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WO2004020465A2 - Proteines intervenant dans la regulation de l'homeostasie energetique - Google Patents

Proteines intervenant dans la regulation de l'homeostasie energetique Download PDF

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WO2004020465A2
WO2004020465A2 PCT/EP2003/009755 EP0309755W WO2004020465A2 WO 2004020465 A2 WO2004020465 A2 WO 2004020465A2 EP 0309755 W EP0309755 W EP 0309755W WO 2004020465 A2 WO2004020465 A2 WO 2004020465A2
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crossveinless
nucleic acid
polypeptide
acid molecule
frizzled
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PCT/EP2003/009755
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WO2004020465A3 (fr
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Karsten Eulenberg
Günter BRÖNNER
Tri Nguyen
Daria Onichtchouk
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DeveloGen Aktiengesellschaft für entwicklungsbiologische Forschung
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Priority to AU2003271579A priority Critical patent/AU2003271579A1/en
Publication of WO2004020465A2 publication Critical patent/WO2004020465A2/fr
Publication of WO2004020465A3 publication Critical patent/WO2004020465A3/fr

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    • 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
    • C07K14/475—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • This invention relates to the use of nucleic acid sequences encoding crossveinless 2, frizzled, and crossveinless homologous proteins, to the use of polynucleotides encoding these, and to the use of modulators/effectors of the proteins and polynucleotides in the diagnosis, study, prevention, and treatment of diseases and disorders related to body-weight regulation, for example, but not limited to, metabolic diseases or dysfunctions such as obesity, metabolic disorder, diabetes mellitus, eating disorder, cachexia, pancreatitis, hypertension, coronary heart disease, hypercholesterolemia, dyslipidemia, osteoarthritis, gallstones, or liver fibrosis, or to the use in regeneration of tissues such as pancreatic tissues and others.
  • metabolic diseases or dysfunctions such as obesity, metabolic disorder, diabetes mellitus, eating disorder, cachexia, pancreatitis, hypertension, coronary heart disease, hypercholesterolemia, dyslipidemia, osteoarthritis, gallstones, or liver fibrosis, or to the use
  • Obesity is one of the most prevalent metabolic disorders in the world. It is still a poorly understood human disease that becomes more and more relevant for western society. Obesity is defined as a body weight more than 20% in excess of the ideal body weight, frequently resulting in a significant impairment of health. Obesity may be measured by body mass index, an indicator of adiposity or fatness. Further parameters for defining obesity are waist circumferences, skinfold thickness and bioimpedance. Obesity is associated with an increased risk for cardiovascular disease, hypertension, diabetes, hyperlipidaemia and an increased mortality rate.
  • Obesity is influenced by genetic, metabolic, biochemical, psychological, and behavioral factors, and can be caused by different reasons such as non-insulin dependent diabetes, increase in triglycerides, increase in carbohydrate bound energy and low energy expenditure. As such, it is a complex disorder that must be addressed on several fronts to achieve lasting positive clinical outcome. Since obesity is not to be considered as a single disorder but as a heterogeneous group of conditions with (potential) multiple causes, it is also characterized by elevated fasting plasma insulin and an exaggerated insulin response to oral glucose intake (Koltermann O.G., (1980) J. Clin. Invest 65: 1272-1284). A clear involvement of obesity in type 2 diabetes mellitus can be confirmed (Kopelman P.G., (2000) Nature 404: 635-643).
  • Diabetes is a very disabling disease, because medications do not control blood sugar levels well enough to prevent swinging between high and low blood sugar levels.
  • Patients with diabetes are at risk for major complications, including diabetic ketoacidosis, end-stage renal disease, diabetic retinopathy and amputation.
  • diabetic ketoacidosis a host of related conditions, such as metabolic syndrome, obesity, hypertension, heart disease, peripheral vascular disease, and infections, for which persons with diabetes are at substantially increased risk.
  • the treatment of these complications contributes to a considerable degree to the enormous cost which is imposed by diabetes on health care systems world wide.
  • the technical problem underlying the present invention was to provide for means and methods for modulating/effecting (pathological) metabolic conditions influencing body-weight regulation and/or energy homeostatic circuits.
  • the solution to said technical problem is achieved by providing the embodiments characterized in the claims.
  • the present invention relates to novel functions of proteins and nucleic acids encoding these in body-weight regulation, energy homeostasis, metabolism, obesity, and regeneration and/or development of pancreatic tissues. Further new compositions are provided that are useful in diagnosis, treatment, and prognosis of metabolic diseases and disorders as described.
  • the present invention discloses that crossveinless-2 (GadFly Accession Number CG15671 ), frizzled (GadFly Accession Number CG17697), or crossveinless (GadFly Accession Number CG 12410) homologous proteins (herein referred to as “proteins of the invention” or “a protein of the invention”) are regulating the energy homeostasis and fat metabolism, especially the metabolism and storage of triglycerides, and polynucleotides, which identify and encode the proteins disclosed in this invention.
  • the invention also relates to vectors, host cells, and recombinant methods for producing the polypeptides and polynucleotides of the invention.
  • the invention also relates to the use of these compounds and effectors/modulators thereof, e.g. antibodies, biologically active nucleic acids, such as antisense molecules, RNAi molecules or ribozymes, aptamers, peptides or low-molecular weight organic compounds recognizing said polynulceotides or polypeptides, in the diagnosis, study, prevention, and treatment of metabolic diseases or dysfunctions, including metabolic syndrome, obesity, and/or diabetes as well as related disorders such as eating disorder, cachexia, hypertension, coronary heart disease, hypercholesterolemia, dyslipidemia, osteoarthritis, gallstones, or liver fibrosis.
  • metabolic diseases or dysfunctions including metabolic syndrome, obesity, and/or diabetes as well as related disorders such as eating disorder, cachexia, hypertension, coronary heart disease, hypercholesterolemia, dyslipidemia, osteoarthritis, gallstones, or liver fibrosis.
  • this invention describes a secreted protein, twisted gastulation homolog 1 , which is specifically expressed in pancreatic tissues early in the development and is also found to regulate energy homeostasis. Therefore, twisted gastulation homolog 1 is involved in pancreas development, regeneration, and in the regulation of energy homeostasis.
  • the invention relates to the use of this gene, its homologues and encoded proteins and effectors/modulators thereof in the diagnosis, prevention and/or treatment of pancreatic dysfunctions, such as diabetes, and other related diseases such as obesity and/or metabolic syndrome. These compounds are especially useful in regeneration processes, such as regeneration of the pancreas cells or tissues, e.g.
  • cells having exocrinous functions such as acinar cells, centroacinar cells and/or ductal cells, and/or cells having endocrinous functions, particularly cells in Langerhans islets such as alpha-, beta-, delta- and/or PP-cells, more particularly beta-cells.
  • the compounds are especially useful in the modulation, e.g. stimulation of pancreatic development.
  • TSG twisted gastrulation protein
  • the tsg proteins display strong homology among themselves and they are composed of a putative signal peptide at the N-terminus followed by a cysteine rich (CR) region, a conserved domain devoid of cysteines, a variable midregion, and a C-terminal CR region (see, Graf et al., (2001 ) Mamm Genome 12: 554-560; Vilmos et al., (2001 ) Mol Pathol 54: 317-323; Scott et al., (2001 ) Nature 410: 475-478).
  • CR cysteine rich
  • Bone morphogenetic proteins are important regulators of early vertebrate and invertebrate dorsal-ventral development.
  • An evolutionarily conserved BMP regulatory mechanism operates from fly to fish, frog and mouse to control the dorsal-ventral axis determination.
  • Several secreted factors modulate the activity of BMPs.
  • twisted gastrulation is also involved in dorsal-ventral patterning, yet the extact mechanism of its function is unclear.
  • TSG binds both the vertebrate member of the TGF-beta family of secreted proteins, the Decapentaplegic orthologue BMP4, and the BMP antagonist, the vertebrate short gastrulation orthologue chordin, and that these interactions have multiple effects. It was suggested that TSG acts as a cofactor in chordin's antagonism of BMP signalling. BMP-2 and -4 are crucial for gastrulation and neural induction, and aberrant signaling by BMPs and other TGF-beta family members results in developmental defects.
  • TSG vertebrate TSG homologues
  • TSG can block BMP function in Xenopus embryonic explants, and it was suggested that TSG could function as a BMP inhibitor in Xenopus; furthermore, TSG may have additional functions during frog embryogenesis.
  • the BMP-like signaling is required to reinforce the development of most veins in the Drosophila wing.
  • the formation of the cross veins is especially sensitive to reductions in BMP-like signaling.
  • Conley et al., (2000, Development 127: 3947-3959) have shown that the formation of the definitive cross veins occurs in a process that requires localized BMP-like activity.
  • the authors suggest that the product of the crossveinless 2 gene is a novel member of the BMP-like signaling pathway required to potentiate signaling in the cross veins.
  • the Crossveinless 2 protein contains a putative signal or transmembrane sequence, and a partial Von Willebrand Factor D domain similar to those known to regulate the formation of intramolecular and intermolecular bonds. It also contains five cysteine-rich domains, similar to the cysteine-rich domains found in Chordin, Short Gastrulation and Procollagen that are known to bind BMP-like ligands.
  • Frizzled proteins belong to a family of cell surface membrane proteins. Molecular cloning of members of the frizzled family revealed hydropathy plots with seven putative, transmembrane-spanning regions, conserved in Frizzleds characterized in mice, humans, flies, and worms. Frizzled receptors are members of the superfamily of G-protein-coupled receptors that possess seven transmembrane-spanning domains (see, for review, Malbon et al., (2001 ) Biochem Biophys Res Commun 287: 589-593). It is well established that many medically significant biological processes are mediated by proteins participating in signal transduction pathways that involve G-proteins and/or second messengers, e.g. cAMP (Lefkowitz, (1991 ) Nature 351 : 353-354).
  • Frizzled-7 In Drosophila, the frizzled gene plays a critical role in the establishment of tissue polarity, but the function of the frizzled family of proteins in mammals is largely unknown (see for review, Adler, (2002) Dev Cell 2: 525-535).
  • Frizzled-7 As over-expression of Frizzled-7 leads to activation of the WNT-beta-catenin-TCF pathway, it was suggested that up-regulation of Frizzled-7 in human gastric cancer might play key roles in carcinogenesis through activation of the WNT-beta-catenin-TCF pathway (Kirikoshi et al., (2001 ) Int J Oncol 19: 1 1 1 -1 15).
  • Frizzled-1 couples via G-proteins (Go and Gq) to a special pathway, the canonical beta-catenin-Lef-Tcf pathway.
  • Frizzled-2 couples via G-proteins (Gq and Gt) to downstream effectors including calcium mobilization. It has been suggested that frizzled-7 signalling controls tissue separation during Xenopus gastrulation (see, Winklbauer et al., (2001 ) Nature 413: 856-860). Frizzled proteins are suggested to play roles in signaling processeces controlling aspects of early development. Recent evidence suggested that frizzled proteins are receptors for the Wnt family of secreted glycoproteins, which are involved in the regulation of diverse developmental processes.
  • Wntl Ob was recently identified as a potent inhibitor of adipogenesis that must be suppressed for preadipocytes to differentiate in vitro. It was suggested that inhibition of adipogenesis by Wnt10b is likely mediated by Wnt receptors, Frizzled 1 , 2, and/or 5, and co-receptors low density lipoprotein receptor-related proteins 5 and 6. These receptors, like Wntl Ob, are highly expressed in preadipocytes and stromal vascular cells.
  • frizzled proteins of the invention and homologous proteins are involved in the regulation of energy homeostasis and body-weight regulation and related disorders, and thus, no functions in metabolic diseases and dysfunctions and other diseases as listed above have been discussed.
  • Crossveinless 2, frizzled, and crossveinless homologous proteins and nucleic acid molecules coding therefore are obtainable from insect or vertebrate species, e.g. mammals or birds.
  • Particularly preferred are homologous nucleic acids, particularly nucleic acids encoding a human protein as described in Table 1 .
  • the invention particularly relates to nucleic acid molecules encoding polypeptides contributing to regulating the energy homeostasis and/or the metabolism of triglycerides, and in regeneration and/or developmental processes, wherein said nucleic acid molecule comprises (a) the nucleotide sequence encoding Drosophila crossveinless 2, frizzled, or crossveinless or human homologous nucleic acids, particularly nucleic acids encoding a human protein as described in
  • (f) a partial sequence of any of the nucleotide sequences of (a) to (e) having a length of 15-25 bases, preferably 25-35 bases, more preferably 35-50 bases and most preferably at least 50 bases.
  • the invention is based on the finding that crossveinless 2, frizzled, or crossveinless and/or homologous proteins and the polynucleotides encoding these, are involved in the regulation of triglyceride storage and therefore energy homeostasis.
  • the invention describes the use of these compositions for the diagnosis, study, prevention, or treatment of metabolic diseases or dysfunctions, including metabolic syndrome, obesity, and/or diabetes, as well as related disorders such as eating disorder, cachexia, hypertension, coronary heart disease, hypercholesterolemia, dyslipidemia, osteoarthritis, liver fibrosis, or gallstones.
  • the present invention relates to genes with novel functions in body-weight regulation, energy homeostasis, metabolism, and obesity, functional fragments of said genes, polypeptides encoded by said genes or functional fragments thereof, and modulators/effectors thereof, e.g. antibodies, biologically active nucleic acids, such as antisense molecules, RNAi molecules, or ribozymes, aptamers, peptides or low-molecular weight organic compounds recognizing said polynucleotides or polypeptides.
  • modulators/effectors thereof e.g. antibodies, biologically active nucleic acids, such as antisense molecules, RNAi molecules, or ribozymes, aptamers, peptides or low-molecular weight organic compounds recognizing said polynucleotides or polypeptides.
  • model organisms such as the fly Drosophila melanogaster
  • Identification of novel gene functions in model organisms can directly contribute to the elucidation of correlative pathways in mammals (humans) and of methods of modulating them.
  • a correlation between a pathology model (such as changes in triglyceride levels as indication for metabolic syndrome including obesity) and the modified expression of a fly gene can identify the association of the human ortholog with the particular human disease.
  • a forward genetic screen was performed in fly displaying a mutant phenotype due to misexpression of a known gene (see, St Johnston D., (2002) Nat Rev Genet 3: 176-188; Rorth P., (1996) Proc Natl Acad Sci U S A 93: 12418-12422).
  • this invention we have used a genetic screen to identify mutations that cause changes in the body weight, which are reflected by a significant change of triglyceride levels. Obese people mainly show a significant increase in the content of triglycerides. Triglycerides are the most efficient storage for energy in cells.
  • the content of triglycerides of a pool of flies with the same genotype was analyzed after prolonged feeding using a triglyceride assay.
  • Male flies homozygous or hemizygous for the integration of vectors for Drosophila EP-lines were analyzed in an assay measuring the triglyceride contents of these flies, illustrated in more detail in the Examples section.
  • the results of the triglyceride content analysis are shown in Figures 1 , 5, and 10.
  • Genomic DNA sequences were isolated that are localized to the EP vector integration. Using those isolated genomic sequences public databases like Berkeley Drosophila Genome Project (GadFly; see also FlyBase, (1999) Nucleic Acids Research 27: 85-88) were screened thereby identifying the integration sites of the vectors, and the corresponding gene, described in more detail in the Examples section. The molecular organization of the genes is shown in Figures 2, 6, and 1 1.
  • Drosophila genes and proteins encoded thereby with functions in the regulation of triglyceride metabolism were further analysed in publicly available sequence databases (see Examples for more detail) and mammalian homologs were identified. Additionally, in this invention, we used a screen for secreted factors expressed in developing mammalian (mouse) pancreas, as described in more detail in the Examples section (see Example 4). This screen identified twisted gastrulation homolog 1 as secreted factor expressed in developing mouse pancreas.
  • Fat cells surrounding the internal organs play an especially important role in the pathophysiology of obesity e.g. Gasteyger C and Tremblay A., (2002) J Endocrinol Invest. 25: 876-883.
  • adipocytes are mesodermally derived cells, it is likely that they originate from the mesenchymal cells located around the organ primordial during embryonic development.
  • pancreatic bud which was used as starting material in the secreted factor screen which lead to the identification of twisted gastrulation homolog 1 most likely contained both pancreatic precursor cells and precursors of visceral adipocytes (or cells controlling their differentiation).
  • the present invention relates to a secreted protein twisted Jerusalemtulation 2 (crossveinless homolog) with novel functions in the human metabolism, regeneration, and pancreatic developmental processes.
  • the present invention discloses specific genes and proteins encoded thereby and effectors/modulators thereof involved in the regulation of pancreatic function and metabolism, especially in pancreas diseases such as diabetes mellitus, e.g. insulin dependent diabetes mellitus and/or non insulin dependent diabetes mellitus, and/or metabolic syndrome, obesity, and/or related disorders such as coronary heart disease, eating disorder, cachexia, hypertension, hypercholesterolemia (dyslipidemia), liver fibrosis, and/or gallstones.
  • the present invention dislcoses specific genes and proteins encoded thereby and effectors/modulators thereof involved in the regeneration of pancreatic cells or tissues, e.g. cells having exocrinous functions such as acinar cells, centroacinar cells and/or ductal cells and/or cells having endocrinous functions, particularly cells in Langerhans islets such as alpha-, beta-, delta- and/or PP-cells, more particularly beta-cells.
  • mice carrying gene knockouts in the leptin pathway for example, ob (leptin) or db (leptin receptor) mice
  • mice developing typical symptoms of diabetes show hepatic lipid accumulation and frequently have increased plasma lipid levels (see Bruning J.C. et al, (1998) Mol. Cell. 2: 559-569).
  • Microarrays are analytical tools routinely used in bioanalysis.
  • a microarray has molecules distributed over, and stably associated with, the surface of a solid support.
  • the term "microarray” refers to an arrangement of a plurality of polynucleotides, polypeptides, antibodies, or other chemical compounds on a substrate.
  • Microarrays of polypeptides, polynucleotides, and/or antibodies have been developed and find use in a variety of applications, such as monitoring gene expression, drug discovery, gene sequencing, gene mapping, bacterial identification, and combinatorial chemistry.
  • One area in particular in which microarrays find use is in gene expression analysis (see Example 6).
  • array technology can be used to explore the expression of a single polymorphic gene or the expression profile of a large number of related or unrelated genes.
  • arrays are employed to detect the expression of a specific gene or its variants.
  • arrays provide a platform for identifying genes that are tissue specific, are affected by a substance being tested in a toxicology assay, are part of a signaling cascade, carry out housekeeping functions, or are specifically related to a particular genetic predisposition, condition, disease, or disorder.
  • Microarrays may be prepared, used, and analyzed using methods known in the art (see for example, Brennan T.M., (1995) U.S. Patent No. US5474796; Schena M. et al., (1996) Proc. Natl. Acad. Sci. USA 93: 10614-10619; Baldeschwieler et al., (1995) PCT application WO95251 16; Shalon T.D. and Brown P.O., (1995) PCT application WO9535505; Heller R.A. et al., (1997) Proc. Natl. Acad. Sci. USA 94: 2150-2155; Heller MJ. and Tu E., (1997) U.S. Patent No. US5605662).
  • Various types of microarrays are well known and thoroughly described in Schena M., ed. (1999); DNA Microarrays: A Practical Approach, Oxford University Press, London.
  • Oligonucleotides or longer fragments derived from any of the polynucleotides described herein may be used as elements on a microarray.
  • the microarray can be used in transcript imaging techniques, which monitor the relative expression levels of large numbers of genes simultaneously as described below.
  • the microarray may also be used to identify genetic variants, mutations, and polymorphisms. This information may be used to determine gene function, to understand the genetic basis of a disorder, to diagnose a disorder, to monitor progression/regression of disease as a function of gene expression, and to develop and monitor the activities of therapeutic agents in the treatment of disease. In particular, this information may be used to develop a pharmacogenomic profile of a patient in order to select the most appropriate and effective treatment regimen for that patient.
  • therapeutic agents which are highly effective and display the fewest side effects may be selected for a patient based on his/her pharmacogenomic profile.
  • crossveinless 2, frizzled homolog 7 (FZD7), frizzled homolog 2 (FZD2), and twisted gastrulation homolog 1 (TWSG1 ) show differential expression in human primary adipocytes.
  • FZD7, FZD2, and TWSG1 are strong candidates for the manufacture of a pharmaceutical composition and a medicament for the treatment of conditions related to human metabolism, such as obesity, diabetes, and/or metabolic syndrome.
  • the invention also encompasses polynucleotides that encode a protein of the invention or a homologous protein. Accordingly, any nucleic acid sequence, which encodes the amino acid sequence of a protein of the invention or a homologous protein, can be used to generate recombinant molecules that express a protein of the invention or a homologous protein.
  • the invention encompasses a nucleic acid encoding Drosophila crossveinless 2, frizzled, or crossveinless, or human crossveinless 2, frizzled, or crossveinless homologs, preferably a human homologous protein as described in Table 1 ; referred to herein as the proteins of the invention.
  • nucleotide sequences encoding the proteins may be produced.
  • the invention contemplates each and every possible variation of nucleotide sequence that can be made by selecting combinations based on possible codon choices.
  • polynucleotide sequences that are capable of hybridizing to the claimed nucleotide sequences, and in particular, those of the polynucleotides encoding crossveinless 2, frizzled, or crossveinless, or a homologous protein, preferably a human homologous protein as described in Table 1 , under various conditions of stringency.
  • Hybridization conditions are based on the melting temperature (Tm) of the nucleic acid binding complex or probe, as taught in Wahl G.M. et al., (1987) Methods Enzymol. 152: 399-407 and Kimmel A.R., (1987) Methods Enzymol. 152: 507-51 1 , and may be used at a defined stringency.
  • hybridization under stringent conditions means that after washing for 1 h with 1 x SSC and 0.1 % SDS at 50°C, preferably at 55°C, more preferably at 62°C and most preferably at 68°C, particularly for 1 h in 0.2 x SSC and 0.1 % SDS at 50°C, preferably at 55°C, more preferably at 62°C and most preferably at 68°C, a positive hybridization signal is observed.
  • Altered nucleic acid sequences encoding the proteins which are encompassed by the invention include deletions, insertions or substitutions of different nucleotides resulting in a polynucleotide that encodes the same or a functionally equivalent protein.
  • the encoded proteins may also contain deletions, insertions or substitutions of amino acid residues, which produce a silent change and result in functionally equivalent proteins. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the biological activity of the protein is retained.
  • the invention relates to peptide fragments of the proteins or derivatives thereof such as cyclic peptides, retro-inverso peptides or peptide mimetics having a length of at least 4, preferably at least 6 and up to 50 amino acids.
  • an 'allele' or 'allelic sequence' is an alternative form of the gene, which may result from at least one mutation in the nucleic acid sequence. Alleles may result in altered mRNAs or polypeptides whose structures or function may or may not be altered. Any given gene may have none, one or many allelic forms. Common mutational changes, which give rise to alleles, are generally ascribed to natural deletions, additions or substitutions of nucleotides. Each of these types of changes may occur alone or in combination with the others, one or more times in a given sequence.
  • nucleic acid sequences encoding a protein of the invention or a homologous protein may be extended utilizing a partial nucleotide sequence and employing various methods known in the art to detect upstream sequences such as promoters and regulatory elements.
  • one method which may be employed, 'restriction-site' PCR uses universal primers to retrieve unknown sequence adjacent to a known locus (Sarkar G. et al., (1993) PCR Methods Applic. 2: 318-322).
  • Inverse PCR may also be used to amplify or extend sequences using divergent primers based on a known region (Triglia T. et al., (1988) Nucleic Acids Res. 16: 8186).
  • Another method which may be used is capture PCR which involves PCR amplification of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA (Lagerstrom M. et al., (1991 ) PCR Methods Applic. 1 : 1 1 1 -1 19). Another method which may be used to retrieve unknown sequences is that of Parker J.D. et al., (1991 ) Nucleic Acids Res. 19: 3055-3060. Additionally, one may use PCR, nested primers, and PROMOTERFINDER libraries to walk in genomic DNA (Clontech, Palo Alto, Calif.). This process avoids the need to screen libraries and is useful in finding intron/exon junctions.
  • nucleotide sequences encoding the proteins or functional equivalents may be inserted into appropriate expression vectors, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence.
  • appropriate expression vectors i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence.
  • Methods which are well known to those skilled in the art, may be used to construct expression vectors containing sequences encoding the proteins and the appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described in Sambrook, J. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, NN. and Ausubel, F.M. et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y.
  • natural, modified or recombinant nucleic acid sequences encoding a protein of the invention or a homologous protein may be ligated to a heterologous sequence to encode a fusion protein.
  • Heterologous sequences are preferably located at the N-and/or C-terminus of the fusion protein.
  • a variety of expression vector/host systems may be utilized to contain and express sequences encoding the proteins or fusion proteins. These include, but are not limited to, micro-organisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus, adenovirus, adeno-associated virus, lentiverus, retrovirus); plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or PBR322 plasmids); or animal cell systems.
  • micro-organisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus, aden
  • polynucleotide sequences encoding a protein of the invention or a homologous protein in a sample can be detected by DNA-DNA or DNA-RNA hybridization or amplification using probes or portions or fragments of said polynucleotides.
  • Nucleic acid amplification based assays involve the use of oligonucleotides or oligomers based on the sequences specific for the gene to detect transformants containing DNA or RNA encoding the corresponding protein.
  • 'oligonucleotides' or 'oligomers' refer to a nucleic acid sequence of at least about 10 nucleotides and as many as about 60 nucleotides, preferably about 15 to 30 nucleotides, and more preferably about 20-25 nucleotides, which can be used as a probe or amplimer.
  • labels and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid and amino acid assays.
  • Means for producing labeled hybridization or PCR probes for detecting polynucleotide sequences include oligo-labeling, nick translation, end-labeling of RNA probes, PCR amplification using a labeled nucleotide, or enzymatic synthesis. These procedures may be conducted using a variety of commercially available kits (Pharmacia & Upjohn, (Kalamazoo, Mich.); Promega (Madison Wis.); and U.S. Biochemical Corp., (Cleveland, Ohio).
  • Suitable reporter molecules or labels which may be used for nucleic acid and protein assays, include radionuclides, enzymes, fluorescent, chemiluminescent or chromogenic agents as well as substrates, co-factors, inhibitors, magnetic particles, and the like.
  • Host cells transformed with nucleotide sequences encoding a protein of the invention may be cultured under conditions suitable for the expression and recovery of said protein from cell culture.
  • the protein produced by a recombinant cell may be secreted or contained intracellularly depending on the sequence and/or the vector used.
  • expression vectors containing polynucleotides, which encode the protein may be designed to contain signal sequences, which direct secretion of the protein through a prokaryotic or eukaryotic cell membrane.
  • Other recombinant constructions may be used to join sequences encoding the protein to nucleotide sequence encoding a polypeptide domain, which will facilitate purification of soluble proteins.
  • Such purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAG extension/affinity purification system (Immunex Corp., Seattle, Wash.)
  • metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals
  • protein A domains that allow purification on immobilized immunoglobulin
  • the domain utilized in the FLAG extension/affinity purification system Immunex Corp., Seattle, Wash.
  • cleavable linker sequences such as those specific for Factor XA or Enterokinase (Invitrogen, San Diego, Calif.) between the purification domain and the desired protein may be used to facilitate purification.
  • nucleic acids and proteins of the invention and effectors/modulators thereof are useful in diagnostic and therapeutic applications implicated, for example but not limited to, metabolic diseases or dysfunctions, including metabolic syndrome, obesity, and/or pancreatic diseases e.g. diabetes mellitus such as insulin dependent diabetes mellitus or non insulin dependent diabetes mellitus, as well as related disorders such as eating disorder, cachexia, hypertension, coronary heart disease, hypercholesterolemia, dyslipidemia, osteoarthritis, gallstones, or liver fibrosis.
  • metabolic diseases or dysfunctions including metabolic syndrome, obesity, and/or pancreatic diseases e.g. diabetes mellitus such as insulin dependent diabetes mellitus or non insulin dependent diabetes mellitus, as well as related disorders such as eating disorder, cachexia, hypertension, coronary heart disease, hypercholesterolemia, dyslipidemia, osteoarthritis, gallstones, or liver fibrosis.
  • pancreatic cells or tissue are e.g. cells having exocrinous functions such as acinar cells, cenracinar cells and/or ductal cells and/or cells having endocrine functions, particularly cells in Langerhans islets such as alpha-, beta-, delta-, and/or PP-cells.
  • diagnostic and therapeutic uses for the proteins and nucleic acids of the invention are, for example but not limited to, the following: (i) tissue regeneration in vitro and in vivo (regeneration for all these tissues and cell types composing these tissues and cell types derived from these tissues), (ii) small molecule drug target, (iii) antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) protein therapy, (v) diagnostic and/or prognostic marker, (vi) gene therapy (gene delivery/gene ablation), and (vii) research tools.
  • compositions of the present invention will have efficacy for treatment of patients suffering from, for example, pancreatic diseases (e.g. diabetes), obesity, and/or metabolic syndrome as described above.
  • DG177 nucleic acids and proteins and/or effectors/modulators thereof in a pharmaceutical composition to a subject in need thereof, particularly a human patient lead to an at least partial regeneration of, for example, pancreas cells.
  • the composition will then at least partially restore normal pancreatic function.
  • these cells are beta cells of the islets which then start producing insulin on their own.
  • the beta cells of a diabetic subject will grow back to approach the normal size and number present in a nondiabetic person. This effect upon the body reverses the condition of diabetes. As the subject's blood sugar level improves, the dosage administered may be reduced in strength.
  • further administration can be discontinued entirely and the subject continues to produce a normal amount of insulin without further treatment.
  • the subject is thereby not only treated but cured entirely of a diabetic condition.
  • beta cells or precursors thereof may be treated in vitro and implanted or reimplanted into a subject in need thereof.
  • other cells of the pancreas can be regenerated in vivo and/or in vitro to cure a certain condition.
  • compositions of the present invention will also have efficacy for treatment of patients with other pancreatic diseases such as acute and chronic pancreatitis, complications from pancreatitis, pancreatic insufficiency, pancreatic cancer including pancreatic endocrine tumors (such as insulinomas, gastrinomas, glucagonoma, carcinoid, multiple endocrine neoplasia (MEN) syndrome, dysplasia, systemic diseases affecting the pancreas such as cystic fibrosis, or hemochromatosis.
  • pancreatic diseases such as acute and chronic pancreatitis, complications from pancreatitis, pancreatic insufficiency, pancreatic cancer including pancreatic endocrine tumors (such as insulinomas, gastrinomas, glucagonoma, carcinoid, multiple endocrine neoplasia (MEN) syndrome, dysplasia, systemic diseases affecting the pancreas such as cystic fibrosis, or hemochromat
  • nucleic acids and proteins of the invention and modulators/effectors thereof are useful in diagnostic and therapeutic applications implicated in various applications as described below.
  • cDNAs encoding a protein of the invention and particularly their human homologues may be useful in gene therapy, and the proteins of the invention and particularly their human homologues may be useful when administered to a subject in need thereof.
  • the compositions of the present invention will have efficacy for treatment of patients suffering from, for example, but not limited to, in metabolic disorders as described above.
  • nucleic acids encoding a protein of the invention, a homologous protein, or a functional fragment thereof may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acids or the proteins are to be assessed. These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention may be used in therapeutic or diagnostic methods.
  • antibodies which are specific for a protein of the invention or a homologous protein, may be used directly as a modulator/effector, e.g. an antagonist or agonist or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissue which express the protein.
  • the antibodies may be generated using methods that are well known in the art. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric single chain, Fab fragments, and fragments produced by a Fab expression library.
  • Neutralising antibodies i.e., those which inhibit dimer formation are especially preferred for therapeutic use.
  • various hosts including goats, rabbits, rats, mice, humans, and others, may be immunized by injection with the protein or any fragment or oligopeptide thereof which has immunogenic properties.
  • various adjuvants may be used to increase immunological response. It is preferred that the peptides, fragments or oligopeptides used to induce antibodies to the protein have an amino acid sequence consisting of at least five amino acids, and more preferably at least 10 amino acids.
  • Monoclonal antibodies to the proteins may be prepared using any technique that provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique (Kohler G. and Milstein C. (1975) Nature 256: 495-497; Kozbor D. et al. (1985) J. Immunol. Methods 81 : 31 -42; Cote R.J. et al., (1983) Proc. Natl. Acad. Sci. 80: 2026-2030; Cole S.P. et al., (1984) Mol Cell Biochem. 62: 109-120).
  • Antibodies with related specificity, but of distinct idiotypic composition may be generated by chain shuffling from random combinatorial immunoglobulin libraries (Karig A.S. et al., (1991 ) Proc. Natl. Acad. Sci. 88: 1 1 120-1 1 123). Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening recombinant immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature (Orlandi R. et al., (1989) Proc. Natl. Acad. Sci. 86: 3833-3837; Winter G. and Milste ⁇ n C, (1991 ) Nature 349: 293-299).
  • Antibody fragments which contain specific binding sites for the proteins may also be generated.
  • fragments include, but are not limited to, the F(ab') 2 fragments which can be produced by Pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of F(ab') 2 fragments.
  • Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse W.D. et al., (1989) Science 246: 1275-1281 ).
  • immunoassays may be used for screening to identify antibodies having the desired specificity.
  • Numerous protocols for competitive binding and immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are well known in the art.
  • Such immunoassays typically involve the measurement of complex formation between the protein and its specific antibody.
  • a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reacive to two non-interfering protein epitopes are preferred, but a competitive binding assay may also be employed (Maddox D.E. et al., (1983) J. Exp. Med. 158: 121 1-1216).
  • the polynucleotides of the invention or fragments thereof or nucleic acid modulator/effector molecules such as aptamers, antisense molecules, RNAi molecules, or ribozymes may be used for therapeutic purposes.
  • nucleic acid modulator/effector molecules such as aptamers, antisense molecules, RNAi molecules, or ribozymes
  • aptamers i.e. nucleic acid molecules, which are capable of binding to a protein of the invention and modulating its activity, may be generated by a screening and selection procedure involving the use of combinatorial nucleic acid libraries.
  • antisense molecules may be used in situations in which it would be desirable to block the transcription of the mRNA.
  • cells may be transformed with sequences complementary to polynucleotides encoding a protein of the invention or a homologous protein.
  • antisense molecules may be used to modulate/effect protein activity or to achieve regulation of gene function.
  • sense or antisense oligomers or larger fragments can be designed from various locations along the coding or control regions of sequences encoding the proteins.
  • Expression vectors derived from retroviruses, adenovirus, herpes or vaccinia viruses or from various bacterial plasmids may be used for delivery of nucleotide sequences to the targeted organ, tissue or cell population. Methods, which are well known to those skilled in the art, can be used to construct recombinant vectors, which will express antisense molecules complementary to the polynucleotides of the genes encoding a protein of the invention or a homologous protein. These techniques are described both in Sambrook et al. (supra) and in Ausubel et al. (supra).
  • Genes encoding a protein of the invention or a homologous protein can be turned off by transforming a cell or tissue with expression vectors, which express high levels of polynucleotides that encode a protein of the invention or a homologous protein or a functional fragment thereof.
  • Such constructs may be used to introduce untranslatable sense or antisense sequences into a cell. Even in the absence of integration into the DNA, such vectors may continue to transcribe RNA molecules until they are disabled by endogenous nucleases. Transient expression may last for a month or more with a non-replicating vector and even longer if appropriate replication elements are part of the vector system.
  • modifications of gene expression can be obtained by designing antisense molecules, e.g.
  • DNA, RNA or PNA to the control regions of the genes encoding a protein of the invention or a homologous protein, i.e., the promoters, enhancers, and introns.
  • Oligonucleotides derived from the transcription initiation site e.g., between positions -10 and + 10 from the start site, are preferred.
  • inhibition can be achieved using "triple helix" base-pairing methodology. Triple helix pairing is useful because it cause inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors or regulatory molecules. Recent therapeutic advances using triplex DNA have been described in the literature (Gee, J. E. et al. (1994) In; Huber, B. E. and B. I. Carr, Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco, N.Y.).
  • the antisense molecules may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
  • Ribozymes enzymatic RNA molecules, may also be used to catalyze the specific cleavage of RNA.
  • the mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Examples, which may be used, include engineered hammerhead motif ribozyme molecules that can be specifically and efficiently catalyze endonucleolytic cleavage of sequences encoding a protein of the invention or a homologous protein.
  • Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences: GUA, GUU, and GUC.
  • RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for secondary structural features which may render the oligonucleotide inoperable.
  • the suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.
  • Nucleic acid effector/modulator molecules, e.g. antisense molecules and ribozymes of the invention may be prepared by any method known in the art for the synthesis of nucleic acid molecules. These include techniques for chemically synthesizing oligonucleotides such as solid phase phosphoramidite chemical synthesis.
  • RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences. Such DNA sequences may be incorporated into a variety of vectors with suitable RNA polymerase promoters such as T7 or SP6. Alternatively, these cDNA constructs that synthesize antisense RNA constitutively or inducibly can be introduced into cell lines, cells or tissues. RNA molecules may be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends of the molecule or modifications in the nucleobase, sugar and/or phosphate moieties, e.g.
  • vectors may be introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient. Delivery by transfection and by liposome injections may be achieved using methods, which are well known in the art. Any of the therapeutic methods described above may be applied to any suitable subject including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.
  • An additional embodiment of the invention relates to the administration of a pharmaceutical composition, in conjunction with a pharmaceutically acceptable carrier, for any of the therapeutic effects discussed above.
  • compositions may consist of the nucleic acids or proteins of the invention or homologous nucleic acids or proteins, antibodies to a protein of the invention or a homologous protein, mimetics, agonists, antagonists or inhibitors of a protein of the invention or homologous proteins or nucleic acids.
  • the compositions may be administered alone or in combination with at least one other agent, such as stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water.
  • the compositions may be administered to a patient alone or in combination with other agents, drugs or hormones.
  • compositions utilized in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual or rectal means.
  • these pharmaceutical compositions may contain suitable pharmaceutically-acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active compounds into preparations, which can be used pharmaceutically. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, Pa.).
  • compositions of the present invention may be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • the pharmaceutical composition may be provided as a salt and can be formed with many acids. After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition. For administration of proteins, such labeling would include amount, frequency, and method of administration.
  • compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose.
  • the determination of an effective dose is well within the capability of those skilled in the art.
  • the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of preadipocyte cell lines or in animal models, usually mice, rabbits, dogs or pigs.
  • the animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • a therapeutically effective dose refers to that amount of active ingredient, for example the nucleic acid or protein of the invention or a functional fragment thereof, or an antibody, which is sufficient for treating a specific condition.
  • Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population).
  • the dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50.
  • Pharmaceutical compositions, which exhibit large therapeutic indices, are preferred.
  • the data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use.
  • the dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage varies within this range depending upon the dosage from employed, sensitivity of the patient, and the route of administration.
  • Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors, which may be taken into account, include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy.
  • Long-acting pharmaceutical compositions may be administered every 3 to 4 days, every week or once every two weeks depending on half-life and clearance rate of the particular formulation. Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of administration.
  • compounds e.g. antibodies which specifically bind to the proteins may be used for the diagnosis of conditions or diseases characterized by or associated with over- or underexpression of a protein of the invention or a homologous protein or in assays to monitor patients being treated with a protein of the invention or a homologous protein, or modulators/effectors thereof, e.g. agonists, antagonists, or inhibitors.
  • Diagnostic assays include methods which utilize the antibody and a label to detect the protein in human body fluids. or extracts of cells or tissues.
  • the antibodies may be used with or without modification, and may be labeled by joining them, either covalently or non-covalently, with a reporter molecule.
  • a wide variety of reporter molecules which are known in the art may be used several of which are described above.
  • Compounds that bind DG177 proteins are useful for the identification or enrichment of cells, which are positive for the expression of the proteins of the invention, from complex cell mixtures.
  • Such cell populations are useful in transplantation, for experimental evaluation, and as source of lineage and cell specific products, including mRNA species useful in identifying genes specifically expressed in these cells, and as target for the identification of factors of molecules that can affect them.
  • Cells expressing the protein of the invention or which have been treated with the protein of the invention are useful in transplantation to provide a recipient with pancreatic islet cells, including insulin producing beta cells; for drug screening; experimental models of islet differentiation and interaction with other cell types; in vitro screening assays to define growth and differentiation factors, and to additionally characterize genes involved in islet development and regulation; and the like.
  • the native cells may be used for these purposes, or they may be genetically modified to provide altered capabilities.
  • Cells from a regenerating pancreas, from embryonic foregut, stomach and duodenum, or other sources of pancreatic progenitor cells may be used as a starting population.
  • the progenitor cells may be obtained from any mammalian species, e.g. equine, bovine, porcine, canine, feline, rodent, e.g. mice, rats, hamster, primate, etc. particularly human.
  • the cells are transfected with a DNA construct, e.g. a viral or non-viral vector containing a reporter gene, e.g. the lacZ gene or the GFP gene, under regulatory control of a promoter of a gene involved in for example beta-cell differentiation, e.g. a promoter of a gene stimulation beta-cell differentiation, preferably a Pax4 promoter.
  • a promoter of a gene involved in for example beta-cell differentiation e.g. a promoter of a gene stimulation beta-cell differentiation, preferably a Pax4 promoter.
  • the transfected cells are divided into aliquots and each aliquot is contacted with a test substance, e.g., candidate 1 , candidate 2 and candidate 3.
  • the activity of the reporter gene corresponds to the capability of the test compound to induce beta-cell differentiation.
  • a medium throughput validation is carried out.
  • the test compound is added to stem cells being cultivated and the insulin production is determined.
  • an initial high throughput assay such as the cell based assay outlined above where for example a Pax4 promoter is used as marker for beta-cell regeneration
  • the activity of candidate molecules to induce beta-cell differentiation is tested in a validation assay comprising adding said compounds to the culture media of the embryoid bodies. Differentiation into insulin-producing cells is then evaluated, e.g. by comparison to wild type and/or Pax4 expressing ES cells to assess the effectiveness of a compound.
  • a variety of protocols including ELISA, RIA, and FACS for measuring proteins are known in the art and provide a basis for diagnosing altered or abnormal levels of gene expression.
  • Normal or standard values for gene expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, preferably human, with antibodies to the protein under conditions suitable for complex formation. The amount of standard complex formation may be quantified by various methods, but preferably by photometry, means. Quantities of protein expressed in control and disease, samples from biopsied tissues are compared with the standard values. Deviation between standard and subject values establishes the parameters for diagnosing disease.
  • the polynucleotides specific for a protein of the invention or a homologous protein may be used for diagnostic purposes.
  • the polynucleotides, which may be used include oligonucleotide sequences, antisense RNA and DNA molecules, and PNAs.
  • the polynucleotides may be used to detect and quantitate gene expression in biopsied tissues in which gene expression may be correlated with disease.
  • the diagnostic assay may be used to distinguish between absence, presence, and excess gene expression, and to monitor regulation of protein levels during therapeutic intervention.
  • hybridization with probes which are capable of detecting polynucleotide sequences may be used to identify nucleic acid sequences which encode the respective protein.
  • the hybridization probes of the subject invention may be DNA or RNA and derived from the nucleotide sequence of the polynucleotide encoding the proteins of the invention or from a genomic sequence including promoter, enhancer elements, and introns of the naturally occurring gene.
  • Hybridization probes may be labeled by a variety of reporter groups, for example, radionuclides such as 32 P or 35 S or enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.
  • reporter groups for example, radionuclides such as 32 P or 35 S or enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.
  • Polynucleotide sequences specific for the proteins of the invention and homologous nucleic acids may be used for the diagnosis of conditions or diseases, which are associated with the expression of the proteins.
  • Examples of such conditions or diseases include, but are not limited to, metabolic diseases and disorders, including obesity and diabetes.
  • Polynucleotide sequences specific for a protein of the invention or a homologous protein may also be used to monitor the progress of patients receiving treatment for metabolic diseases and disorders, including obesity and diabetes.
  • the polynucleotide sequences may be used qualitative or quantitative assays, e.g. in Southern or Northern analysis, dot blot or other membrane-based technologies; in PCR technologies; or in dip stick, pin, ELISA or chip assays utilizing fluids or tissues from patient biopsies to detect altered gene expression. Such qualitative or quantitative methods are well known in the art.
  • nucleotide sequences specific for the proteins of the invention and homologous nucleic acids may be useful in assays that detect activation or induction of various metabolic diseases or dysfunctions, including metabolic syndrome, obesity, and/or diabetes as well as related disorders such as eating disorder, cachexia, hypertension, coronary heart disease, hypercholesterolemia, dyslipidemia, osteoarthritis, gallstones, or liver fibrosis.
  • the nucleotide sequences may be labeled by standard methods, and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantitated and compared with a standard value.
  • the amount of signal in the biopsied or extracted sample is significantly altered from that of a comparable have hybridized with nucleotide sequences in the sample, and the presence of altered levels of nucleotide sequences encoding a protein of the invention or a homologous protein in the sample indicates the presence of the associated disease.
  • Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials or in monitoring the treatment of an individual patient.
  • a normal or standard profile for expression is established. This may be accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with a sequence or a fragment thereof , which is specific for the nucleic acids encoding the proteins of the invention and homologous nucleic acids, under conditions suitable for hybridization or amplification. Standard hybridization may be quantified by comparing the values obtained from normal subjects with those from an experiment where a known amount of a substantially purified polynucleotide is used. Standard values obtained from normal samples may be compared with values obtained from samples from patients who are symptomatic for disease.
  • Deviation between standard and subject values is used to establish the presence of disease. Once disease is established and a treatment protocol is initiated, hybridization assays may be repeated on a regular basis to evaluate whether the level of expression in the patient begins to approximate that, which is observed in the normal patient. The results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months.
  • the presence of an unusual amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development of the disease or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms.
  • a more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the metabolic diseases and disorders.
  • oligonucleotides designed from the sequences encoding a protein of the invention or a homologous protein may involve the use of PCR.
  • Such oligomers may be chemically synthesized, generated enzymatically or produced from a recombinant source.
  • Oligomers will preferably consist of two nucleotide sequences, one with sense orientation (5'.fwdarw.3 # ) and another with antisense (3'.rarw.5'), employed under optimized conditions for identification of a specific gene or condition.
  • the same two oligomers, nested sets of oligomers or even a degenerate pool of oligomers may be employed under less stringent conditions for detection and/or quantification of closely related DNA or RNA sequences.
  • Methods which may also be used to quantitate the expression of a protein of the invention or a homologous protein include radiolabeling or biotinylating nucleotides, coamplification of a control nucleic acid, and standard curves onto which the experimental results are interpolated (Melby P.C. et al., (1993) J. Immunol. Methods, 159: 235-244; Duplaa C. et al., (1993) Anal. Biochem. 212: 229-236).
  • the speed of quantification of multiple samples may be accelerated by running the assay in an ELISA format where the oligomer of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantification.
  • the nucleic acid sequences may also be used to generate hybridization probes, which are useful for mapping the naturally occurring genomic sequence.
  • the sequences may be mapped to a particular chromosome or to a specific region of the chromosome using well known techniques.
  • Such techniques include FISH, FACS or artificial chromosome constructions, such as yeast artificial chromosomes, bacterial artificial chromosomes, bacterial P1 constructions or single chromosome cDNA libraries as reviewed in Price CM., (1993) Blood Rev. 7: 127-134, and Trask B.J., (1991 ) Trends Genet. 7: 149-154.
  • FISH is described in Verma R.S.
  • the nucleotide sequences of the subject invention may be used to detect differences in gene sequences between normal, carrier or affected individuals. For example, an analysis of polymorphisms, e.g. single nucleotide polymorphisms may be carried out. Further, in situ hybridization of chromosomal preparations and physical mapping techniques such as linkage analysis using established chromosomal markers may be used for extending genetic maps. Often the placement of a gene on the chromosome of another mammalian species, such as mouse, may reveal associated markers even if the number or arm of a particular human chromosome is not known. New sequences can be assigned to chromosomal arms or parts thereof, by physical mapping.
  • any sequences mapping to that area may represent associated or regulatory genes for further investigation.
  • the nucleotide sequences of the subject invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc. among normal, carrier or affected individuals.
  • the proteins of the invention can be used for screening libraries of compounds in any of a variety of drug screening techniques.
  • modulators/effectors e.g. receptors, ligands or substrates that bind to, modulate or mimic the action of one or more of the proteins of the invention.
  • the protein or fragment thereof employed in such screening may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. The formation of binding complexes, between the proteins of the invention and the agent tested, may be measured. Agents could also, either directly or indirectly, influence the activity of a protein of the invention.
  • crossveinless 2 frizzled, or crossveinless homologous proteins against their physiological substrate(s) or derivatives thereof could be measured in cell-based or cell-free assays.
  • Agents may also interfere with posttranslational modifications of a protein of the invention, such as phosphorylation and dephosphorylation, farnesylation, palmitoylation, acetylation, alkylation, ubiquitination, proteolytic processing, subcellular localization or degradation.
  • agents could influence the dimerization or oligomerization of a protein of the invention or, in a heterologous manner, of a protein of the invention with other proteins, for example, but not exclusively, docking proteins, enzymes, receptors, ion channels, uncoupling proteins, or translation factors. Agents could also act on the physical interaction of the proteins of this invention with other proteins, which are required for protein function, for example, but not exclusively, their downstream signaling.
  • binding of a fluorescently labeled peptide derived from a protein of the invention to the interacting protein could be detected by a change in polarisation.
  • binding partners which can be either the full length proteins as well as one binding partner as the full length protein and the other just represented as a peptide are fluorescently labeled
  • binding could be detected by fluorescence energy transfer (FRET) from one fluorophore to the other.
  • FRET fluorescence energy transfer
  • the two or more binding partners can be different proteins with one being the protein of the invention, or in case of dimerization and/or oligomerization the protein of the invention itself.
  • Proteins of the invention, for which one target mechanism of interest, but not the only one, would be such protein/protein interactions are crossveinless 2, frizzled, or crossveinless homologous proteins.
  • Assays for determining enzymatic, carrier, or ion channel activity of the proteins of the invention are well known in the art. Well known in the art are also a variety of assay formats to measure receptor-ligand binding.
  • GPCRs G protein-coupled receptors
  • secondary assays suitable for detection of agonist and antagonist activities on GPCRs measure levels of cAMP produced upon modulation of adenylate cyclase activity by GPCRs.
  • the assays are based on the competition between endogenous cAMP and exogenously added labeled cAMP. (e.g. AlphaScreen; PerkinElmer).
  • a number of response element-based reporter systems have been developed that enable the study of GPCR function. For example, a colorimetric assay for measuring activation of Gs- and Gq-coupled signaling pathways was described by Chen W. et al. (1995, Anal Biochem. 226: 349-354). The assay utilizes a beta-galactosidase (lacZ) gene fused to five copies of the cyclic AMP response element (CRE) to detect the activation of CRE-binding protein that results from an increase in intracellular cAMP or calcium. Alternatively, but comparable assay systems are well known in the art, e.g. luciferase reporter genes linked to CREs.
  • lacZ beta-galactosidase
  • CRE cyclic AMP response element
  • the calcium signalling could be the basis for a screening assay, in which calcium ion flux can be measured as an end point for GPCR stimulated signal transduction.
  • a screening assay in which calcium ion flux can be measured as an end point for GPCR stimulated signal transduction.
  • widely applicated is a fluorescence-based assay system for the measurement of intracellular calcium developed by Molecular Devices. This application is, for example, described in Chambers C. et al., (2003) Comb Chem High Throughput Screen. 6: 355-362.
  • a further assay system uses the secondary regulation of the receptor by binding to a secondary regulatory molecule, particularly on a cytoplasmic domain of the receptor (e.g.. Beta arrestin).
  • the GPCR activity can be monitored by detecting movement of Beta arrestin in a cell (e.g. Transfluor assay; Cellomics).
  • agent as used herein describes any molecule, e.g. protein or pharmaceutical, with the capability of altering or mimicking the physiological function of one or more of the proteins of the invention.
  • Candidate agents encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 50 and less than about 2,500 Daltons.
  • Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups.
  • the candidate agents often comprise carbocyclic or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
  • Candidate agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, nucleic acids and derivatives, structural analogs or combinations thereof.
  • Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides.
  • libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced.
  • natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries.
  • pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs.
  • the screening assay is a binding assay
  • one or more of the molecules may be joined to a label, where the label can directly or indirectly provide a detectable signal.
  • Another technique for drug screening provides for high throughput screening of compounds having suitable binding affinity to the protein of interest as described in published PCT application WO84/03564.
  • large numbers of different small test compounds e.g. aptamers, peptides, low-molecular weight compounds etc.
  • the test compounds are reacted with the proteins or fragments thereof, and washed. Bound proteins are then detected by methods well known in the art.
  • Purified proteins can also be coated directly onto plates for use in the aforementioned drug screening techniques. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.
  • the antibodies can be used to detect the presence of any peptide, which shares one or more antigenic determinants with crossveinless 2, frizzled, or crossveinless homologous protein.
  • the nucleic acids encoding a protein of the invention can be used to generate transgenic animals or site-specific gene modifications in cell lines. These transgenic non-human animals are useful in the study of the function and regulation of said protein in vivo.
  • Transgenic animals, particularly mammalian transgenic animals can serve as a model system for the investigation of many developmental and cellular processes common to humans.
  • a variety of non-human models of metabolic disorders can be used to test modulators/effectors of the proteins of the invention.
  • Misexpression for example, overexpression or lack of expression
  • a protein of the invention particular feeding conditions, and/or administration of biologically active compounds can create models of metablic disorders.
  • such assays use mouse models of insulin resistance and/or diabetes, such as mice carrying gene knockouts in the leptin pathway (for example, ob (leptin) or db (leptin receptor) mice).
  • leptin pathway for example, ob (leptin) or db (leptin receptor) mice.
  • Such mice develop typical symptoms of diabetes, show hepatic lipid accumulation and frequently have increased plasma lipid levels (see Bruning J.C. et al, (1998) Mol. Cell. 2: 559-569).
  • Susceptible wild type mice for example C57BI/6) show similiar symptoms if fed a high fat diet.
  • mice could be used to test whether administration of a candidate modulator/effector alters for example lipid accumulation in the liver, in plasma, or adipose tissues using standard assays well known in the art, such as FPLC, colorimetric assays, blood glucose level tests, insulin tolerance tests and others.
  • standard assays well known in the art, such as FPLC, colorimetric assays, blood glucose level tests, insulin tolerance tests and others.
  • Transgenic animals may be made through homologous recombination in non-human embryonic stem cells, where the normal locus of the gene encoding a protein of the invention is altered.
  • a nucleic acid construct encoding a protein of the invention is injected into oocytes and is randomly integrated into the genome.
  • Vectors for stable integration include plasmids, retroviruses and other animal viruses, yeast artificial chromosomes (YACs), and the like.
  • the modified cells or animal are useful in the study of the function and regulation of the proteins of the invention. For example, a series of small deletions and/or substitutions may be made in the gene that encodes a protein of the invention to determine the role of particular domains of the protein, functions in pancreatic differentiation, etc.
  • variants of the genes of the invention like specific constructs of interest include anti-sense molecules, which will block the expression of the proteins of the invention, or expression of dominant negative mutations.
  • a detectable marker such as for example lac-Z or luciferase may be introduced in the locus of a gene of the invention, where up regulation of expression of the genes of the invention will result in an easily detected change in phenotype.
  • genes of the invention or variants thereof in cells or tissues where it is not normally expressed or at abnormal times of development.
  • proteins of the invention in cells in which they are not normally produced, one can induce changes in cell behavior.
  • DNA constructs for homologous recombination will comprise at least portions of the genes of the invention with the desired genetic modification, and will include regions of homology to the target locus. DNA constructs for random integration do not need to contain regions of homology to mediate recombination. Conveniently, markers for positive and negative selection are included. DNA constructs for random integration will consist of the nucleic acids encoding the proteins of the invention, a regulatory element (promoter), an intron and a poly-adenylation signal. Methods for generating cells having targeted gene modifications through homologous recombination are known in the art. For non-human embryonic stem (ES) cells, an ES cell line may be employed, or embryonic cells may be obtained freshly from a host, e.g. mouse, rat, guinea pig, etc. Such cells are grown on an appropriate fibroblast-feeder layer and are grown in the presence of leukemia inhibiting factor (LIF).
  • LIF leukemia inhibiting factor
  • non-human ES or embryonic cells or somatic pluripotent stem cells When non-human ES or embryonic cells or somatic pluripotent stem cells have been transfected, they may be used to produce transgenic animals. After transfection, the cells are plated onto a feeder layer in an appropriate medium. Cells containing the construct may be selected by employing a selective medium. After sufficient time for colonies to grow, they are picked and analyzed for the occurrence of homologous recombination or integration of the construct. Those colonies that are positive may then be used for embryo transfection and morula aggregation. Briefly, morulae are obtained from 4 to 6 week old superovulated females, the Zona Pellucida is removed and the morulae are put into small depressions of a tissue culture dish.
  • the ES cells are trypsinized, and the modified cells are placed into the depression closely to the morulae.
  • the aggregates are transfered into the uterine horns of pseudopregnant females.
  • Females are then allowed to go to term.
  • Chimeric offsprings can be readily detected by a change in coat color and are subsequently screened for the transmission of the mutation into the next generation (F1 -generation).
  • Offspring of the F1 -generation are screened for the presence of the modified gene and males and females having the modification are mated to produce homozygous progeny. If the gene alterations cause lethality at some point in development, tissues or organs can be maintained as allogenic or congenic grafts or transplants, or in vitro culture.
  • the transgenic animals may be any non-human mammal, such as laboratory animal, domestic animals, etc., for example, mouse, rat, guinea pig, sheep, cow, pig, and others.
  • the transgenic animals may be used in functional studies, drug screening, and other applications and are useful in the study of the function and regulation of the proteins of the invention in vivo.
  • the invention also relates to a kit comprising at least one of
  • the kit may be used for diagnostic or therapeutic purposes or for screening applications as described above.
  • the kit may further contain user instructions.
  • Figure 1 shows the triglyceride content of Drosophila crossveinless 2 (GadFly Accession Number CG 15671 ) mutants. Shown is the change of triglyceride content HD-EP(2)21045 and HD-EP(2)26773 flies caused by homozygous viable integration of the P-vector into the annotated transcription unit (referred to as 'HD-EP21045' in column 2 and 'HD-EP26773' in column 3, respectively) in comparison to controls containing all flies of the EP collection (referred to as 'EP-control', column 1 ).
  • Figure 2 shows the molecular organization of the mutated crossveinless 2 gene locus.
  • Figure 3 shows the expression of the crossveinless 2 homolog in mammalian (mouse) tissues.
  • Figure 3A shows the real-time PCR analysis of hypothetical protein, Genbank Ace. No. AK014221 (crossveinless 2) expression in wild-type mouse tissues.
  • Figure 3B shows the real-time PCR analysis of crossveinless 2 expression in different mouse models.
  • Figure 3C shows the real-time PCR analysis of crossveinless 2 expression in mice fed with a high fat diet compared to mice fed with a standard diet.
  • Figure 3D shows the real-time PCR analysis of crossveinless 2 expression during the differentiation of 3T3-L1 cells from preadipocytes to mature adipocytes.
  • Figure 4 shows the expression of the crossveinless 2 homolog in mammalian (human) tissue. Shown is the quantitative analysis of crossveinless 2 (LOC168667) expression in human primary abdominal adipocyte cells (PAAT; columns 1 and 2) and in human primary adipose mammary gland cells (PAMG; columns 3 and 4), during the differentiation from preadipocytes to mature adipocytes.
  • PAAT human primary abdominal adipocyte cells
  • PAMG human primary adipose mammary gland cells
  • Figure 5 shows the triglyceride content of Drosophila frizzled (GadFly Accession Number CG17697) mutants. Shown is the change of triglyceride content of HD-EP(3)35082 and HD-EP(3)37405 flies caused by homozygous viable integration of the P-vector into the annotated transcription unit (referred to as 'HD-EP35082' in column 2 and 'HD-EP37405' in column 3, respectively) in comparison to controls containing all flies of the EP collection (referred to as 'EP-control', column 1 ).
  • Figure 6 shows the molecular organization of the mutated frizzled gene locus.
  • Figure 7 shows the multiple sequence alignment of Drosophila Frizzled-1 with human and mouse Frizzled-1 , Frizzled-2, and Frizzled-7 proteins.
  • 'Frizzled l Hs' refers to Genbank Accession Number NP 003496 (human Frizzled homolog 1);
  • 'Frizzled_1_Mm' refers to Genbank Accession Number XP 124462.1 (mouse Frizzled homolog 1 );
  • 'Frizzled 7_Hs' refers to Genbank Accession Number NP_003498.1 (human frizzled homolog 7);
  • 'Frizzled_7_Mm' refers to Genbank Accession Number XP 123681 .1 (mouse frizzled homolog 7);
  • 'Frizzled_2_Mm' refers to Genbank Accession Number NP_065256.1 (mouse frizzled homolog
  • Figure 8 shows the expression of two frizzled homologs in mammalian (mouse) tissues.
  • Figure 8A shows the real-time PCR analysis of frizzled homolog 7 (Fzd7) expression in wild-type mouse tissues.
  • Figure 8B shows the real-time PCR analysis of Fzd7 expression in different mouse models.
  • Figure 8C shows the real-time PCR analysis of Fzd7 expression in mice fed with a high fat diet compared to mice fed with a standard diet.
  • Figure 8D shows the real-time PCR analysis of Fzd7 expression during the differentiation of 3T3-L1 cells from preadipocytes to mature adipocytes.
  • Figure 8E shows the real-time PCR analysis of frizzled homolog 2 (Fzd2) expression in wild-type mouse tissues.
  • Figure 8F shows the real-time PCR analysis of Fzd2 expression in different mouse models.
  • Figure 8G shows the real-time PCR analysis of Fzd2 expression in mice fed with a high fat diet compared to mice fed with a standard diet.
  • Figure 8H shows the real-time PCR analysis of Fzd2 expression during the differentiation of 3T3-L1 cells from preadipocytes to mature adipocytes.
  • Figure 9 shows the expression of two frizzled homologs in mammalian (human) tissue.
  • Figure 9A shows the quantitative analysis of Frizzled homolog 7 (FZD7) expression in human primary abdominal adipocyte cells (PAAT; columns 1 and 2), in human primary adipose mammary gland cells (PAMG; columns 3 and 4), and in a human preadipocyte cell line (SGBS; columns 5 and 6), during the differentiation from preadipocytes to mature adipocytes.
  • Figure 9B shows the quantitative analysis of Frizzled homolog 2 (FZD2) expression in human primary abdominal adipocyte cells (PAAT; columns 1 and 2), in human primary adipose mammary gland cells (PAMG; columns 3 and 4), and in a human preadipocyte cell line (SGBS; columns 5 and 6), during the differentiation from preadipocytes to mature adipocytes.
  • FZD7 Frizzled homolog 7
  • Figure 9B shows the quantitative analysis of Frizzled homolog 2 (FZD2) expression in human primary abdominal adipocyte cells (PAAT; columns 1 and 2), in human primary adipos
  • Figure 10 shows the triglyceride content of Drosophila crossveinless (GadFly Accession Number CG12410) mutants. Shown is the change of triglyceride content of HD-EP(X) 10646 flies caused by integration of the P-vector into the annotated transcription unit (referred to as 'HD-EP10646' in column 2) in comparison to controls containing all flies of the EP collection (referred to as 'EP-control', column 1 ).
  • Figure 1 1 shows the molecular organization of the mutated crossveinless gene locus.
  • Figure 12 shows the expression of the crossveinless homolog in mammalian (mouse) tissues.
  • Figure 12A shows the real-time PCR analysis of twisted gastrulation homolog 1 (Drosophila) (TwsgD expression in wild-type mouse tissues.
  • Figure 12B shows the real-time PCR analysis of Twsgl expression in different mouse models.
  • Figure 12C shows the real-time PCR analysis of Twsgl expression in mice fed with a high fat diet compared to mice fed with a standard diet.
  • Figure 12D shows the real-time PCR analysis of Twsgl expression during the differentiation of 3T3-L1 cells from preadipocytes to mature adipocytes.
  • Figure 13 shows the expression of a crossveinless homolog in mammalian (human) tissue. Shown is the quantitative analysis of twisted gastrulation homolog 1 (Drosophila) (TWSG1 ) expression in human primary abdominal adipocyte cells (PAAT; columns 1 and 2), in human primary adipose mammary gland cells (PAMG; columns 3 and 4), and in a human preadipocyte cell line (SGBS; columns 5 and 6), during the differentiation from preadipocytes to mature adipocytes.
  • PAT twisted gastrulation homolog 1
  • PAMG human primary abdominal adipocyte cells
  • PAMG human primary adipose mammary gland cells
  • SGBS human preadipocyte cell line
  • Example 1 Measurement of triglyceride content in Drosophila
  • Mutant flies are obtained from a proprietary fly mutation stock collection. The flies are grown under standard conditions known to those skilled in the art. In the course of the experiment, additional feedings with bakers yeast (Saccharomyces cerevisiae) are provided. The average change of triglyceride content of Drosophila containing the EP-vectors in homozygous viable or hemizygous viable integration, was investigated in comparison to control flies (see Figures 1 , 5, and 10). For determination of triglyceride, flies were incubated for 5 min at 90°C in an aqueous buffer using a waterbath, followed by hot extraction.
  • bakers yeast Sacharomyces cerevisiae
  • the triglyceride content of the flies extract was determined using Sigma Triglyceride (INT 336-10 or -20) assay by measuring changes in the optical density according to the manufacturer's protocol.
  • the protein content of the same extract was measured using BIO-RAD DC Protein Assay according to the manufacturer's protocol. The assays were repeated several times.
  • the average triglyceride level of all flies of the EP collection (referred to as 'EP-control') is shown as 100% in the first columns in Figures 1 , 5, and 10. Standard deviations of the measurements are shown as thin bars.
  • HD-EP(2)21045 homozygous flies (column 2 in Figure 1 ), HD-EP(2)26773 homozygous flies (column 3 in Figure 1), and HD-EP(X) 10646 hemizygous flies (column 2 in Figure 10) show constantly a higher triglyceride content than the controls.
  • HD-EP(3)35082 homozygous flies (column 2 in Figure 5) and HD-EP(3)37405 homozygous flies (column 3 in Figure 5) show constantly a lower triglyceride content than the controls.
  • the loss of gene activity in the loci, where the EP-vectors are integrated, are responsible for changes in the metabolism of the energy storage triglycerides.
  • Example 2 Identification of Drosophila genes associated with triglyceride metabolism
  • Nucleic acids encoding the proteins of the present invention were identified using a plasmid-rescue technique. Genomic DNA sequences were isolated that are localized adjacent to the EP vector (herein HD-EP(2)21045, HD-EP(2)26773, HD-EP(3)35082, HD-EP(3)37405, and HD-EP(X) 10646) integration. Using those isolated genomic sequences public databases like Berkeley Drosophila Genome Project (GadFly) were screened, thereby identifying the integration sites of the vectors, and the corresponding genes. The molecular organization of these gene loci is shown in Figures 2, 6, and 1 1 .
  • genomic DNA sequence is represented by the assembly as a dotted black line in the middle that includes the integration sites of the vectors for lines HD-EP(2)21045, HD-EP(2)26773, or
  • HD-EP(X) 10646 Numbers represent the coordinates of the genomic DNA.
  • the upper parts of the figures represent the sense strand " + ", the lower parts represent the antisense strand "-”.
  • the insertion sites of the P-elements in the Drosophila lines are shown as triangles or boxes in the "P-elements + “ and/or "P-elements -” lines.
  • Transcribed DNA sequences ESTs
  • cDNAs are shown as bars in the "cDNA + " and/ or "cDNA -” lines.
  • Predicted exons of the cDNAs are shown as dark grey bars and predicted introns are shown as light grey bars (see also legend at the bottom of the figures).
  • the HD-EP(2)21045 and HD-EP(2)26773 vectors are homozygous viable integrated into EST clone RE66648 at base pair 3 in sense orientation and confirming the homozygous viable integration site of the HD-EP(2) 26773 vector 42 base pairs 5' of EST clone RE66648 in sense orientation.
  • RE66648 represents a transcribed sequence overlapping with the sequence of a Drosophila gene in sense orientation, identified as crossveinless 2 (GadFly Accession Number CG15671 ).
  • the chromosomal localization site of integration of the vectors EP(2)21045 and HD-EP(2)26773 is at gene locus 2R,57D12.
  • the HD-EP(X) 10646 vector is hemizygous viable integrated into EST clone SD27025 at base pair 100/101 in antisense orientation.
  • SD27025 represents a transcribed sequence overlapping with the sequence of a Drosophila gene in antisense orientation, identified as crossveinless (synonyms tsg2, TSG2, cv-1 , twisted gastrulation, GadFly Accession Number CG 12410).
  • the chromosomal localization site of integration of the vector of HD-EP(X) 10646 is at gene locus X, 5B10-C1. In Figure 1 1 , the coordinates of the genomic DNA start at position 5497500 on chromosome X, ending at position 5502500.
  • the insertion site of the P-element in Drosophila HD-EP10646 line is shown as a triangle in the "P-elements -" line and is labeled.
  • the predicted cDNA of the crossveinless gene shown in the "cDNA + " line is labeled, the corresponding ESTs are shown in the "EST + " and the "IPI + " lines.
  • the HD-EP(3)35082 vector is homozygous viable integrated 85 base pairs 5' antisense of CG17697-RA and 57 base pairs 5' antisense of CG17697-RB, a transcribed sequence overlapping with the sequence of a Drosophila gene in sense orientation, identified as frizzled (GadFly Accession Number CG17697).
  • a second homozygous viable integration site of the HD-EP(3)37405 vector 42 base pairs 5' sense of CG17697-RA and 14 base pairs 5' sense of CG17697-RB.
  • the chromosomal localization site of integration of the vectors of HD-EP(3)35082 and HD-EP(3)37405 is at gene locus 3L, 70D4-5.
  • genomic DNA sequence is represented by the assembly as a black scaled double-headed arrow in the lower half of the figure that includes the integration site of the vectors for lines HD-EP(3)35082 or HD-EP(3)37405.
  • Ticks represent the coordinates of the genomic DNA ( 10000 base pairs per tick).
  • the grey arrows in the upper part of the figures represent BAC clones, the black arrow in the topmost part of the figure represents the section of the chromosome.
  • the insertion sites of the P-elements in the Drosophila lines are shown as triangles and are labeled.
  • the cDNA sequence of the predicted gene (as predicted by the Berkeley Drosophila Genome Project, GadFly and by Magpie) are shown as dark grey bars (exons), linked by dark grey lines (introns), and are labeled (see also key at the bottom of the figures).
  • the insertion site of the P-element in Drosophila HD-EP(3)37405 line is shown as a triangle in the upper half of the figure, and the insertion site of the P-element in Drosophila HD-EP(3)35082 line is shown as a triangle in the lower half of the figure, both are labeled.
  • the predicted cDNAs of the frizzled gene shown in the middle of the figure are labeled ('fz').
  • Drosophila genes and proteins encoded thereby with functions in the regulation of triglyceride metabolism were further analysed using the BLAST algorithm searching in publicly available sequence databases and mammalian homologs were identified (see Table 1 and Figure 7).
  • polynucleotide comprising the nucleotide sequence as shown in GenBank Accession number relates to the expressible gene of the nucleotide sequences deposited under the corresponding GenBank Accession number.
  • GenBank Accession number relates to NCBI GenBank database entries (Ref.: Benson D.A. et al., (2000) Nucleic Acids Res. 28: 15-18). Sequences homologous to Drosphila crossveinless 2, frizzled, or crossveinless were identified using the publicly available program BLASTP 2.2.3 of the non-redundant protein data base of the National Center for Biotechnology Information (NCBI) (see, Altschul S.F. et al., (1997) Nucleic Acids Res. 25: 3389-3402).
  • crossveinless 2, frizzled, or crossveinless homologous proteins and nucleic acid molecules coding therefore are obtainable from insect or vertebrate species, e.g. mammals or birds.
  • Particularly preferred are nucleic acids as described in Table 1.
  • mouse homologous cDNAs encoding the polypeptides of the invention were identified as GenBank Acc. No. AK014221 (for the mouse homolog to crossveinless 2; Willebrand factor type C domain containing protein, Mm cv-2), XM_123681 (for the mouse homolog to Frizzled 7; Mm Fzd7), NM_020510 (for the mouse homolog to Frizzled 2; Mm Fzd2), NM_021457 (for the mouse homolog to Frizzled 1 ; Mm Fzd1 ), and NM_023053 (for the mouse homolog to Crossveinless; Mm TSG1 , twisted gastrulation protein).
  • Human twisted gastrulation homolog 1 is also referred in patent applications WO 99/38976, WO 98/46641 , and WO 98/46641 .
  • a screen for secreted factors expressed in developing mouse pancreas was carried out according to methods known by those skilled in the art (see, for example Pera E.M. and De Robertis E.M., (2000) Mech Dev 96(2): 183-195) with several modifications.
  • a mouse embryonic stage 9.5-15 pancreatic bud library was prepared in pCMVSPORT-6 vector using SUPERSCRIPT Plasmid System from Invitrogen according to the manufacturer's instructions.
  • the non-amplified library was electroporated into MaxEff DH10B cells (Invitrogen).
  • Bacterial clones were picked with sterile toothpicks from agar plates and cultured in 96-deep-well microtiter plates in LB-ampicillin (see Sambrook et al., supra). Aliquots of 8 cultures were pooled, and plasmid DNA was isolated using the BioRobot_9600 apparatus according to the manufacturer's instructions (Qiagen; QIAprep(r) Turbo BioRobot Kit. Human 293 cell culture cells were cultured in 75 ml tissue culture flasks in DMEM and 10% fetal calf serum. At 90-99% confluence, the cells were splitted at 1 :3 ratio and plated onto poly-D-lysine (Sigma) coated 96-well plates.
  • Cells were transfected with 100-500 ng plasmid using lipofectamine 2000 (Invitrogen). After 6 hours, the medium was exchanged for fresh complete growth medium. 24 hours after transfection, the cells were washed twice with DMEM without cysteine and methionine (Invitrogen), supplemented with 1 % dialysed Bovine serum (Sigma) with 50 ⁇ g per ml heparin (Sigma) and glutamine. The cells were labeled radioactively ('S35 Met-label', from Hartmann Analytic GmbH).
  • Example 5 Expression of the polypeptides in mammalian (mouse) tissues
  • mice strains C57BI/6J, C57BI/6 ob/ob and C57BI/KS db/db which are standard model systems in obesity and diabetes research
  • Harlan Winkelmann 33178 Borchen, Germany
  • constant temperature preferrably 22°C
  • 40 per cent humidity a light / dark cycle of preferrably 14 / 10 hours.
  • the mice were fed a standard chow (for example, from ssniff Spezialitaten GmbH, order number ssniff M-Z V1 126-000).
  • wild type mice For the fasting experiment (“fasted wild type mice”), wild type mice were starved for 48 h without food, but only water supplied ad libitum (see, for example, Schnetzler B. et al., 1993, J Clin Invest 92: 272-280, Mizuno T.M. et al., 1996, Proc Natl Acad Sci U S A 93: 3434-3438) .
  • wild-type (wt) mice were fed a control diet (preferably Altromin C1057 mod control, 4.5% crude fat) or high fat diet (preferably Altromin C1057mod. high fat, 23.5% crude fat). Animals were sacrificed at an age of 6 to 8 weeks. The animal tissues were isolated according to standard procedures known to those skilled in the art, snap frozen in liquid nitrogen and stored at -80°C until needed.
  • mammalian fibroblast (3T3-L1 ) cells e.g., Green H. and Kehinde O., 1974, Cell 1 : 1 13-1 16
  • 3T3-L1 cells were obtained from the American Tissue Culture Collection (ATCC, Hanassas, VA, USA; ATCC- CL 173).
  • 3T3-L1 cells were maintained as fibroblasts and differentiated into adipocytes as described in the prior art (e.g., Qiu Z. et al., 2001 , J. Biol. Chem. 276: 1 1988-1 1995; Slieker L.J.
  • d4 Four days after confluence (d4), cells were kept in SF medium, containing bovine insulin (5 //g/ml) until differentiation was completed. At various time points of the differentiation procedure, beginning with day 0 (day of confluence) and day 2 (hormone addition; for example, dexamethasone and 3-isobutyl-1 -methylxanthine), up to 10 days of differentiation, suitable aliquots of cells were taken every two days.
  • Trizol Reagent for example, from Invitrogen, Düsseldorf, Germany
  • RNeasy Kit for example, from Qiagen, Germany
  • Taqman analysis was performed preferably using the following primer/probe pairs:
  • mouse crossveinless 2 forward primer (SEQ ID NO: 1): 5'-AGC CCA TCG AGA ATG CCA-3'
  • mouse crossveinless 2 reverse primer (SEQ ID NO: 2): 5'-GAC CGG TAG AAA GTG GTA TAG TCC A-3'
  • mouse crossveinless 2 Taqman probe (SEQ ID NO: 3): (5/6-FAM)- AAC TCA AGT CCT GGG AGT TCC AGA CCT GC-(5/6-TAMRA).
  • mouse frizzled homolog 7 (Fzd7) sequence (GenBank Accession Number XM 123681 ): Mouse Fzd7 forward primer (SEQ ID NO: 4): 5'-GTC GCG AGT GCA GCG G-3'; mouse Fzd7 reverse primer (SEQ ID NO: 5): 5'-TCC CTG TAT CCA AGC CTC TCC-3'; mouse Fzd7 Taqman probe (SEQ ID NO: 6): (5/6-FAM)- CTC AGC TGG AGG AAA AAG ACG AGG CG- (5/6-TAMRA).
  • Fzd7 forward primer (SEQ ID NO: 4): 5'-GTC GCG AGT GCA GCG G-3'
  • mouse Fzd7 reverse primer (SEQ ID NO: 5): 5'-TCC CTG TAT CCA AGC CTC TCC-3'
  • mouse Fzd7 Taqman probe (SEQ ID NO: 6): (5/6-FAM)- CTC AGC TGG AGG AAA
  • Mouse Fzd2 forward primer (SEQ ID NO: 7): 5'-AAT AAA ATA GGA GTC CGT AGG GTG C-3'; mouse Fzd2 reverse primer (SEQ ID NO: 8): 5'-CCC TCT CCC AGG TCC GG-3'; mouse Fzd2 Taqman probe (SEQ ID NO: 9): (5/6-FAM)- CTG AGT GAA GGA GGG CAC GGT GC- (5/6-TAMRA).
  • mice twisted gastrulation homolog 1 (Drosophila) (Twsgl ) sequence (GenBank Accession Number NM_023053): Mouse Twsgl forward primer (SEQ ID NO: 10): 5'-GCT GCA CCA CCA AAA CGT G-3'; mouse Twsgl reverse primer (SEQ ID NO: 1 1): 5'- ACA TGC GCT CTT TGT CGC T -3'; mouse Twsgl Taqman probe (SEQ ID NO: 12): (5/6-FAM)- TTC CCA GCA ACA ATG TCC ACG CC-(5/6-TAMRA).
  • RNA-expression is shown on the Y-axis.
  • the tissues tested are given on the X-axis.
  • WAT white adipose tissue
  • BAT brown adipose tissue.
  • the X-axis represents the time axis.
  • dO refers to day 0 (start of the experiment)
  • d2 -
  • d10 refers to day 2 - day 10 of adipocyte differentiation.
  • the function of the proteins of the invention in metabolism was further validated by analyzing the expression of the transcripts in different tissues and by analyzing the role in adipocyte differentiation.
  • mice carrying gene knockouts in the leptin pathway (for example, ob/ob (leptin) or db/db (leptin receptor/ligand) mice) to study the expression of the proteins of the invention.
  • leptin pathway for example, ob/ob (leptin) or db/db (leptin receptor/ligand) mice
  • Such mice develop typical symptoms of diabetes, show hepatic lipid accumulation and frequently have increased plasma lipid levels (see Bruning J.C. et al, (1998) Mol. Cell. 2: 559-569).
  • mRNAs encoding the proteins of the invention were also examined in susceptible wild type mice (for example, C57BI/6) that show symptoms of diabetes, lipid accumulation, and high plasma lipid levels, if fed a high fat diet.
  • crossveinless 2 As regulator of energy metabolism in mammals. Taqman analysis revealed that crossveinless 2 is expressed in several mammalian tissues, showing highest level of expression in white adipose tissue (WAT) and higher levels in further tissues, e.g. brown adipose tissue (BAT), hypothalamus, and lung. Furthermore crossveinless 2 is expressed on lower but still robust levels in muscle, brain, testis, heart, and spleen of wild type mice as depicted in Figure 3A. We found, for example, that the expression of crossveinless 2 is strongly up regulated in liver of genetically induced obese mice (ob/ob) compared to wild type mice.
  • WAT white adipose tissue
  • BAT brown adipose tissue
  • ob/ob genetically induced obese mice
  • crossveinless 2 is up regulated in liver of fasted mice compared to wild type mice (see Figure 3B).
  • WAT wild type mice fed a high fat diet
  • BAT slightly upregulated in BAT
  • spleen down regulated in spleen
  • crossveinless 2 in metabolic active tissues of wild type mice, as well as the up regulation of crossveinless 2 in different animal models used to study metabolic disorders, suggests that this gene plays a central role in energy homeostasis. This hypothesis is supported by the regulation during the differentiation from preadipocytes to mature adipocytes.
  • Fzd7 frizzled homolog 7
  • Taqman analysis revealed highest level of Fzd7 expression in the muscle of wild type mice and on high levels in of Fzd7 in WAT. Furthermore Fzd7 is expressed on lower but still robust levels in BAT, brain, kidney and lung of wild type mice as depicted in Figure 8A.
  • Fzd7 is up regulated in the pancreas and down regulated in the bone marrow of genetically induced obese mice (ob/ob) compared to wild type mice, furthermore Fzd7 is down regulated in WAT, pancreas, and bone marrow of fasted mice compared to wild type mice (see Figure 8B).
  • WAT wild type mice fed a high fat diet
  • Fzd7 is up regulated in WAT as depicted in Figure 8C.
  • Figure 8D shows in this invention (see Figure 8D) that the Fzd7 mRNA is expressed and down regulated during the differentiation into mature adipocyctes. Therefore, the Fzd7 protein might play an essential role in adipogenesis.
  • Fdz2 frizzled homolog 2
  • Taqman analysis revealed that Fdz2 is expressed in several mammalian tissues, showing highest level of expression in lung and higher levels in further tissues, e.g. WAT, hypothalamus, brain, and colon.
  • Fdz2 is expressed on lower but still robust levels in BAT, muscle, testis, small intestine, heart, spleen, and kidney of wild type mice as depicted in Figure 8E.
  • BAT BAT of genetically induced obese mice (ob/ob) compared to wild type mice.
  • Fdz2 is down regulated in small intestine and bone marrow of fasted mice compared to wild type mice (see Figure 8F).
  • the expression of Fdz2 is significantly up regulated in muscle and down regulated in liver as depicted in Figure 8G.
  • Figure 8H we show in this invention (see Figure 8H) that the Fdz2 mRNA is expressed and regulated during the differentiation into mature adipocyctes. Therefore, the Fdz2 protein might play an essential role in adipogenesis.
  • Fdz2 is regulated in metabolic active tissues (e.g. BAT, muscle and liver) of different animal models used to study metabolic disorders, together with the regulation during the differentiation from preadipocytes to mature adipocytes, suggests that this gene plays a central role in energy homeostasis.
  • crossveinless as regulator of energy metabolism in mammals.
  • Taqman analysis revealed highest level of crossveinless expression in the lung of wild type mice.
  • crossveinless is expressed on high levels in WAT, heart, kidney, BAT, muscle, liver, hypothalamus, brain, testis, colon, small intestine, and spleen of wild type mice.
  • crossveinless is expressed on lower but still robust levels in pancreas and bone marrow of wild type mice as depicted in Figure 12A. We found, for example, that the expression of crossveinless is down regulated in the bone marrow of fasted mice, compared to wild type mice (see Figure 12B).
  • crossveinless In wild type mice fed a high fat diet, the expression of crossveinless is up regulated in muscle as depicted in Figure 12C.
  • Figure 12D We show in this invention (see Figure 12D) that the crossveinless mRNA is expressed and regulated during the differentiation into mature adipocyctes. Therefore, the crossveinless protein might play an essential role in adipogenesis.
  • Example 6 Analysis of the differential expression of transcripts of the proteins of the invention in human tissues
  • RNA preparation from human primary adipose tissues was done as described in Example 5.
  • the hybridization and scanning was performed as described in the manufactures manual (see Affymetrix Technical Manual, 2002, obtained from Affmetrix, Santa Clara, USA).
  • the X-axis represents the time axis, shown are day 0 and day 12 of adipocyte differentiation.
  • the Y-axis represents the flourescent intensity.
  • FZD2 twisted gastrulation homolog 1
  • TWSG1 twisted gastrulation homolog 1
  • TWSG1 transcripts are most abundant at day
  • the crossveinless 2 protein has to be significantly increased and the FZD7, FZD2, and TWSG1 proteins have to be significantly decreased in order for the preadipocyctes to differentiate into mature adipocycte.
  • crossveinless 2 in preadipocyctes has the potential to enhance, and the FZD7, FZD2, and TWSG1 proteins in preadipocyctes have the potential to inhibit adipose differentiation. Therefore, crossveinless 2, FZD7, FZD2, and TWSG1 proteins might play an essential role in the regulation of human metabolism, in particular in the regulation of adipogenesis and thus it might play an essential role in obesity, diabetes, and/or metabolic syndrome.

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Abstract

L'invention concerne de nouvelles utilisations de protéines de régulation de l'homéostasie énergétique et de polynucléotides codant ces protéines dans le diagnostic, l'étude, la prévention et le traitement de maladies et de troubles du métabolisme.
PCT/EP2003/009755 2002-09-02 2003-09-02 Proteines intervenant dans la regulation de l'homeostasie energetique WO2004020465A2 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005025590A3 (fr) * 2003-09-15 2005-11-03 Develogen Ag Utilisation d'un produit proteique dg280 dans la prevention et le traitement de troubles metaboliques
EP2289908A1 (fr) * 2003-07-11 2011-03-02 DeveloGen Aktiengesellschaft Utilisation de produits des protéines DG177 sécretées pour la prévention et le traitement de maladies du pancreas et/ou de l'obésité et/ou du syndrome métabolique

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CA2249251A1 (fr) * 1996-03-20 1997-09-25 Human Genome Sciences, Inc. Polypeptides cytokines humains
EP1251863A4 (fr) * 2000-01-31 2005-03-02 Human Genome Sciences Inc 22 proteines humaines secretees
WO2002053592A1 (fr) * 2000-12-28 2002-07-11 Shionogi & Co., Ltd. Nouveau polypeptide et son adn
US20040198651A1 (en) * 2001-04-20 2004-10-07 Klammer Aaron A. Secreted proteins

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2289908A1 (fr) * 2003-07-11 2011-03-02 DeveloGen Aktiengesellschaft Utilisation de produits des protéines DG177 sécretées pour la prévention et le traitement de maladies du pancreas et/ou de l'obésité et/ou du syndrome métabolique
WO2005025590A3 (fr) * 2003-09-15 2005-11-03 Develogen Ag Utilisation d'un produit proteique dg280 dans la prevention et le traitement de troubles metaboliques

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