129402A priority patent/IL161294A0/xx
Priority to JP2003536252A priority patent/JP2005510214A/ja
Publication of WO2003033513A1 publication Critical patent/WO2003033513A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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 present invention relates generally to a nucleic acid molecule which expressed in at least the stomach, hypothalamus or liver identified using differential display techniques under differing physiological conditions. It is proposed that the nucleic acid molecules encode expression products associated with the modulation of obesity, anorexia, weight maintenance, diabetes and/or metabolic energy levels. More particularly, the present invention is directed to nucleic acid molecules and expression products produced by recombinant means from the nucleic acid molecule or isolated from natural sources and their use in therapeutic and diagnostic protocols for conditions such as obesity, anorexia, weight maintenance, diabetes and/or energy imbalance.
  • nucleic acid molecule and expression products and their derivatives, homologs, analogs and mimetics are proposed to be useful, therefore, as therapeutic and diagnostic agents for obesity, anorexia, weight maintenance, diabetes and/or energy imbalance or as targets for the design and/or identification of modulators of their activity and/or function.
  • Obesity is defined as a pathological excess of body fat and is the result of an imbalance between energy intake and energy expenditure for a sustained period of time.
  • Obesity is the most common metabolic disease found in affluent nations. The prevalence of obesity in these nations is alarmingly high, ranging from 10% to upwards of 50% in some subpopulations (Bouchard, The genetics of obesity. Boca Raton: CRC Press, 1994). Of particular concern is the fact that the prevalence of obesity appears to be rising consistently in affluent societies and is now increasing rapidly in less mature nations as they become more affluent and/or adopt cultural practices from the more affluent countries (Zimmet, Diabetes Care 15(2): 232-247, 1992).
  • Obesity is a complex and heterogeneous disorder and has been identified as a key risk indicator of preventable morbidity and mortality since obesity increases the risk of a number of other metabolic conditions including type 2 diabetes mellitus and cardiovascular disease (Must et al, JAMA. 282(16): 1523-1529, 1999; Kopelman, Nature 404: 635-643, 2000).
  • type 2 diabetes mellitus and cardiovascular disease Malt et al, JAMA. 282(16): 1523-1529, 1999; Kopelman, Nature 404: 635-643, 2000.
  • the prevalence of diabetes continues to increase rapidly. It has been estimated that there were about 700,000 persons with diabetes in Australia in 1995 while in the US, diabetes prevalence increased from 4.9% in 1990 to 6.9% in 1999 (Mokdad, Diabetes Care 24(2): 412, 2001).
  • hypothalamus A number of organs/tissues have been implicated in the pathophysiology of obesity and type 2 diabetes, and of particular interest are the hypothalamus, stomach and liver.
  • the hypothalamus has long been recognized as a key brain area in the regulation of energy intake (Stellar, Psychol Rev 61: 5-22, 1954) and it is now widely accepted that the hypothalamus plays a central role in energy homeostasis, integrating and co-ordinating a large number of factors produced by and/or acting on the hypothalamus.
  • a number of these factors have been investigated for their role in energy balance and body weight regulation, including neuropeptide Y, corticotropin-releasing hormone, melanin-concentrating hormone, leptin and insulin.
  • the role of the stomach in regulating food intake is thought to involve two types of signals: the degree of distension of the stomach and the activation of chemoreceptors in the gastric or intestinal wall (Koopmans, Experimental studies on the control of food intake. In: Handbook of Obesity, Eds. GA Bray, C Bouchard, WPT lames pp 273-312, 1998).
  • the gut is the largest endocrine organ in the body and after a meal a large number of gastrointestinal hormones are released. Some examples are gastrin, somatostatin, cholecystokinin, gastric inhibitory polypeptide and neurotensin.
  • stomach provides part of the signal that restricts food intake during a single meal
  • the nature of this signal or how it is transmitted to the brain remains to be determined.
  • the information relating to the degree of distension of the stomach or the presence of nutrients in the gastrointestinal walls is transmitted to the brain through either nerves or hormones.
  • the role of the gut hormones identified to date in the regulation of food intake remains to be equivocally determined.
  • the liver also plays a significant role in a number of important physiological pathways. It has a major role in the regulation of metabolism of glucose, amino acids and fat. In addition the liver is the only organ (other than the gut) that comes into direct contact with a large volume of ingested, absorbed food via the portal vein and, therefore, the liver is able to "sense” or monitor the level of nutrients entering the body, particularly the amounts of protein and carbohydrate. It has been proposed that the liver may also have a role in the regulation of food intake through the transmission of unidentified signals relaying information to the brain about nutrient absorption from the gut and metabolic changes throughout the body (Russek, Nature 200: 176, 1963).
  • the liver also plays a crucial role in maintaining circulating glucose concentrations by regulating pathways such as gluconeogenesis and glycogenolysis. Alterations in glucose homeostasis are important factors in the pathophysiology of impaired glucose tolerance and the development of type 2 diabetes mellitus.
  • genes were sought which are differentially expressed in particular vertebrate animal tissues or organs between either fed, re-fed or fasting conditions, or between diabetic and non-diabetic conditions. Novel genes are identified which are differentially expressed at least in the stomach, liver and/or hypothalamus under one or both of the above-mentioned conditions.
  • the inventors have isolated genes which are proposed to be associated with one or more biological functions associated with disease conditions such as but not limited to obesity, anorexia, diabetes or energy balance.
  • SEQ ID NO: Nucleotide and amino acid sequences are referred to by a sequence identifier number (SEQ ID NO:).
  • the SEQ ID NOs: correspond numerically to the sequence identifiers ⁇ 400>1 (SEQ ID NO:l), ⁇ 400>2 (SEQ ID NO:2), etc.
  • SEQ ID NO:1 sequence identifiers ⁇ 400>1
  • SEQ ID NO:2 sequence identifiers
  • Group A lean animals (normoglycemic; normoinsulinemic);
  • Group B obese, non-diabetic animals (normoglycemic; hyperinsulinemic); and
  • Group C obese, diabetic animals (hyperglycemic; hyperinsulinemic).
  • AGT-119 [SEQ TD NO:l]
  • AGT-120 [SEQ ID NO:2]
  • AGT- 121 [SEQ ID NO:3]
  • AGT-122 [SEQ ID NO:5]
  • AGT-422 [SEQ ID NO:6]
  • AGT-123 [SEQ TD NO:7]
  • AGT-504 [SEQ D NO:8 and SEQ ID N0:9].
  • SEQ ID NO:9 is a genomic form of AGT-504 and is also represented as SEQ TD NO:8.
  • AGT-119 was detected initially in stomach tissue using differential display PCR and its expression was elevated in fed animals compared to fasted or re-fed animals.
  • AGT-120 was initially detected in stomach tissue using differential display PCR and its expression was elevated in fed animals compared to fasted or re-fed animals.
  • AGT-121 was initially identified in the hypothalamus using differential display PCR and its expression levels were elevated in fasted diabetic, non-diabetic and lean animals compared to fed animals when separated by genotype.
  • AGT- 122 was initially identified in the liver using differential display PCR and was shown to have elevated expression levels in fasted compared to fed diabetic or non-diabetic animals.
  • AGT-422 was identified suppression subtractive hybridization in liver tissue and its expression was elevated in fed lean animals compared to fed diabetic animals and elevated in fed, lean animals compared to fasted lean animals, in fed non-diabetic animals compared to fasted non-diabetic animals and fed diabetic animals compared to fasted diabetic animals.
  • AGT-123 was identified in the hypothalamus tissue using differential display PCR and its expression was found to be elevated in fasted lean, non-diabetic animals and diabetic animals compared to fed animals.
  • AGT-504 was identified using amplified fragment length polymorphism analysis in genomic DNA and its expression in liver tissue was elevated in diabetic animals compared to lean or non-diabetic animals. A summary of the AGT genes is provided in Table 1.
  • the present invention provides, therefore, a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding an expression product or a derivative, homolog, analog or mimetic thereof wherein said nucleic acid molecule is differentially expressed in one or more of stomach, liver or hypothalamus tissue under fed or unfed or diabetic or non-diabetic conditions.
  • the present invention further provides a nucleic acid molecule comprising a nucleotide sequence encoding or complementary to a sequence encoding an expression product or a derivative, homolog, analog or mimetic thereof wherein the nucleotide sequence is as substantially set forth in SEQ ID NO:l or SEQ ID NO:2 or SEQ TD NO:3 or SEQ TD NO:5 or SEQ TD NO:6 or SEQ ID NO:7 or SEQ TD NO:8 or SEQ ID NO:9 or a nucleotide sequence having at least about 30% similarity to all or part of SEQ LD NO:l or SEQ ID NO:2 or SEQ TD NO:3 or SEQ ID NO:4 or SEQ TD NO:5 or SEQ ID NO:6 or SEQ ID NO:7 or SEQ TD NO:8 or SEQ ID NO:9 and/or is capable of hybridizing to one or more of SEQ ID NO:l or SEQ TD NO:2 or SEQ TD NO:3 or SEQ ID NO:
  • the present invention also provides an isolated expression product or a derivative, homolog, analog or mimetic thereof which expression product is encoded by a nucleotide sequence which is differentially expressed in one or more of stomach, liver or hypothalamus tissue under fed or unfed or diabetic or non-diabetic conditions.
  • the present invention is directed to an isolated expression product or a derivative, homolog, analog or mimetic thereof wherein the expression product is encoded by a nucleotide sequence substantially as set forth in SEQ ID NO:l, SEQ ID NO:2 or SEQ ID NO:3 or SEQ TD NO:5 or SEQ ID NO:6 or SEQ ID NO:7 or SEQ TD NO:8 or SEQ TD NO:9 or a nucleotide sequence having at least 30% similarity to all or part of SEQ ID NO:l, SEQ TD NO:2 or SEQ TD NO:3 or SEQ TD NO:5 or SEQ ID NO:6 or SEQ ID NO:7 or SEQ ID NO:8 or SEQ TD NO:9 and/or is capable of hybridizing to SEQ ID NO:l, SEQ TD NO:2 or SEQ ID NO:3 or SEQ ID NO:5 or SEQ TD NO:6 or SEQ TD NO:7 or SEQ ID NO: 8 or SEQ ID NO: 9 or their
  • the preferred genetic sequence of the present invention are referred to herein as AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504.
  • the expression products encoded by AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504 are referred to herein as AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504, respectively.
  • the expression product may be an RNA (e.g. mRNA) or a protein. Where the expression product is an RNA, the present invention extends to RNA- related molecules associated thereto such as RNAi or intron or exon sequences therefrom.
  • Even yet another aspect of the present invention relates to a composition
  • a composition comprising AGT- 119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and/or AGT-504 or its derivatives, homologs, analogs or mimetics or agonists or antagonists of AGT-119, AGT- 120, AGT-121, AGT-122, AGT-422, AGT-123 and/or AGT-504 together with one or more pharmaceutically acceptable carriers and/or diluents.
  • Another aspect of the present invention contemplates a method for treating a subject comprising administering to said subject a treatment effective amount of AGT-119, AGT- 120, AGT-121, AGT-122, AGT-422, AGT-123 and/or AGT-504 or a derivative, homolog, analog or mimetic thereof or a genetic sequence encoding same or an agonist or antagonist of AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and/or AGT-504 activity ox AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and/or AGT-504 gene expression for a time and under conditions sufficient to effect treatment.
  • treatments contemplated herein include but are not limited to obesity, anorexia, weight maintenance, energy imbalance and diabetes. Treatment may be by the administration of a pharmaceutical composition or genetic sequences via gene therapy. Treatment is contemplated for human subjects as well as animals such as animals important to livestock industry.
  • a further aspect of the present invention is directed to a diagnostic agent for use in monitoring or diagnosing conditions such as but not limited to obesity, anorexia, weight maintenance, energy imbalance and/or diabetes, said diagnostic agent selected from an antibody to AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 or AGT-504 or its derivatives, homologs, analogs or mimetics and a genetic sequence comprising or capable of annealing to a nucleotide strand associated with AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 or AGT-504 useful inter alia in PCR, hybridization, RFLP analysis or AFLP analysis.
  • diagnostic agent selected from an antibody to AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 or AGT-504 or its derivatives, homologs, analogs or mimetics and a genetic sequence comprising or capable of annea
  • Figure 1 is a graphical representation showing AGT-119 stomach gene expression in fed, fasted and re-fed Psammomys obesus.
  • Figure 2 is a graphical representation of AGT-120 stomach gene expression in fasted, fed and re- fed Psammomys obesus.
  • Figure 3 is a graphical representation showing the distribution of tissue in which AGT-121 is expressed.
  • Figure 4 is a photographic representation showing Northern analysis of AGT-121 expression in (1) heart; (2) brain; (3) placenta; (4) lung; (5) liver; (6) skeletal muscle); (7) kidney and (8) pancreas.
  • Figure 5 is a graphical representation showing AGT-121 expression in energy restricted hypothalamus.
  • Figure 6 is a graphical representation showing expression of AGT-121 versus level of body weight.
  • Figure 7 is a graphical representation showing expression of AGT-121 versus change in glucose levels.
  • Figure 8 graphical representation showing expression of AGT-121 versus scap fat.
  • Figure 9 is a graphical representation of AGT-122 hepatic gene expression in Groups A, B and C fed and fasted Psammomys obesus.
  • Figure 10 is a graphical representation of AGT-122 hepatic gene expression for fed and fasted Psammomys ohesus(pooled data) .
  • Figure 11 is a graphical representation of the association between A GT-122 hepatic gene expression and body weight in fed Psammomys obesus.
  • Figure 12 is a graphical representation of AGT-422 hepatic gene expression data from Groups A, B and C fed and fasted Psammomys obesus.
  • Figure 13 is a graphical representation of AGT-422 hepatic gene expression in all fed and fasted Psammomys obesus (pooled data).
  • Figure 14 is a graphical representation of AGT-123 hypothalamic gene expression in Groups A, B and C fed and fasted Psammomys obesus.
  • Figure 15 is a graphical representation of AGT-504 hepatic gene expression in Groups A, B and C Psammomys obesus.
  • the present invention is predicated in part on the identification of novel genes associated inter alia with regulation of obesity, anorexia, weight maintenance, diabetes and/or metabolic energy levels.
  • the genes are identified following differential screening of mRNA from one or more of stomach, liver or hypothalamus tissue in fed, re-fed and fasted mammals or in diabetic and non-diabetic mammals. The selection of stomach, liver and hypothalamus is not intended to imply that differential expression does not occur in other tissue.
  • one aspect of the present invention provides a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding an expression product or a derivative, homolog, analog or mimetic thereof wherein said nucleic acid molecule is differentially expressed in one or more of stomach, liver or hypothalamus tissue under fed (or re-fed) or unfed or diabetic or non-diabetic conditions.
  • the term "differentially expressed” is used in its most general sense and includes elevated levels of an expression product such as mRNA or protein or a secondary product such as cDNA in one tissue compared to another tissue or in the same tissue but under different conditions. Examples of different conditions includes differential expression in tissue from fed, re-fed and fasting animals or diabetic and non-diabetic animals. Differential expression is conveniently determined by a range of techniques including polymerase chain reaction (PCR) such as real-time PCR. Other techniques include suppression subtractive hyridization (SSH) and amplified fragment length polymorphism (AFLP) analysis.
  • PCR polymerase chain reaction
  • SSH suppression subtractive hyridization
  • AFLP amplified fragment length polymorphism
  • an animal model may be employed to study the differences in gene expression in animal tissues under different conditions.
  • the present invention is exemplified using the Psammomys obesus (the Israeli Sand Rat) animal model of dietary-induced obesity and type 2 diabetes. In their natural desert habitat, an active lifestyle and saltbush diet ensure that they remain lean and normoglycemic (Shafrir and Gutman, J. Basic Clin. Physiol. Pharm. 4: 83-99, 1993).
  • Psammomys obesus exhibit a range of bodyweight and blood glucose and insulin levels which form a continuous curve that closely resembles the patterns found in human populations, including the inverted U-shaped relationship between blood glucose and insulin levels known as "Starling's curve of the pancreas" (Barnett et al, 1994a, supra). It is the heterogeneity of the phenotypic response of Psammomys obesus which makes it an ideal model to study the etiology and pathophysiology of obesity and type 2 diabetes.
  • the animals are conveniently classified into three groups designated Groups A, B and C:
  • Group A animals are lean
  • Group B animals are obese and non-diabetic.
  • Group C animals are obese and diabetic.
  • a number of differentially expressed genetic sequences were identified in stomach, liver or hypothalamus tissue in Psammomys obesus under different feeding regimes (i.e. fed, re-fed or fasting) or under diabetic or non- diabetic conditions.
  • nucleic acid molecule comprising a nucleotide sequence encoding or complementary to a sequence encoding an expression product or a derivative, homolog, analog or mimetic thereof wherein said nucleotide sequence is as substantially set forth in SEQ TD NO:l (AGT-119) or SEQ TD NO:2 (AGT- 120) or SEQ TD NO:3 (AGT-121) or SEQ ID NO:5 (AGT-122) or SEQ TD NO:6 (AGT- 422) or SEQ TD NO:7 (AGT-123) or SEQ TD NO:8 (AGT-504 genomic) or SEQ TD NO:9 (AGT-504 cDNA) or a nucleotide sequence having at least about 30% similarity to all or part of SEQ ID NO:l or SEQ TD NO:2 or SEQ TD NO:3 or SEQ ID NO:5 or SEQ TD NO:6 or SEQ ID NO:7 or SEQ TD NO:8 or SEQ
  • An expression product includes an RNA molecule such as an mRNA transcript as well as a protein.
  • Some genes are non-protein encoding genes and produce mRNA or other RNA molecules and are involved in regulation by RNA:DNA, RNA:RNA or RNA:protein interaction.
  • the RNA e.g. mRNA
  • the RNA may act directly or via the induction of other molecules such as RNAi or via products mediated from splicing events (e.g. exons or introns).
  • Other genes encode mRNA transcripts which are then translated into proteins.
  • a protein includes a polypeptide.
  • the differentially expressed nucleic acid molecules therefore, may encode mRNAs only or, in addition, proteins. Both mRNAs and proteins are forms of "expression products".
  • Reference herein to similarity is generally at a level of comparison of at least 15 consecutive or substantially consecutive nucleotides. It is particularly convenient, however, to determine similarity by comparing a total or complete sequence, after optimal alignment.
  • similarity includes exact identity between compared sequences at the nucleotide level. Where there is non-identity at the nucleotide level, "similarity" includes differences between sequences which may encode different amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels, i a particularly preferred embodiment, nucleotide sequence comparisons are made at the level of identity rather than similarity.
  • sequence relationships between two or more polynucleotides include “reference sequence”, “comparison window”, “sequence similarity”, “sequence identity”, “percentage of sequence similarity”, “percentage of sequence identity”, “substantially similar” and “substantial identity”.
  • a “reference sequence” is at least 12 but frequently 15 to 18 and often at least 25 or above, such as 30 monomer units in length. Because two polynucleotides may each comprise (1) a sequence (i.e.
  • sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity.
  • a “comparison window” refers to a conceptual segment of typically 12 contiguous residues that is compared to a reference sequence.
  • the comparison window may comprise additions or deletions (i.e. gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment (i.e. resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected.
  • GAP Garnier et al.
  • Altschul et al. Nucl Acids Res. 25: 3389, 1997.
  • a detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al. ("Current Protocols in Molecular Biology" John Wiley & Sons Inc, Chapter 15, 1994-1998).
  • a range of other algorithms may be used to compare the nucleotide and amino acid sequences such as but not limited to PILEUP, CLUSTALW, SEQUENCHER or VectorNTI.
  • sequence similarity and “sequence identity” as used herein refers to the extent that sequences are identical or functionally or structurally similar on a nucleotide-by- nucleotide basis over a window of comparison.
  • a “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g. A, T, C, G, I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • sequence identity will be understood to mean the “match percentage” calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA) using standard defaults as used in the reference manual accompanying the software. Similar comments apply in relation to sequence similarity.
  • Reference herein to a low stringency includes and encompasses from at least about 0 to at least about 15% v/v formamide and from at least about 1 M to at least about 2 M salt for hybridization, and at least about 1 M to at least about 2 M salt for washing conditions.
  • low stringency is at from about 25-30°C to about 42°C. The temperature may be altered and higher temperatures used to replace formamide and/or to give alternative stringency conditions.
  • Alternative stringency conditions may be applied where necessary, such as medium stringency, which includes and encompasses from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5 M to at least about 0.9 M salt for hybridization, and at least about 0.5 M to at least about 0.9 M salt for washing conditions, or high stringency, which includes and encompasses from at least about 31% v/v to at least about 50% v/v formamide and from at least about 0.01 M to at least about 0.15 M salt for hybridization, and at least about 0.01 M to at least about 0.15 M salt for washing conditions.
  • medium stringency which includes and encompasses from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5 M to at least about 0.9 M salt for hybridization, and at least about 0.5 M to at least about 0.9 M salt for washing conditions
  • high stringency which includes and encompasses from at least about 31% v/v to at least about 50% v/v form
  • T m of a duplex DNA decreases by 1°C with every increase of 1% in the number of mismatch base pairs (Bonner and Laskey, Eur. J. Biochem. 46: 83, 1974).
  • Formamide is optional in these hybridization conditions. Accordingly, particularly preferred levels of stringency are defined as follows: low stringency is 6 x SSC buffer, 0.1% w/v SDS at 25-42°C; a moderate stringency is 2 x SSC buffer, 0.1% w/v SDS at a temperature in the range 20°C to 65°C; high stringency is 0.1 x SSC buffer, 0.1% w/v SDS at a temperature of at least 65°C.
  • nucleotide sequence or amino acid sequence of the present invention may correspond to exactly the same sequence of the naturally occurring gene (or corresponding cDNA) or protein or other expression product or may carry one or more nucleotide or amino acid substitutions, additions and/or deletions.
  • SEQ ID NO:l The nucleotide sequences set forth in SEQ ID NO:l (AGT-119), SEQ ID NO:2 (AGT-120) and SEQ TD NO:3 (AGT-121) or SEQ ID NO:5 (AGT-122) or SEQ ID NO:6 (AGT-422) or SEQ ID NO:7 (AGT-123) or SEQ ID NO:8 (AGT-504 genomic) or SEQ ID NO:9 (AGT-504 cDNA) correspond to novel genes referred to in parenthesis.
  • the corresponding expression products are AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504.
  • references herein to AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504 includes, where appropriate, reference to the genomic gene or cDNA as well as any naturally occurring or induced derivatives.
  • a genomic form of AGT-504 is represented as SEQ ID NO: 8.
  • the present invention further encompasses mutants, fragments, parts and portions of the nucleotide sequence corresponding to AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504.
  • nucleic acid molecule or derivative, homolog or analog thereof comprising a nucleotide sequence encoding, or a nucleotide sequence complementary to a sequence encoding an expression product wherein said nucleotide sequence is substantially as set forth in SEQ ID NO:l (AGT-119) or a derivative, homolog or mimetic thereof or having at least about 30% similarity to all or part of SEQ ID NO:l or a nucleotide sequence capable of hybridizing to SEQ TD NO:l or its complementary form under low stringency conditions.
  • nucleic acid molecule or derivative, homolog or analog thereof comprising a nucleotide sequence encoding, or a nucleotide sequence complementary to a sequence encoding an expression product wherein said nucleotide sequence is substantially as set forth in SEQ ID NO. "2 (AGT-120) or a derivative, homolog or mimetic thereof or having at least about 30% similarity to all or part of SEQ LD NO:2 or a nucleotide sequence capable of hybridizing to SEQ ID NO:2 or its complementary form under low stringency conditions.
  • Still yet another aspect of the present invention provides a nucleic acid molecule or derivative, homolog or analog thereof comprising a nucleotide sequence encoding, or a nucleotide sequence complementary to a sequence encoding an expression product wherein said nucleotide sequence is substantially as set forth in SEQ ID NO:3 (AGT-121) or a derivative, homolog or mimetic thereof or having at least about 30% similarity to all or part of SEQ ID NO:3 or a nucleotide sequence capable of hybridizing to SEQ LD NO:3 or their complementary forms under low stringency conditions.
  • nucleic acid molecule or derivative, homolog or analog thereof comprising a nucleotide sequence encoding, or a nucleotide sequence complementary to a sequence encoding an expression product wherein said nucleotide sequence is substantially as set forth in SEQ ID NO: 5 (AGT-122) or a derivative, homolog or mimetic thereof or having at least about 30%> similarity to all or part of SEQ ID NO:5 or a nucleotide sequence capable of hybridizing to SEQ ID NO:5 or its complementary form under low stringency conditions.
  • nucleic acid molecule or derivative, homolog or analog thereof comprising a nucleotide sequence encoding, or a nucleotide sequence complementary to a sequence encoding an expression product wherein said nucleotide sequence is substantially as set forth in SEQ ID NO: 6 (AGT-422) or a derivative, homolog or mimetic thereof or having at least about 30% similarity to all or part of SEQ LD NO:6 or a nucleotide sequence capable of hybridizing to SEQ ID NO:6 or its complementary fonn under low stringency conditions.
  • nucleic acid molecule or derivative, homolog or analog thereof comprising a nucleotide sequence encoding, or a nucleotide sequence complementary to a sequence encoding an expression product wherein said nucleotide sequence is substantially as set forth in SEQ ID NO:7 (AGT-123) or a derivative, homolog or mimetic thereof or having at least about 30% similarity to all or part of SEQ ID NO:7 or a nucleotide sequence capable of hybridizing to SEQ ID NO:7 or its complementary form under low stringency conditions.
  • a further aspect of the present invention provides a nucleic acid molecule or derivative, homolog or analog thereof comprising a nucleotide sequence encoding, or a nucleotide sequence complementary to a sequence encoding an expression product wherein said nucleotide sequence is substantially as set forth in SEQ TD NO:8 (AGT-504 genomic) or a derivative, homolog or mimetic thereof or having at least about 30% similarity to all or part of SEQ ID NO:8 or a nucleotide sequence capable of hybridizing to SEQ ID NO:8 or its complementary form under low stringency conditions.
  • nucleic acid molecule or derivative, homolog or analog thereof comprising a nucleotide sequence encoding, or a nucleotide sequence complementary to a sequence encoding an expression product wherein said nucleotide sequence is substantially as set forth in SEQ TD NO:9 (AGT-504 cDNA) or a derivative, homolog or mimetic thereof or having at least about 30% similarity to all or part of SEQ ID NO:9 or a nucleotide sequence capable of hybridizing to SEQ ID NO:9 or its complementary form under low stringency conditions.
  • AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504 has been determined, inter alia, to indicate an involvement in the regulation of one or more of obesity, anorexia, weight maintenance, diabetes and/or energy metabolism.
  • AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504 in one or more of stomach, liver or hypothalamus tissue of fed versus fasted or diabetic versus non-diabetic animals these genes may also be expressed in other tissues including but in no way limited to brain, muscle, adipose tissue, pancreas and gastrointestinal trait.
  • the nucleic acid molecule encoding each of AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 or AGT-504 is preferably a DNA such as a cDNA sequence or a genomic DNA.
  • a genomic sequence may also comprise exons and introns.
  • a genomic sequence may also include a promoter region or other regulatory regions.
  • a homolog is considered to be a gene from another animal species which has the same or greater than 30% similarity to one of AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504 and/or which has a similar function.
  • the above-mentioned genes are exemplified herein from Psammomys obesus stomach, liver or hypothalamus.
  • the present invention extends, however, to the homologous gene, as determined by nucleotide sequence and/or function, from humans, primates, livestock animals (e.g. cows, sheep, pigs, horses, donkeys), laboratory test animals (e.g. mice, guinea pigs, hamsters, rabbits), companion animals (e.g. cats, dogs) and captured wild animals (e.g. rodents, foxes, deer, kangaroos).
  • livestock animals e.g. cows, sheep, pigs, horses
  • the nucleic acids of the present invention and in particular AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504 and their derivatives and homologs may be in isolated or purified form and/or may be ligated to a vector such as an expression vector.
  • Expression may be in a eulcaryotic cell line (e.g. mammalian, insect or yeast cells) or in prokaryote cells (e.g. E. coli) or in both.
  • isolated is meant a nucleic acid molecule having undergone at least one purification step and this is conveniently defined, for example, by a composition comprising at least about 10% subject nucleic acid molecule, preferably at least about 20%, more preferably at least about 30%, still more preferably at least about 40-50%, even still more preferably at least about 60-70%, yet even still more preferably 80-90% or greater of subject nucleic acid molecule relative to other components as determined by molecular weight, encoding activity, nucleotide sequence, base composition or other convenient means.
  • the nucleic acid molecule of the present invention may also be considered, in a preferred embodiment, to be biologically pure.
  • the nucleic acid molecule may be ligated to an expression vector capable of expression in a prokaryotic cell (e.g. E. coli) or a eukaryotic cell (e.g. yeast cells, fungal cells, insect cells, mammalian cells or plant cells).
  • the nucleic acid molecule may be ligated or fused or otherwise associated with a nucleic acid molecule encoding another entity such as, for example, a signal peptide. It may also comprise additional nucleotide sequence information fused, linked or otherwise associated with it either at the 3' or 5' terminal portions or at both the 3' and 5' terminal portions.
  • the nucleic acid molecule may also be part of a vector, such as an expression vector.
  • the derivatives of the nucleic acid molecule of the present invention include oligonucleotides, PCR primers, antisense molecules, molecules suitable for use in co- suppression and fusion nucleic acid molecules.
  • Ribozymes and DNAzymes are also contemplated by the present invention directed to AGT-119, AGT-120, AGT-121, AGT- 122, AGT-422, AGT-123 and AGT-504 or their mRNAs.
  • Derivatives and homologs of AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504 are conveniently encompassed by those nucleotide sequences capable of hybridizing to one or more of S ⁇ Q ID NO:l, S ⁇ Q TD NO:2 or S ⁇ Q ID NO:3 or S ⁇ Q ID NO:5 or S ⁇ Q ID NO:6 or S ⁇ Q ID NO:7 or S ⁇ Q ID NO:8 or S ⁇ Q ID NO:9 or their complementary forms under low stringency conditions.
  • Derivatives include fragments, parts, portions, mutants, variants and mimetics from natural, synthetic or recombinant sources including fusion nucleic acid molecules. Derivatives may be derived from insertion, deletion or substitution of nucleotides.
  • Another aspect of the present invention provides an isolated expression product or a derivative, homolog, analog or mimetic thereof which is produced in larger or lesser amounts in one or more of stomach, liver or hypothalamus tissue in obese animals compared to lean animals or in fed (including re-fed) compared to fasted animals or in animals under diabetic compared to non-diabetic conditions.
  • An expression product may be RNA or protein.
  • derivatives include amino acid insertional derivatives such as amino and/or carboxylic terminal fusions as well as intra-sequence insertions of single or multiple amino acids.
  • Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in a protein although random insertion is also possible with suitable screening of the resulting product.
  • Deletional variants are characterized by the removal of one or more amino acids from the sequence.
  • Substitutional amino acid variants are those in which at least one residue in the sequence has been removed and a different residue inserted in its place.
  • An example of substitutional amino acid variants are conservative amino acid substitutions.
  • Conservative amino acid substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine and leucine; aspartic acid and glutamic acid; asparagine and glutamine; serine and threonine; lysine and arginine; and phenylalanine and tyrosine. Additions to amino acid sequences include fusions with other peptides, polypeptides or proteins.
  • Chemical and functional equivalents of protein forms of the expression products AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 or AGT-504 should be understood as molecules exhibiting any one or more of the functional activities of these molecules and may be derived from any source such as being chemically synthesized or identified via screening processes such as natural product screening or screening of chemical libraries.
  • the derivatives include fragments having particular epitopes or parts of the entire protein fused to peptides, polypeptides or other proteinaceous or non-proteinaceous molecules.
  • references herein to AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 or AGT-504 includes reference to isolated or purified naturally occurring AGT-119, AGT- 120, AGT-121, AGT-122, AGT-422, AGT-123 or AGT-504 as well as any derivatives, homologs, analogs and mimetics thereof.
  • Derivatives include parts, fragments and portions of AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504 as well as single and multiple amino acid substitutions, deletions and/or additions to AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504 when the expression products are proteins.
  • a derivative of AGT-119, AGT-120, AGT-121, AGT-122, AGT- 422, AGT-123 or AGT-504 is conveniently encompassed by molecules encoded by a nucleotide sequence capable of hybridizing to SEQ ID NO:l or SEQ ID NO:2 or SEQ TD NO:3 or SEQ ID NO:5 or SEQ ID NO:6 or SEQ TD NO:7 or SEQ TD NO:8 or SEQ ID NO: 9 under low stringency conditions.
  • AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504 include chemical analogs.
  • Analogs of AGT-119, AGT-120, AGT-121, AGT- 122, AGT-422, AGT-123 and AGT-504 contemplated herein include, but are not limited to, modifications to side chains, incorporation of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose confirmational constraints on the proteinaceous molecule or their analogs.
  • side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH ; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulfonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH .
  • amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH ; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulfonic acid (TNBS); acylation
  • the guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.
  • the carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitization, for example, to a corresponding amide.
  • Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4- chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid, phenylmercury chloride, 2- chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.
  • Tryptophan residues may be modified by, for example, oxidation with N- bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides.
  • Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to fonn a 3-nitrotyrosine derivative.
  • Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate.
  • Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3- hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids.
  • a list of unnatural amino acid, contemplated herein is shown in Table 3. TABLE 3
  • Non-conventional Code Non-conventional Code amino acid amino acid
  • D- ⁇ -methylcysteine Dmcys N-(4-aminobutyl)glycine Nglu D- ⁇ -methylglutamine Dmgln N-(2-aminoethyl)glycine Naeg
  • peptides can be conformationally constrained by, for example, incorporation of C ⁇ and N c rmethylamino acids, introduction of double bonds between C a and C ⁇ atoms of amino acids and the formation of cyclic peptides or analogs by introducing covalent bonds such as forming an amide bond between the N and C termini, between two side chains or between a side chain and the N or C terminus.
  • the expression product may be a RNA or protein.
  • protein should be understood to encompass peptides, polypeptides and proteins.
  • the protein may be glycosylated or unglycosylated and/or may contain a range of other molecules fused, linked, bound or otherwise associated to the protein such as amino acids, lipids, carbohydrates or other peptides, polypeptides or proteins.
  • Reference hereinafter to a "protein” includes a protein comprising a sequence of amino acids as well as a protein associated with other molecules such as amino acids, lipids, carbohydrates or other peptides, polypeptides or proteins.
  • the expression product is encoded by a sequence of nucleotides comprising SEQ LD NO:l or a derivative, homolog or analog thereof including a nucleotide sequence having at least about 30% similarity to SEQ ID NO:l or a nucleotide sequence capable of hybridizing to SEQ ID NO:l or its complementary form under low stringency conditions.
  • the expression product is encoded by a sequence of nucleotides comprising SEQ TD NO:2 or a derivative, homolog or analog thereof including a nucleotide sequence having at least about 30% similarity to SEQ TD NO:2 or a nucleotide sequence capable of hybridizing to SEQ TD NO:2 or its complementary form under low stringency conditions.
  • the expression product is encoded by a sequence of nucleotides comprising SEQ TD NO: 3 or a derivative homolog or analog thereof including a nucleotide sequence having at least about 30%> similarity to SEQ ID NO:3 or a nucleotide sequence capable of hybridizing to SEQ ID NO:3 or their complementary form under low stringency conditions.
  • the expression product is encoded by a sequence of nucleotides comprising SEQ ID NO: 5 or a derivative homolog or analog thereof including a nucleotide sequence having at least about 30%> similarity to SEQ TD NO:5 or a nucleotide sequence capable of hybridizing to SEQ LD NO:5 or its complementary form under low stringency conditions.
  • the expression product is encoded by a sequence of nucleotides comprising SEQ TD NO: 6 or a derivative homolog or analog thereof including a nucleotide sequence having at least about 30%> similarity to SEQ ID NO:6 or a nucleotide sequence capable of hybridizing to SEQ ID NO:6 or its complementary form under low stringency conditions.
  • the expression product is encoded by a sequence of nucleotides comprising SEQ ID NO:7 or a derivative homolog or analog thereof including a nucleotide sequence having at least about 30%) similarity to SEQ ID NO:7 or a nucleotide sequence capable of hybridizing to SEQ ID NO:7 or its complementary form under low stringency conditions.
  • the expression product is encoded by a sequence of nucleotides comprising SEQ ID NO: 8 or a derivative homolog or analog thereof including a nucleotide sequence having at least about 30% similarity to SEQ ID NO:8 or a nucleotide sequence capable of hybridizing to SEQ ID NO:8 or its complementary form under low stringency conditions.
  • the expression product is encoded by a sequence of nucleotides comprising SEQ ID NO:9 or a derivative homolog or analog thereof including a nucleotide sequence having at least about 30%) similarity to SEQ ID NO:9 or a nucleotide sequence capable of hybridizing to SEQ TD NO:9 or its complementary form under low stringency conditions.
  • Another aspect of the present invention is directed to an isolated expression product selected from the list consisting of:-
  • a protein comprising an amino acid sequence substantially as set forth in SEQ ID NO:4 or a derivative, homolog or analog thereof or a sequence encoding an amino acid sequence having at least about 30%> similarity to these sequences or a derivative, homolog, analog, chemical equivalent or mimetic of said protein;
  • (x) a protein encoded by a nucleotide sequence substantially comprising SEQ ID NO:6 or a derivative, homolog or analog thereof or a sequence encoding an amino acid sequence having at least about 30%> similarity to this sequence or a derivative, homolog, analog, chemical equivalent or mimetic of said protein;
  • xii a protein encoded by a nucleotide sequence substantially comprising SEQ ID NO: 8 or a derivative, homolog or analog thereof or a sequence encoding an amino acid sequence having at least about 30% similarity to this sequence or a derivative, homolog, analog, chemical equivalent or mimetic of said protein;
  • xiii a protein encoded by a nucleotide sequence substantially comprising SEQ ID NO:9 or a derivative, homolog or analog thereof or a sequence encoding an amino acid sequence having at least about 30% similarity to this sequence or a derivative, homolog, analog, chemical equivalent or mimetic of said protein;
  • a protein encoded by a nucleic acid molecule capable of hybridizing to a nucleotide sequence comprising SEQ ID NO:l or its complementary form or a derivative, homolog or analog thereof under low stringency conditions;
  • xv a protein encoded by a nucleic acid molecule capable of hybridizing to a nucleotide sequence comprising SEQ ID NO:2 or its complementary form or a derivative, homolog or analog thereof under low stringency conditions
  • xvi a protein encoded by a nucleic acid molecule capable of hybridizing to a nucleotide sequence comprising SEQ ID NO:3 or their complementary forms or a derivative, homolog or analog thereof under low stringency conditions
  • a protein encoded by a nucleic acid molecule capable of hybridizing to a nucleotide sequence comprising SEQ ID NO:5 or its complementary form or a derivative, homolog or analog thereof under low stringency conditions;
  • a protein encoded by a nucleic acid molecule capable of hybridizing to a nucleotide sequence comprising SEQ ID NO:6 or its complementary form or a derivative, homolog or analog thereof under low stringency conditions;
  • (xix) a protein encoded by a nucleic acid molecule capable of hybridizing to a nucleotide sequence comprising SEQ LD NO: 7 or its complementary form or a derivative, homolog or analog thereof under low stringency conditions;
  • a protein encoded by a nucleic acid molecule capable of hybridizing to a nucleotide sequence comprising SEQ LD NO: 9 or its complementary form or a derivative, homolog or analog thereof under low stringency conditions.
  • An example of an expression product is the amino acid sequence set forth in SEQ LD NO:4 (AGT-121).
  • the protein of the present invention is preferably in isolated form.
  • isolated is meant a protein having undergone at least one purification step and this is conveniently defined, for example, by a composition comprising at least about 10% subject protein, preferably at least about 20%, more preferably at least about 30%, still more preferably at least about 40-50%o, even still more preferably at least about 60-10%, yet even still more preferably 80-90%) or greater of subject protein relative to other components as dete ⁇ nined by molecular weight, amino acid sequence or other convenient means.
  • the protein of the present invention may also be considered, in a preferred embodiment, to be biologically pure.
  • AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and/or AGT-504 is thought to relate to regulation of body weight and glucose homeostasis. Modulation of expression of these genes is thought inter alia to regulate energy balance via effects on energy intake and also effects on carbohydrate/fat metabolism. The energy intake effects are likely to be mediated via the central nervous system but peripheral effects on the metabolism of both carbohydrate and fat are possible. The expression of these genes may also be regulated by fasting and feeding. Accordingly, regulating the expression and/or activity of these genes or their expression products provides a mechanism for regulating both body weight and energy metabolism, including carbohydrate and fat metabolism.
  • AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504 permits the generation of a range of therapeutic molecules capable of modulating expression of AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504 or modulating the activity of AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT- 123 and AGT-504.
  • Modulators contemplated by the present invention include agonists and antagonists of AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504 expression.
  • Antagonists of AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504 expression include antisense molecules, ribozymes and co-suppression molecules (including any molecules which induce RNAi).
  • Agonists include molecules which increase promoter activity or which interfere with negative regulatory mechanisms.
  • Antagonists of AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT- 504 include antibodies and inhibitor peptide fragments. All such molecules may first need to be modified to enable such molecules to penetrate cell membranes.
  • viral agents may be employed to introduce genetic elements to modulate expression of AGT- 119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504.
  • AGT- 119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504 act in association with other genes such as the ob gene which encodes leptin
  • the therapeutic molecules may target AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504 and ob genes or their translation products.
  • the present invention contemplates, therefore, a method for modulating expression of AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504 in a mammal, said method comprising contacting the AGT-119, AGT-120, AGT-121, AGT-122, AGT- 422, AGT-123 and AGT-504 gene with an effective amount of a modulator of AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504 expression for a time and under conditions sufficient to up-regulate or down-regulate or otherwise modulate expression of AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504.
  • a nucleic acid molecule encoding AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504 or a derivative or homolog thereof may be introduced into a cell to enhance the ability of that cell to produce AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504, conversely, AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504 sense and/or antisense sequences such as oligonucleotides may be introduced to decrease expression of the genes at the level of transcription, post-transcription or translation.
  • Sense sequences preferably encode hair pin RNA molecules or double-stranded RNA molecules.
  • Another aspect of the present invention contemplates a method of modulating activity of AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504 in a mammal, said method comprising administering to said mammal a modulating effective amount of a molecule for a time and under conditions sufficient to increase or decrease AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504 activity.
  • the molecule may be a proteinaceous molecule or a chemical entity and may also be a derivative of AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT- 504 or its ligand. Modulating levels of AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504 expression or AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and/or AGT-504 activity or function is important in the treatment of a range of conditions such as obesity, anorexia, energy imbalance, diabetes, metabolic syndrome, dyslipidemia, hypertension and insulin resistance.
  • mammals contemplated by the present invention include but are not limited to humans, primates, livestock animals (e.g. pigs, sheep, cows, horses, donkeys), laboratory test animals (e.g. mice, rats, guinea pigs, hamsters, rabbits), companion animals (e.g. dogs, cats) and captured wild animals (e.g. foxes, kangaroos, deer).
  • livestock animals e.g. pigs, sheep, cows, horses, donkeys
  • laboratory test animals e.g. mice, rats, guinea pigs, hamsters, rabbits
  • companion animals e.g. dogs, cats
  • captured wild animals e.g. foxes, kangaroos, deer.
  • a particularly preferred host is a human, primate or livestock animal.
  • the present invention contemplates therapeutic and prophylactic use of AGT- 119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and/or AGT-504 expression products or AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504 genetic mutants and/or agonists or antagonists agents thereof.
  • the present invention contemplates, therefore, a method of modulating expression of AGT- 119, AGT-120 AGT-121, AGT-122, AGT-422, AGT-123 and/or AGT-504 in a mammal, said method comprising contacting the AGT-119, AGT-120, AGT-121, AGT-122, AGT- 422, AGT-123 and! or AGT-504 genes with an effective amount of an agent for a time and under conditions sufficient to up-regulate, down-regulate or otherwise module expression of AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504.
  • Another aspect of the present invention contemplates a method of modulating activity of AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and/or AGT-504 in a subject, said method comprising administering to said subject a modulating effective amount of an agent for a time and under conditions sufficient to increase or decrease AGT- 119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and/or AGT-504 activity or function.
  • Modulation of activity by the administration of an agent to a mammal can be achieved by one of several techniques, including, but in no way limited to, introducing into a mammal a proteinaceous or non-proteinaceous molecule which:
  • (iii) functions as an agonist of AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and/or AGT-504.
  • the molecules which may be administered to a mammal in accordance with the present invention may also be linked to a targeting means such as a monoclonal antibody, which provides specific delivery of these molecules to the target cells.
  • a targeting means such as a monoclonal antibody, which provides specific delivery of these molecules to the target cells.
  • a further aspect of the present invention relates to the use of the invention in relation to mammalian disease conditions.
  • the present invention is particularly useful in a therapeutic or prophylactic treatment of obesity, anorexia, diabetes or energy imbalance.
  • another aspect of the present invention relates to a method of treating a mammal suffering from a condition characterized by one or more symptoms of obesity, anorexia, diabetes and/or energy imbalance, said method comprising administering to said mammal an effective amount of an agent for a time and under conditions sufficient to modulate the expression of AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and/or AGT-504 or sufficient to modulate the activity of AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and/or AGT-504.
  • the present invention relates to a method of treating a mammal suffering from a disease condition characterized by one or more symptoms of obesity, anorexia, diabetes or energy imbalance, said method comprising administering to said mammal an effective amount of AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and/or AGT-504 or AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and/or AGT-504.
  • An agent includes proteinaceous or non-proteinaceous molecules such as antibodies, natural products, chemical entities or nucleic acid molecules (including antisense molecules, sense molecules, ribozymes, ds-RNA molecules or DNA-targeting molecules).
  • an “effective amount” means an amount necessary at least partly to attain the desired immune response (e.g. against AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 or AGT-504) or to delay the onset or inhibit progression or halt altogether the onset or progression of a particular condition.
  • AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and/or AGT-504 or AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT- 123 and/or AGT-504 or agents capable of modulating the expression or activity of said molecules may be co-administered with one or more other compounds or other molecules.
  • co-administered is meant simultaneous administration in the same formulation or in two different formulations via the same or different routes or sequential administration by the same or different routes.
  • quential administration is meant a time difference of from seconds, minutes, hours or days between the administration of the two types of molecules. These molecules may be administered in any order.
  • the present invention relates to the use of an agent capable of modulating the expression of AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT- 123 and/or AGT-504 or a derivative, homolog or analog thereof in the manufacture of a medicament for the treatment of a condition characterized by obesity, anorexia, weight maintenance, diabetes and/or energy imbalance.
  • the present invention relates to the use of an agent capable of modulating the activity of AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and/or AGT-504 or a derivative, homolog, analog, chemical equivalent or mimetic thereof in the manufacture of a medicament for the treatment of a condition characterized by obesity, anorexia, weight maintenance, diabetes and/or energy imbalance.
  • a further aspect of the present invention relates to the use of AGT-119, AGT-120, AGT- 121, AGT-122, AGT-422, AGT-123 and/or AGT-504 or derivative, homolog or analog thereof or AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and/or AGT- 504 or derivative, homolog, analog, chemical equivalent or mimetic thereof in the manufacture of a medicament for the treatment of a condition characterized by obesity, anorexia, weight maintenance, diabetes and/or energy imbalance.
  • Still yet another aspect of the present invention relates to agents for use in modulating the expression of AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and/or AGT- 504 or a derivative, homolog or analog thereof.
  • a further aspect relates to agents for use in modulating AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and/or AGT-504 activity or a derivative, homolog, analog, chemical equivalent or mimetic thereof.
  • Still another aspect of the present invention relates to AGT-119, AGT-120, AGT-121, AGT- 122, AGT-422, AGT-123 and/or AGT-504 or derivative, homolog or analog thereof or AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and/or AGT-504 or derivative, homolog, analog, chemical equivalent or mimetic thereof for use in treating a condition characterized by one or more symptoms of obesity, anorexia, weight maintenance, diabetes and/or energy imbalance.
  • the mammal undergoing treatment may be a human or an animal in need of therapeutic or prophylactic treatment.
  • the present invention contemplates in one embodiment a composition comprising a modulator of AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504 expression or AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT- 123 and AGT-504 activity and one or more pharmaceutically acceptable carriers and/or diluents
  • the composition comprises AGT-119, AGT-120, AGT- 121, AGT-122, AGT-422, AGT-123 and AGT-504 or a derivative, homolog, analog or mimetic thereof and one or more pharmaceutically acceptable carriers and/or diluents.
  • the compositions may also comprise leptin or modulations of leptin activity or ob expression.
  • active components all such components of such a composition are referred to as "active components”.
  • compositions of active components in a form suitable for injectable use include sterile aqueous solutions (where water soluble) and sterile powders for the extemporaneous preparation of sterile injectable solutions.
  • sterile aqueous solutions where water soluble
  • sterile powders for the extemporaneous preparation of sterile injectable solutions.
  • the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or other medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • solvent or other medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the preventions of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thirmerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged abso ⁇ tion of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active components in the required amount in the appropriate solvent with optionally other ingredients, as required, followed by sterilization by, for example, filter sterilization, irradiation or other convenient means. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.
  • AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504 or AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504 are suitably protected, they may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet.
  • the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • Such compositions and preparations should contain at least 1%> by weight of active compound.
  • the percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit.
  • the amount of active compound in such therapeutically useful compositions is such that a suitable dosage will be obtained.
  • Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 0.1 ⁇ g and 2000 mg of active compound.
  • the tablets, troches, pills, capsules and the like may also contain the following: A binder such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such a sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring.
  • a binder such as gum tragacanth, acacia, corn starch or gelatin
  • excipients such as dicalcium phosphate
  • a disintegrating agent such as corn starch, potato starch, alginic acid and the like
  • a lubricant such as magnesium stearate
  • a sweetening agent such as sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint
  • tablets, pills, or capsules may be coated with shellac, sugar or both.
  • a syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour.
  • any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed.
  • the active compound may be incorporated into sustained-release preparations and formulations.
  • Pharmaceutically acceptable carriers and/or diluents include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.
  • the use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active material for the treatment of disease in living subjects having a diseased condition in which bodily health is impaired as herein disclosed in detail.
  • the principal active component may be compounded for convenient and effective administration in sufficient amounts with a suitable pharmaceutically acceptable carrier in dosage unit form.
  • a unit dosage form can, for example, contain the principal active component in amounts ranging from 0.5 ⁇ g to about 2000 mg. Expressed in proportions, the active compound is generally present in from about 0.5 ⁇ g to about 2000 mg/ml of carrier.
  • the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.
  • effective amounts of AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504 or AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504 will range from 0.01 ng/kg/body weight to above 10,000 mg/kg/body weight.
  • Alternative amounts range from 0.1 ng/kg/body weight to above 1000 mg/kg/body weight.
  • the active ingredients may be administered per minute, hour, day, week, month or year depending on the condition being treated.
  • the route of administration may vary and includes intravenous, intraperitoneal, sub-cutaneous, intramuscular, intranasal, via suppository, via infusion, via drip, orally or via other convenient means.
  • the pharmaceutical composition may also comprise genetic molecules such as a vector capable of transfecting target cells where the vector carries a nucleic acid molecule capable of modulating AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504 expression or AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT- 504 activity.
  • the vector may, for example, be a viral vector.
  • Still another aspect of the present invention is directed to antibodies to AGT-119, AGT- 120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504 and their derivatives and homologs insofar as AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504 are proteins.
  • Such antibodies may be monoclonal or polyclonal and may be selected from naturally occurring antibodies to AGT-119, AGT-120.
  • AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504 may be specifically raised to AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504 or derivatives or homologs thereof.
  • AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504 or their derivatives or homologs may first need to be associated with a carrier molecule.
  • the antibodies and/or recombinant AGT-119, AGT-120, AGT- 121, AGT-122, AGT-422, AGT-123 and AGT-504 or their derivatives of the present invention are particularly useful as therapeutic or diagnostic agents.
  • An antibody "to" a molecule includes an antibody specific for said molecule.
  • AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504 and their derivatives can be used to screen for naturally occurring antibodies to AGT-119, AGT- 120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504 which may occur in certain autoimmune diseases.
  • specific antibodies can be used to screen for AGT- 119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504.
  • Techniques for such assays are well known in the art and include, for example, sandwich assays and ELISA.
  • Antibodies to AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT- 504 of the present invention may be monoclonal or polyclonal and may be selected from naturally occurring antibodies to the AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504 or may be specifically raised to the AGT-119, AGT-120 and AGT-121or their derivatives, hi the case of the latter, the AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504 protein may need first to be associated with a carrier molecule.
  • fragments of antibodies may be used such as Fab fragments.
  • the present invention extends to recombinant and synthetic antibodies and to antibody hybrids.
  • a "synthetic antibody” is considered herein to include fragments and hybrids of antibodies.
  • the antibodies of this aspect of the present invention are particularly useful for immunotherapy and may also be used as a diagnostic tool or as a means for purifying AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504.
  • specific antibodies can be used to screen for AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504 proteins.
  • the latter would be important, for example, as a means for screening for levels of AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504 in a cell extract or other biological fluid or purifying AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504 made by recombinant means from culture supernatant fluid.
  • Techniques for the assays contemplated herein are known in the art and include, for example, sandwich assays and ELISA.
  • any second antibodies (monoclonal, polyclonal or fragments of antibodies) directed to the first mentioned antibodies discussed above. Both the first and second antibodies may be used in detection assays or a first antibody may be used with a commercially available anti-immunoglobulin antibody.
  • An antibody as contemplated herein includes any antibody specific to any region of AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504.
  • Both polyclonal and monoclonal antibodies are obtainable by immunization with the enzyme or protein and either type is utilizable for immunoassays.
  • the methods of obtaining both types of sera are well known in the art.
  • Polyclonal sera are less preferred but are relatively easily prepared by injection of a suitable laboratory animal with an effective amount of AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504, or antigenic parts thereof, collecting serum from the animal, and isolating specific sera by any of the known immunoadsorbent techniques.
  • antibodies produced by this method are utilizable in virtually any type of immunoassay, they are generally less favoured because of the potential heterogeneity of the product.
  • the use of monoclonal antibodies in an immunoassay is particularly preferred because of the ability to produce them in large quantities and the homogeneity of the product.
  • the preparation of hybridoma cell lines for monoclonal antibody production derived by fusing an immortal cell line and lymphocytes sensitized against the immunogenic preparation can be done by techniques which are well known to those who are skilled in the art. (See, for example, Douillard and Hoffman, Basic Facts about Hybridomas, in Compendium of Immunology Vol. II, ed. by Schwartz, 1981; Kohler and Milstein, Nature 256: 495-499, 1975; Kohler and Milstein, European Journal of Immunology 6: 511-519, 1976.)
  • Another aspect of the present invention contemplates a method for detecting AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504 or a derivative or homolog thereof in a biological sample from a subject, said method comprising contacting said biological sample with an antibody specific for AGT-119, AGT-120, AGT-121, AGT- 122, AGT-422, AGT-123 and AGT-504 or their antigenic derivatives or homologs for a time and under conditions sufficient for a complex to fonn, and then detecting said complex.
  • the presence of the complex is indicative of the presence of AGT-119, AGT-120, AGT- 121, AGT-122, AGT-422, AGT-123 and AGT-504.
  • This assay may be quantitated or semi-quantitated to determine a propensity to develop obesity or other conditions or to monitor a therapeutic regimen.
  • AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504 may be accomplished in a number of ways such as by Western blotting and ELISA procedures.
  • a wide range of immunoassay techniques are available as can be seen by reference to U.S. Patent Nos. 4,016,043, 4,424,279 and 4,018,653. These, of course, include both single-site and two-site or "sandwich" assays of the non-competitive types, as well as in the traditional competitive binding assays. These assays also include direct binding of a labelled antibody to a target.
  • Sandwich assays are among the most useful and commonly used assays. A number of variations of the sandwich assay technique exist, and all are intended to be encompassed by the present invention. Briefly, in a typical forward assay, an unlabelled antibody is immobilized on a solid substrate and the sample to be tested brought into contact with the bound molecule.
  • a second antibody specific to the AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504 complex is then added and incubated, allowing time sufficient for the formation of another complex of antibody-AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504-labelled antibody.
  • AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504 is determined by observation of a signal produced by the reporter molecule.
  • the results may either be qualitative, by simple observation of the visible signal, or may be quantitated by comparing with a control sample containing known amounts of hapten.
  • Variations on the forward assay include a simultaneous assay, in which both sample and labelled antibody are added simultaneously to the bound antibody.
  • the sample is one which might contain AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT- 123 and AGT-504 including cell extract, tissue biopsy or possibly serum, saliva, mucosal secretions, lymph, tissue fluid and respiratory fluid.
  • the sample is, therefore, generally a biological sample comprising biological fluid but also extends to fermentation fluid and supernatant fluid such as from a cell culture.
  • the solid surface is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
  • the solid supports may be in the form of tubes, beads, discs or microplates, or any other surface suitable for conducting an immunoassay.
  • the binding processes are well-known in the art and generally consist of cross-linking covalently binding or physically adsorbing, the polymer-antibody complex to the solid surface which is then washed in preparation for the test sample. An aliquot of the sample to be tested is then added to the solid phase complex and incubated for a period of time sufficient (e.g. 2-40 minutes or overnight if more convenient) and under suitable conditions (e.g.
  • the antibody subunit solid phase is washed and dried and incubated with a second antibody specific for a portion of AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504.
  • the second antibody is linked to a reporter molecule which is used to indicate the binding of the second antibody to AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504.
  • An alternative method involves immobilizing the target molecules in the biological sample and then exposing the immobilized target to specific antibody which may or may not be labelled with a reporter molecule. Depending on the amount of target and the strength of the reporter molecule signal, a bound target may be detectable by direct labelling with the antibody.
  • a second labelled antibody specific to the first antibody is exposed to the target-first antibody complex to form a target-first antibody-second antibody tertiary complex. The complex is detected by the signal emitted by the reporter molecule.
  • reporter molecule as used in the present specification, is meant a molecule which, by its chemical nature, provides an analytically identifiable signal which allows the detection of antigen-bound antibody. Detection may be either qualitative or quantitative.
  • the most commonly used reporter molecules in this type of assay are either enzymes, fluorophores or radionuclide containing molecules (i.e. radioisotopes) and chemiluminescent molecules.
  • an enzyme is conjugated to the second antibody, generally by means of glutaraldehyde or periodate.
  • glutaraldehyde or periodate As will be readily recognized, however, a wide variety of different conjugation techniques exist, which are readily available to the skilled artisan.
  • Commonly used enzymes include horseradish peroxidase, glucose oxidase, ⁇ -galactosidase and alkaline phosphatase, amongst others.
  • the substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable colour change. Examples of suitable enzymes include alkaline phosphatase and peroxidase.
  • fluorogenic substrates which yield a fluorescent product rather than the chromogenic substrates noted above
  • the enzyme-labelled antibody is added to the first antibody hapten complex, allowed to bind, and then the excess reagent is washed away.
  • a solution containing the appropriate substrate is then added to the complex of antibody-antigen- antibody.
  • the substrate will react with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an indication of the amount of hapten which was present in the sample.
  • a "reporter molecule” also extends to use of cell agglutination or inhibition of agglutination such as red blood cells on latex beads, and the like.
  • fluorescent compounds such as fluorescein and rhodamine
  • fluorescein and rhodamine may be chemically coupled to antibodies without altering their binding capacity.
  • the fluorochrome-labelled antibody When activated by illumination with light of a particular wavelength, the fluorochrome-labelled antibody absorbs the light energy, inducing a state to excitability in the molecule, followed by emission of the light at a characteristic colour visually detectable with a light microscope.
  • the fluorescent-labelled antibody is allowed to bind to the first antibody- hapten complex. After washing off the unbound reagent, the remaining tertiary complex is then exposed to the light of the appropriate wavelength. The fluorescence observed indicates the presence of the hapten of interest.
  • Immunofluorescence and EIA techniques are both very well established in the art and are particularly preferred for the present method. However, other reporter molecules, such as radioisotope, chemiluminescent or bioluminescent molecules, may also be employed.
  • the present invention also contemplates genetic assays such as involving, for example, PCR analysis to detect AGT-119, AGT-120, AGT-121, AGT-122, AGT-422, AGT-123 and AGT-504 or their derivatives.
  • Real-time PCR is also particularly useful for assaying for particular genetic molecules.
  • Psammomys obesus rats were used for differential expression studies under different conditions.
  • the rats are divided into three groups, based on metabolic phenotype, as follows:-
  • AGT-119 was identified using differential display PCR of stomach cDNA from fed, fasted and re-fed Psammomys obesus.
  • the partial nucleotide sequence is as follows :-
  • the AGT-119 sequence does not show significant homology with anything on the public database (BLASTN version 2.2.1).
  • AGT-120 was identified using differential display PCR of stomach cDNA from fed, fasted and re-fed Psammomys obesus.
  • the partial nucleotide sequence is as follows:-
  • the AGT-120 sequence shows homology to a Lactobacillus gasseri hypothetical protein (NZ_AAAB01000011). A human homolog is yet to be identified.
  • AGT-121 was identified using differential display PCR of hypothalamic cDNA from diabetic and non-diabetic Psammomys obesus.
  • AGT-121 is a hypothalamic gene that was initially identified by differential display. From primary gene expression data in fed/fasted hypothalamus, it was ofinterest because ofthe large increase in its expression in Group B and C animals, as well as the large disparity in signal between the animals.
  • the nucleotide sequence is as follows:-
  • Tissue distribution of AGT-121 was investigated by Taqman PCR in multiple tissues of P. obesus ( Figure 3). Highest levels were seen in the brain with very low levels also evident in the spleen.
  • AGT-121 Tissue distribution of AGT-121 in human tissues was also examined by Northern analysis of a Clontech multiple tissue RNA blot. A specific band of approximately 6 kb was seen in the brain ( Figure 4). AGT-121 is thought to be brain-specific.
  • AGT-121 alleles are associated with obesity
  • the insertion and deletion alleles described in the original patent are associated with obesity. Eighty lean and obese individual Psammomys obesus were genotyped for the presence of the deletion, or the insertion, or both. Diabetic animals were not considered. The genetoype is significantly associated with the obesity phenotype seen in P. obesus. The results are shown in Tables 4 and 5.
  • Oligonuclotide primers were designed in the coding sequence of Psammomys obesus AGT-121. Expression of AGT-121 was analyzed in energy restricted hypothalamus. Positive conelations were seen with body weight in control animals, change in glucose in all animals and subscapular fat mass in all animals ( Figures 5-8).
  • the AGT-121 The ISR protein shows strong homology at both the nucleotide and protein level to human hypothetical protein DKFZp761D221 (DKFZp761D221) [Accession: NP_115667]. This protein is predicted to contain the pfam00928.5, Adap_comp_sub domain which is identified as adaptor complexes medium subunit family. This family also contains members which are cocatomer subunits. This gene has been localized to human chromosome lp31.2.
  • AGT-122 was identified using differential display PCR of liver cDNA from diabetic and non-diabetic Psammomys obesus.
  • the partial nucleotide sequence is as follows:-
  • AGT-122 in grouped fasted animals showed no association with body weight, glucose, or insulin.
  • the AGT-122 sequence shows significant homology with a number of regions on different mouse chromosomes. There was no homologous sequence on the public database (BLASTN version 2.2.1).
  • EXAMPLE 17 Partial sequence of Psammomys obesus AGT-422
  • AGT-422 was identified by Suppression Subtractive Hybridization (SSH) [also referred to as Representational Difference Analysis (RDA)] of liver cDNA from diabetic and non- diabetic Psammomys obesus.
  • SSH Suppression Subtractive Hybridization
  • RDA Representational Difference Analysis
  • the partial nucleotide sequence is as follows :-
  • AGT-422 was normally distributed.
  • An independent samples T test found AGT-422 gene expression greater in fed animals than fasted animals when group data were combined (pO.OOl; Figure 13).
  • the AGT-422 sequence shows sequence homology to Rattus norvegicus clone CH230- 213K1.
  • the full gene and open reading frame are yet to be identified and a human homolog is also as yet undetermined.
  • AGT-123 was identified using differential display PCR of hypothalamic cDNA from diabetic and non-diabetic Psammomys obesus.
  • the partial nucleotide sequence is as follows:-
  • the AGT-123 sequence matches three mouse expressed sequence tags (gb
  • EXAMPLE 23 Partial sequence of Psammomys obesus AGT-504
  • AGT-504 was identified using Amplified Fragment Length Polymo ⁇ hism (AFLP) screening of genomic DNA from diabetic and non-diabetic Psammomys obesus. It was found to be expressed in the liver of diabetic and non-diabetic Psammomys obesus.
  • AFLP Amplified Fragment Length Polymo ⁇ hism
  • the AFLP technique is based on the amplification of subsets of genomic restriction fragments using polymerase chain reaction (PCR).
  • DNA is cut with restriction enzymes (EcoRI and Mse ⁇ ) and double-stranded adapters are ligated to the ends of the DNA fragments to generate template DNA for amplification.
  • restriction enzymes EcoRI and Mse ⁇
  • the sequences of adapters and the adjacent restriction site serve as primer binding sites for subsequent amplification of the restriction fragments.
  • Selective nucleotides are included at the 3' ends of the PCR primers, which therefore prime DNA amplification only from a subset of the restriction fragments. This method will identify sequence differences in the restriction sites that are associated with the obesity/diabetes phenotype.
  • AFLP was performed using the GibcoBRL "AFLP Analysis System I” and "AFLP Starter Primer” Kits according to the manufacturer's instructions.
  • the PCRs were performed in 20 ⁇ l under the following conditions: one cycle at 94°C for 30 s; 65°C for 30 s; and 72°C for 60 s, then for each consecutive cycle the annealing temperature was lowered by 0.7°C for 12 cycles. This gives a touchdown phase of 13 cycles. This was followed by 23 cycles at 94°C for 30 s, 56°C for 30 s and 72°C for 60 s.
  • the fragments were separated on a 6% (w/v) polyacrylamide gel to visualize the (typically) 50-150 bands. Those bands that were deemed to be different between the groups of animals were excised from the gel. Re-amplification of the band of interest was performed using the following PCR conditions: 94°C 2 min, 40 cycles of (94°C 30 s; 54°C 30 s; 72°C 60 s); 72°C 5 min and the following primers: F 5'-GTA GAC TGC GTA CCA ATT C-3' [SEQ LD NO: 10] and R 5'-GAC GAT GAG TCC TGA GTA A-3' [SEQ LD NO:l 1]. The amplified bands were sequenced with Applied Biosystems Big Dye sequencing kit.
  • Primer and probe sequences for amplification and analysis of each gene (shown in the 5' to 3' direction).
  • AGT-422 Forward: cagtcaccagtgggctagca [SEQ LD NO:23] Reverse: cctggcagatggaagagctt [SEQ LD NO:24]
  • Russek M., A hypothesis on the participation of hepatic glucoreceptors in the control of food intake. Nature 200: 176, 1963.

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Abstract

L'invention concerne des molécules d'acides nucléiques codant des produits d'expression associés à la modulation de l'obésité, l'anorexie, du maintien du poids, du diabète et/ou des niveaux d'énergie métabolique. Les molécules d'acides nucléiques et les produits d'expression de l'invention sont produits à l'aide de moyens de recombinaison ou isolés dans des ressources naturelles. Les molécules d'acides nucléiques et les produits d'expression ainsi que leurs dérivés, homologues, analogues et mimétiques sont utilisés comme agents thérapeutiques et diagnostiques pour l'obésité, l'anorexie, le maintien du poids, le diabète et/ou le déséquilibre d'énergie ou comme cibles pour la conception et/ou l'identification de modulateurs de leur activité et/ou fonction. Les molécules d'acides nucléiques et les produits d'expression de l'invention sont identifiés à l'aide de techniques d'affichage différentiel.
PCT/AU2002/001405 2001-10-16 2002-10-16 Genes exprimes de maniere differentielle, associes a l'obesite et au diabete de type 2 WO2003033513A1 (fr)

Priority Applications (6)

Application Number Priority Date Filing Date Title
CA002463578A CA2463578A1 (fr) 2001-10-16 2002-10-16 Genes exprimes de maniere differentielle, associes a l'obesite et au diabete de type 2
MXPA04003635A MXPA04003635A (es) 2001-10-16 2002-10-16 Genes diferencialmente expresados asociados con obesidad y diabetes tipo 2.
EP02801234A EP1446414A4 (fr) 2001-10-16 2002-10-16 Genes exprimes de maniere differentielle, associes a l'obesite et au diabete de type 2
KR10-2004-7005672A KR20040088019A (ko) 2001-10-16 2002-10-16 비만 및 제 2 형 당뇨병과 관련된 차별적으로 발현된 유전자
IL16129402A IL161294A0 (en) 2001-10-16 2002-10-16 Differentially expressed genes associated with obesity and type 2 diabetes
JP2003536252A JP2005510214A (ja) 2001-10-16 2002-10-16 肥満と２型糖尿病に関連のある差異的に発現する遺伝子

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US33014901P 2001-10-16 2001-10-16
US60/330,149 2001-10-16

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CA (1) CA2463578A1 (fr)
IL (1) IL161294A0 (fr)
MX (1) MXPA04003635A (fr)
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ZA (1) ZA200402859B (fr)

Cited By (5)

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WO2004063218A1 (fr) * 2003-01-13 2004-07-29 Autogen Research Pty Ltd Genes relatifs a l'obesite
WO2005045054A2 (fr) * 2003-11-07 2005-05-19 Forschungsinstitut Für Die Biologie Landwirtschaftlicher Nutztiere Procedes pour identifier des principes actifs ayant une influence sur la masse corporelle et l'accumulation de graisse
WO2005061711A1 (fr) * 2003-12-24 2005-07-07 Samsung Electronics Co., Ltd. Polynucleotide associe au diabete sucre de type ii comprenant un polymorphisme a un seul nucleotide, micro-reseau et kit de diagnostic comprenant celui-ci et procede d'analyse du polynucleotide utilisant le micro-reseau et le kit de diagnostic
WO2005090378A1 (fr) * 2004-03-16 2005-09-29 Agt Biosciences Limited Ligands de la molecule fit (agt-121) et leur utilisation pharmaceutique
EP1824518A2 (fr) * 2004-12-07 2007-08-29 Ohio University Diagnostic de l'hyperinsulinemie et du diabete de type ii et protection contre ceux-ci sur la base de proteines exprimees differemment dans le serum

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CN115851931A (zh) * 2021-06-28 2023-03-28 广东医科大学附属第二医院 一种肝癌分子标志物在制备肝癌检测或疗效评价产品中的应用
CN114958875B (zh) * 2022-05-15 2023-08-18 赣南师范大学 柑橘黄龙病菌内参基因metG筛选和应用

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See also references of EP1446414A4 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004063218A1 (fr) * 2003-01-13 2004-07-29 Autogen Research Pty Ltd Genes relatifs a l'obesite
WO2005045054A2 (fr) * 2003-11-07 2005-05-19 Forschungsinstitut Für Die Biologie Landwirtschaftlicher Nutztiere Procedes pour identifier des principes actifs ayant une influence sur la masse corporelle et l'accumulation de graisse
WO2005045054A3 (fr) * 2003-11-07 2005-09-22 Forsch Die Biolog Landw Licher Procedes pour identifier des principes actifs ayant une influence sur la masse corporelle et l'accumulation de graisse
WO2005061711A1 (fr) * 2003-12-24 2005-07-07 Samsung Electronics Co., Ltd. Polynucleotide associe au diabete sucre de type ii comprenant un polymorphisme a un seul nucleotide, micro-reseau et kit de diagnostic comprenant celui-ci et procede d'analyse du polynucleotide utilisant le micro-reseau et le kit de diagnostic
US7635559B2 (en) 2003-12-24 2009-12-22 Samsung Electronics Co., Ltd. Polynucleotide associated with a type II diabetes mellitus comprising single nucleotide polymorphism, microarray and diagnostic kit comprising the same and method for analyzing polynucleotide using the same
WO2005090378A1 (fr) * 2004-03-16 2005-09-29 Agt Biosciences Limited Ligands de la molecule fit (agt-121) et leur utilisation pharmaceutique
EP1824518A2 (fr) * 2004-12-07 2007-08-29 Ohio University Diagnostic de l'hyperinsulinemie et du diabete de type ii et protection contre ceux-ci sur la base de proteines exprimees differemment dans le serum
EP1824518A4 (fr) * 2004-12-07 2009-10-28 Univ Ohio Diagnostic de l'hyperinsulinemie et du diabete de type ii et protection contre ceux-ci sur la base de proteines exprimees differemment dans le serum

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EP1446414A1 (fr) 2004-08-18
CA2463578A1 (fr) 2003-04-24
JP2005510214A (ja) 2005-04-21
ZA200402859B (en) 2005-01-24
IL161294A0 (en) 2004-09-27
CN1596264A (zh) 2005-03-16
MXPA04003635A (es) 2004-07-30
EP1446414A4 (fr) 2005-11-23
KR20040088019A (ko) 2004-10-15

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