WO2002002599A2 - Recepteur couple aux proteines g humaines igpcr17, et son utilisation - Google Patents

Recepteur couple aux proteines g humaines igpcr17, et son utilisation Download PDF

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WO2002002599A2
WO2002002599A2 PCT/EP2001/007532 EP0107532W WO0202599A2 WO 2002002599 A2 WO2002002599 A2 WO 2002002599A2 EP 0107532 W EP0107532 W EP 0107532W WO 0202599 A2 WO0202599 A2 WO 0202599A2
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igpcrl
protein
gene
activity
expression
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PCT/EP2001/007532
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WO2002002599A3 (fr
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Frank Wattler
Sigrid Wattler
Paul Trommler
Michael C. Nehls
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Ingenium Pharmaceuticals Ag
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Publication of WO2002002599A3 publication Critical patent/WO2002002599A3/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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70571—Receptors; Cell surface antigens; Cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor
    • 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

Definitions

  • the present invention relates to the field of cellular and molecular biology, protein biochemistry, and pharmacology.
  • the invention relates particularly to the identification of the polynucleotide sequence of a novel G protein-coupled receptor (GPcR) and the characterization of nucleic acids that encode this G protein-coupled receptor, which is referred to herein as IGPcRl 7.
  • the invention further relates to animal orthologs of the human gene encoding IGPcRl 7, to expression of both human and animal proteins, to the function of the gene product and to uses for the receptor, and its ligands in drug screening and in diagnosing, preventing and treating disease, particularly dysfunctions associated with signal processing in the central nervous system (CNS).
  • CNS central nervous system
  • Animal models of such diseases and dysfunctions, in which the IGPcRl 7 gene is mutated, knocked-out or present in the form of a transgene, are also incorporated within the invention.
  • proteins that participate in signal transduction pathways involving G proteins and second messengers e.g. cAMP, diacylglycerol and inositol phosphates (Lefkowitz, 1991, Nature, 351:353-354).
  • cAMP cyclositol phosphates
  • these proteins are referred to as proteins participating in pathways with G protein-coupled receptors, either as the receptors themselves, such as those for adrenergic agents and dopamine (Kobilka,
  • the receptor Upon hormone binding to a GPcR the receptor interacts with the heterotrimeric G protein and induces the dissociation of GDP from the guanine nucleotide-binding site.
  • GTP fills the site immediately. Binding of GTP to the alpha subunit of the G protein causes the dissociation of the G protein from the receptor and the dissociation of the G protein into alpha and beta-gamma subunits.
  • the GTP-carrying form then binds to the generator of an intracellular second messenger: in one common form of signal transduction, activated adenylate cyclase.
  • GTPase activity of the alpha subunit determines the time period during which the G protein is active.
  • the GDP-bound form of the alpha subunit (alpha.GDP) has high affinity for the beta-gamma subunit complex and subsequent re-association of G protein subunits alpha.GDP with beta-gamma returns the G protein to the basal state.
  • the G-protein serves a dual role: as an intermediate that relays the signal from receptor to effector (in this example adenylate cyclase), and as a timer that controls the duration of the signal.
  • Examples of members of the G protein-coupled receptor family gene family include acetylcholine, adenosine, adrenergic, bradykinin, cAMP, calcitonin, capsaicic, CCK, CGRP, CRF, cytomegalo virus, dopamine, endothelial differentiation gene-1, endothelin, FSH, galanin, histamine, kinin, motilin, muscarinic, neurokinin, neuropeptideY, neurotensin, nociceptin, odorant, opsin, rhodopsin, serotonin, somatostatin, thrombin, TSH and NIP receptors.
  • GPcR genes and gene products can cause medical disorders, dysfunctions, or diseases hereafter generally referred to as "diseases".
  • the mechanism of disease may be due to a loss of receptor function or by constitutive receptor activation (reviewed by Coughlin et al, 1994,
  • LHR luteinizing hormone receptor
  • G protein-coupled receptors exhibit seven transmembrane domains which are connected by three hydrophilic extracellular loops alternating with three intracellular loops. Most G protein-coupled receptors have single conserved cysteine residues in each of the first two extracellular loops which form disulfide bonds that are believed to stabilize functional protein structure.
  • the seven transmembrane domains or regions are designated as TM1, TM2, TM3, TM4, TM5, TM6 and TM7.
  • the cytoplasmic loop which connects TM5 and TM6 may be a mayor component of the G protein binding domain.
  • G protein-coupled receptors contain potential phosphorylation sites within the third cytoplasmic loop and/or the carboxyl terminus.
  • GPcRs such as the beta-adrenergic receptor
  • phosphorylation by protein kinase A and/or specific receptor kinases mediates receptor desensitization.
  • G protein-coupled receptors e.g. the calcitonin receptor-like receptor
  • RAMPs receptor-activity-modifying-proteins
  • This interaction of the GPcR with a certain RAMP determines which natural ligands have relevant affinity for the GPcR-RAMP combination and regulate the functional signaling activity of the complex (McLafhie LM, et al, 1998, Nature, 393:333-339).
  • the ligand binding sites of the G protein-coupled receptors are believed to comprise hydrophilic sockets formed by several GPcR transmembrane domains, said sockets being surrounded by hydrophobic residues of the G protein- coupled receptors.
  • each GPcR transmembrane helix is thought to face inward and form a polar ligand-binding site.
  • TM3 has been implicated in several G protein-coupled receptors as having a ligand-binding site, such as the TM3 aspartate residues.
  • TM5 serine residues, and TM6 asparagine and TM6 or TM7 phenylalanine or tyrosine residues are also implicated in ligand binding.
  • G-protein coupled receptors bind to a variety of ligands ranging from small biogens to peptides, small proteins and large glycoproteins (Strader CD, et al, 1994, Annu. Rev. Biochem., 63:101-132).
  • G protein-coupled receptors can be coupled intracellulularly by heterotrimeric G proteins to various intracellular enzymes, ion channels and transporters (see Johnson et al, 1989, Endoc. Rev., 10:317-331). Different G protein alpha-subunits preferentially stimulate particular effectors to modulate various biological functions in a cell. Phosphorylation of cytoplasmic residues of G protein-coupled receptors has been identified as an important mechanism for the regulation of G protein coupling of some G protein-coupled receptors. G protein-coupled receptors are found in numerous sites within animal, and particularly mammalian hosts.
  • a compound that blocks the farnesylation of ras as a tumor inhibitor a JAK-2 blocker as an inhibitor of recurrent pre-B cell acute lymphoblastic leukemia, and a platelet-derived growth factor receptor kinase as a blocker of restenosis (Reviewed in Levitzki A, 1996, Curr. Opin. Cell Biol, 8:239-244).
  • G protein-coupled receptors have been identified and successfully used as targets for several existing drugs; for example, dopamine and serotonin G protein-coupled receptors have been targeted for CNS diseases, angiotensin, muscarinic and adrenergic receptor G protein-coupled receptors have been targeted for cardiovascular diseases, histaminic G protein-coupled receptors have been targeted for respiratory diseases, the prostaglandin GPcR has been targeted for opthalmic purposes, and calcitonin and estrogen for treatment of arthritis.
  • the cerebral cortex of the brain is divided into four distinct sections, the lobes, which are associated with distinct functions: the frontal lobe includes the motor area and is also connected to thinking, planning and spoken language.
  • the parietal lobe includes the somatosensory area.
  • the temporal lobe is active in the processes involved in understanding language and it contains the auditory area.
  • the occipital lobe includes the area responsible for vision.
  • cerebral cortex processes or stores the innumerable sensory stimuli and perceptions that it receives and is able to mark them as memories that can subsequently be recalled from storage, but it is generally agreed that the large association areas of the cerebral cortex are the location of stored memories (reviewed by Duus P, 1998, “Topical Diagnosis in Neurology", 3 rd edition, Thieme).
  • Astrocytes constitute nearly half of the cells in human brain. They ensheath synapses throughout the CNS and are thought of as synaptic support cells, clearing ions and neurotransmitters from the synaptic cleft (see for example Bergles DE, and Jahr CE, 1998, J. Neurosci., 18:7709). Recently Ullian et al reported evidence from in vitro assays that exposure of CNS neurons to astrocytes is able to increase the number of mature, functional synapses on the CNS neurons by sevenfold in comparison to neurons remaining unexposed to astrocytes.
  • astrocytes are required for synaptic maintenance, since within a few days of removing astrocytes from neurons, the neurons begin to shed synapses (Ullian EM, et al, 2001, Science, 291 :657-661). In vivo, neurons send out dendrites and axons to the appropriate parts of the brain very early in development, but they do not form the majority of their synapses with other neurons until many days later - which is approximately the same time period at which astrocytes mature. Ullian et al. suggest that during normal embryonic development, initially formed synapses may be immature and highly plastic. The post-natal appearance of astrocytes increases the number of synapses and locks synaptic circuitry in place.
  • glial cells may also play important roles in adult neural plasticity underlying learning and memory, including short-term memory and long-term memory: a hypothesis supported by many researchers convinced that learning forges stronger connections between neurons.
  • the signal that astrocytes send to trigger synapse formation by neurons has not yet been identified.
  • G protein-coupled receptors Because of the vital role of G protein-coupled receptors in the communication between cells and their environment, such receptors are attractive targets for therapeutic intervention. G protein-coupled receptors have led to more than half of the currently known drugs (Drews, Nature Biotechnology, 1996, 14:1516). Mechanistically, approximately 50% to 60% of all clinically relevant drugs act by modulating the functions of various G protein-coupled receptors, as either agonist (activating activity) or antagonist (blocking activity) of a GPcR (Cudermann et al, 1995, J. Mol. Med., 73:51-63). This indicates that these receptors have an established, proven history as therapeutic targets.
  • the G protein-coupled receptor of the present invention is especially useful for diagnosis, prevention, amelioration or correction of diseases associated with signal processing in the central nervous system (CNS).
  • IGPcRl 7 satisfies a need in the art for identification and characterization of further receptors that can play an important role in diagnosis, prevention, amelioration or correction of psychiatric and central nervous system diseases and disorders, especially learning and memory dysfunctions, disorders or diseases, such as aphasia and Alzheimer's disease; movement dysfunctions, disorders or diseases, such as tics, tremor, Tourette's syndrome, Parkinson's disease, Huntington's disease, dyskinesias, dystonia, pain and spasms.
  • the GPcR of the present invention is especially useful for diagnosis, preventing, ameliorating or correcting of diseases associated with signal processing in the CNS, particularly diseases relating to memory processing and failure: i.e. learning and memory dysfunctions, including long term and short term memory disorders. Modulation of the activity of IGPcRl 7 may also be used for purposes of memory enhancement.
  • Embodiments of the invention include an isolated nucleic acid molecule, wherein said nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:l, or any unique fragment thereof, particularly wherein the nucleotide sequence of the fragment is greater than ten base pairs in length.
  • Embodiments also include an isolated polynucleotide which encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:2, or any unique fragment thereof, particularly wherein the amino acid sequence of the fragment is greater than ten amino acids in length.
  • Embodiments of the invention include any isolated nucleic acid molecule or polynucleotide comprising an allelic variant of a nucleotide sequence or polynucleotide which encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:2, wherein said allelic variant retains at least 70% nucleic acid homology, or in increasing preference at least 80%, 85%, 90%, 95% or 98% nucleic acid homology and hybridizes to the complement of SEQ ID NO:l under stringent conditions (Ausubel FM et al, eds., 1989, Current Protocols in Molecular Biology, Vol.
  • nucleic acid molecules or polynucleotides that comprise a nucleotide sequence which encodes at least one of the group of polypeptides, peptides and fusion proteins, comprising an amino acid sequence at least 70% similar, or in increasing preference at least 75%, 80%, 85%, 90%, 95% or 98% similar, to SEQ ID NO:2.
  • Vectors comprising an isolated nucleic acid molecule or polynucleotide of the invention as previously described are a further embodiment of the invention.
  • Additional embodiments include host cells genetically engineered to contain such a vector or genetically engineered to contain such a nucleic acid molecule or polynucleotide of the invention as described above, and particularly wherein the nucleic acid molecule or polynucleotide of the invention is operatively linked with a nucleotide regulatory sequence that controls expression of said nucleic acid molecule or polynucleotide in the host cell.
  • host cells which are drawn from prokaryotic bacterial cells, or from eukaryotic cells, particularly or yeast, insect or mammalian cells, preferred embodiments employing a mammalian host cell being those in which the host cell is a CHO, BHK, COS, CV1, 293, fibroblast or NERO cell.
  • Embryonic stem cells containing a disrupted endogenous IGPcRl 7 gene are also preferred embodiments of the invention, the most preferred embryonic stem cells being derived from mice.
  • Preferred embodiments of the invention include antibodies to the IGPcRl 7 protein, polypeptides, peptides, isolated domains and fusion proteins.
  • Agonists and antagonists of IGPcRl 7 are preferred embodiments of the invention, including: (a) 'small molecules' of molecular mass less than 6 kDa; (b) molecules of intermediate size, having molecular mass between 5 kDa to 15 kDa; and (c) large molecules of molecular mass greater than 12 kDa; the latter including mutant natural IGPcRl 7 ligand proteins that compete with native natural IGPcRl 7 ligand and which modulate IGPcRl 7 gene expression or gene product activity.
  • Preferred embodiments of the invention are those wherein such molecules bind specifically to the IGPcRl 7 receptor or to the IGPcRl 7 gene.
  • Further embodiments are methods of identifying such compounds which modulate the activity of the IGPcRl 7 receptor or of IGPcRl 7 gene expression, such as anti-sense and ribozyme molecules that can be used to inhibit IGPcRl 7 gene expression, or expression constructs that are capable of enhancing IGPcRl 7 gene expression.
  • non-human animal orthologs of the human sequence in SEQ ID NO: l are preferred embodiments of the invention, particularly ungulate and rodent sequences, and especially those of rat and mouse, and also polynucleotides comprising these sequences or homologous or partially homologous sequences as indicated for the human nucleic acid and polynucleotide.
  • Preferred embodiments include polynucleotides of such non-human animal orthologs comprising a nucleotide sequence which encodes a polypeptide comprising an amino acid sequence at least 70% similar, or in increasing preference at least 75%, 80%, 85%, 90%, 95% or 98% similar, to SEQ ID NO:2; or being at least ten amino acid residues in length and bearing the stated similarity to a unique part of SEQ ID NO:2.
  • Embodiments of the invention include knock-out animals which are non-human animals and which do not express IGPcRl 7. Preferred embodiments are those wherein the endogenous animal ortholog is functionally disrupted by homologous recombination methods such as conditional knock-out and/or null allele knock-out of the IGPcRl 7 gene. Mutated animals that express a non-functional or partially functional form of IGPcRl 7 are further embodiments of the invention.
  • Embodiments of the invention also include progeny of the non-human animals described as being embodiments of the invention, the term 'progeny' including both heterozygous and homozygous offspring.
  • non-human transgenic animal models expressing the human IGPcRl 7 cDNA sequence as shown in SEQ ID NO:l or a modification thereof as described above, operatively linked to a nucleotide regulatory sequence that controls expression of the nucleic acid molecule in the host animal.
  • Particularly preferred embodiments are those non-human animals
  • each embodiment of the invention comprising a non-human animal
  • preferable embodiments are those wherein the non-human animal is a mammal, particularly ungulate or rodent, and preferably wherein the non-human animal is from a genus selected from the group consisting of Mus (e.g., mice), Rattus (e.g., rats), Oryctologus (e.g., rabbits) and Mesocricetus (e.g., hamsters), mouse being the most preferable of this group.
  • Mus e.g., mice
  • Rattus e.g., rats
  • Oryctologus e.g., rabbits
  • Mesocricetus e.g., hamsters
  • Embodiments of the invention include primary cells and cell lines derived from any of the non-human animals of the invention, particularly the non-human transgenic animal models of the invention. Further embodiments include the amino acid sequence of those non-human animal orthologs of IGPcR17 that comprise an amino acid sequence at least 70% similar, or in increasing preference at least 75%, 80%, 85%o, 90%), 95%) or 98% similar, to the sequence of the mouse ortholog provided (SEQ ID NO:8); or a part of said non-human animal sequence which is at least ten amino acid residues in length and bears the stated similarity to a unique part of SEQ ID NO:8.
  • non-human animal or animal model of the invention for the dissection of the molecular mechanisms of the IGPcRl 7 pathway, for the identification and cloning of genes able to modify, reduce or inhibit the phenotype associated with IGPcRl 7 activity or deficiency, constitutes a further embodiment of the invention, as does the use of such non-human animal or animal model for the identification of gene and protein diagnostic markers for diseases, for the identification and testing of compounds useful in the prevention or treatment of symptoms associated with IGPcRl 7 activity or deficiency, in particular but not limited to central nervous system disorders, including neurologic, psychiatric and behavioral disorders, metabolic disorders, visual and olfactory disorders, and especially in the case of IGPcRl 7, visual diseases associated with signal processing in the CNS, particularly diseases relating to memory processing and failure: i.e. learning and memory dysfunctions, including long term and short term memory disorders.
  • Additional embodiments of the invention include methods of identifying compounds suitable for modulating the activity of the protein or polypeptide of the invention, as described above, for treatment of diseases characterized by aberrant expression or activity of IGPcRl 7.
  • Preferred embodiments include methods of prevention, amelioration or treatment of diseases characterized by aberrant expression or activity of IGPcRl 7, by the administration of compounds that bind specifically to the IGPcRl 7 gene or protein and/or which modulate IGPcRl 7 expression or IGPcRl 7 activity; the compounds that that bind specifically to the IGPcRl 7 gene or protein and/or which modulate IGPcRl 7 expression or IGPcRl 7 activity for the prevention, amelioration or treatment of diseases characterized by aberrant expression or activity of IGPcRl 7; and the use of compounds that that bind specifically to the IGPcRl 7 gene or protein and/or which modulate IGPcRl 7 expression or IGPcRl 7 activity for prevention, amelioration or treatment of diseases characterized by aberrant expression or activity of IGPc
  • Further preferred embodiments are gene therapy methods of prevention, amelioration or treatment of diseases characterized by aberrant expression or activity of IGPcRl 7, by the administration of vectors and/or host cells containing nucleotide sequences according to any of claims 1 to 7, that modulate IGPcRl 7 expression or IGPcRl 7 activity; the vectors and/or host cells containing nucleotide sequences according to any of claims 1 to 7 which modulate IGPcRl 7 expression or IGPcRl 7 activity for the prevention, amelioration or treatment of diseases characterized by aberrant expression or activity of IGPcRl 7; and the use of vectors and/or host cells containing nucleotide sequences according to any of claims 1 to 7 which modulate IGPcRl 7 expression or IGPcRl 7 activity for prevention, amelioration or treatment of diseases characterized by aberrant expression or activity of IGPcRl 7.
  • Figure 1 depicts the full-length coding DNA (cDNA) sequence of the human IGPcR17 gene (SEQ ID NO: l).
  • FIG. 2 depicts the amino acid sequence of the human IGPcR17 protein
  • Figure 3 depicts Northern blots detecting human IGPcRl 7 transcript in RNA from several human tissues.
  • FIG. 4a depicts an autoradiogram of a Multi Tissue Expression Array dot blot membrane (described in Fig. 4b) hybridized with a human IGPcRl 7 probe;
  • Fig. 4b depicts the diagram of a Multiple Tissue Expression Array membrane (Clontech Laboratories, Palo Alto, CA; cat no. 7775-1) indicating type and position of human poly A+ RNAs dotted onto a nylon membrane.
  • FIG. 5 depicts the full-length coding DNA (cDNA) sequence of the mouse IGPcR17 gene (SEQ ID NO:8).
  • FIG. 6 depicts the amino acid sequence of the mouse IGPcRl 7 protein
  • FIG. 7 depicts a comparison of the amino acid sequence of the human IGPcRl 7 to the amino acid sequence of the mouse ortholog protein of human IGPcR17;
  • Fig. 7b depicts a comparison of the amino acid sequence of the human IGPcR17 to the amino acid sequence of the human KIAAOOOl protein.
  • Figure 8 depicts hydropathy plots comparing the human IGPcRl 7 receptor, the human KIAAOOOl 7TM receptor and the mouse ortholog protein of the human IGPcRl 7 receptor.
  • Figure 9 depicts Northern blots detecting mouse IGPcRl 7 transcript in RNA from several mouse tissues.
  • FIG. 10 schematically outlines the construction of a mouse IGPcRl 7 targeting vector based on the method described by Wattler S & Nehls M,
  • the present invention relates to the discovery, identification and characterization of nucleic acids that encode IGPcRl 7, a novel G protein-coupled receptor protein that contains regions of homology to the human KIAAOOOl receptor.
  • the invention encompasses nucleotide sequences encoding mammalian forms of IGPcRl 7, including human IGPcRl 7, nucleotides that encode some or all of its functional domains, such as extracellular domains (ECDs), the transmembrane domains (TMs), and the cytoplasmic domains (CDs); mutants of the IGPcRl 7 sequences, and fusion proteins of IGPcRl 7.
  • ECDs extracellular domains
  • TMs transmembrane domains
  • CDs cytoplasmic domains
  • the invention also encompasses host cell expression systems expressing such nucleotides, the host cells and expression products.
  • the invention further encompasses IGPcRl 7 proteins, fusion proteins, antibodies to the receptor, antagonists and agonists of the receptor, transgenic animals that express an IGPcRl 7 transgene, recombinant knock-out animals that do not express the IGPcRl 7, and animal models in which the IGPcRl 7 gene is mutated.
  • the invention also encompasses compounds that modulate IGPcRl 7 gene expression or IGPcRl 7 receptor activity that can be used for drug screening, or for diagnosis, monitoring, preventing or treating visual dysfunctions associated with signal processing in the occipital lobe of the brain.
  • the invention further encompasses the use of IGPcRl 7 nucleotides, IGPcRl 7 proteins and peptides, as well as antibodies to IGPcRl 7, antagonists that inhibit ligand binding, receptor activity or expression, or agonists that increase ligand binding, activate receptor activity, or increase its expression, for the diagnosis and treatment of disorders, including, but not limited to treatment of central nervous system disorders.
  • IGPcRl 7 nucleotides and proteins are useful for the diagnosis of an IGPcRl 7 or pathway abnormality, and for the identification of compounds effective in the treatment of disorders based on the aberrant expression or activity of IGPcRl 7.
  • the invention also relates to host cells and animals genetically engineered to express the human IGPcRl 7 (or mutants thereof) or to inhibit or knock-out expression of the animal's endogenous IGPcRl 7 gene.
  • IGPcRl 7 as a new G protein-coupled receptor, can play a role in diagnosis, preventing, ameliorating and correcting diseases.
  • diseases include, but are not limited to, psychiatric and CNS disorders, including learning and memory dysfunctions, schizophrenia, episodic paroxysmal anxiety (EPA) disorders such as obsessive compulsive disorder (COD), post traumatic stress disorders (PTSD), phobia and panic, major depressive disorder, bipolar disorder, Parkinson's disease, general anxiety disorder, autism, delirium, multiple sclerosis, Alzheimer disease/dementia and other neurodegenerative diseases, severe mental retardation, dyskinesias, Huntington's disease, Gille de la Tourette's syndrome, tics, tremor, dystonia, spasms, anorexia, bulimia, stroke, addiction/dependency/craving, sleep disorders, epilepsy, migraine, attention deficit/hyperactivity disorder (ADHD), cardiovascular diseases, angina pectoris, including heart failure, angina pectoris, arryth
  • dyslipidemias obesity, emesis, gastrointestinal disorders, including irritable bowel syndrome (IBS), inflammatory bowel syndrome (IBD), diarrhoea, gastresophagal reflux disease (GERD), motility disorders and conditions of delayed gastric emptying, such as post operative or diabetic gastroparesis, and diabetis ulcers; other diseases including osteoporosis; inflammations; infections such as bacterial, fungal, protozoan and viral infections, particularly infections caused by HIV-1 or HIN-2; pain; cancers; chemotherapy induced injury; tumor invasion; immune disorders; autoimmune diseases; urinary retention; asthma, allergies; arthritis; benign prostatic hypertrophy; endotoxin shock; sepsis; complication of diabetis mellitus; and gynaecological disorders.
  • IBS irritable bowel syndrome
  • IBD inflammatory bowel syndrome
  • diarrhoea diarrhoea
  • gastresophagal reflux disease GFD
  • motility disorders motility disorders and
  • the new GPcR IGPcRl 7 satisfies a need in the art for identification and characterization of further receptors that can play an important role in diagnosis, preventing, ameliorating or correcting of diseases associated with signal processing in the C ⁇ S, particularly diseases relating to memory processing and failure: i.e. learning and memory dysfunctions, including long term and short term memory disorders.
  • Modulation of the activity of IGPcRl 7 may also be used for purposes of memory enhancement.
  • the signal that astrocytes send to trigger synapse formation by neurons has not yet been identified, it is proposed that a G protein- coupled receptor system recognizing the astrocyte trigger would be capable of generating and accounting for the observed complexity and plasticity of this process.
  • the characteristics of the G protein-coupled receptor IGPcRl 7 disclosed herein are appropriate for performing such a function.
  • IGPcRl 7 nucleotides, sequence or coding sequences - encompass DNA, including genomic DNA (e.g. the IGPcRl 7 gene), cDNA, RNA and include nucleotide sequences encoding IGPcRl 7 protein, peptide fragments, or fusion proteins.
  • IGPcRl 7 - means natural, or mature, IGPcRl 7 receptor protein. Polypeptides or peptide fragments of IGPcRl 7 protein are referred to as IGPcRl 7 polypeptides or IGPcRl 7 peptides. Fusions of IGPcRl 7, or IGPcRl 7 polypeptides or peptide fragments to an unrelated protein are referred to herein as IGPcRl 7 fusion proteins.
  • ECD - means "extracellular domain" of the receptor protein;
  • TM - means
  • transmembrane domain and CD - means "cytoplasmic domain”.
  • a functional IGPcRl 7 refers to a protein which binds natural IGPcRl 7 ligand with high affinity and specificity in vivo or in vitro.
  • Ligand - a molecule that selectively binds to a receptor.
  • Receptor - a plasma membrane protein which binds one or more appropriate ligands and propagates their regulatory signals to target cells, either by direct intracellular effects, or by promoting the synthesis and/or release of another regulatory molecule known as a second messenger.
  • Agonist - a molecule, being a ligand and/or drug, that acts on one or more physiological receptors and mimics the effects of the endogenous regulatory compounds; generally these are compounds that activate the receptor.
  • Antagonist - a molecule being a ligand and/or drug that inhibits a receptor, most acting by inhibiting the action of an agonist, for example by competing for agonist binding sites on a receptor. These are generally themselves devoid of intrinsic regulatory activity, but act to block receptor activation.
  • Transgenic animal a non-human animal containing one or more additional, often foreign genes or "transgenes”, integrated into its genome, that can be used as model systems to determine the phenotypic effects of expressing those genes.
  • Knock-out or knock-out animal a non-human animal wherein a transgene is inserted into the genome to create a partial or complete loss-of-function mutation of an endogenous gene.
  • Endogenous genes are inactivated usually by homologous recombination, using replacement or insertion-type gene targeting vectors.
  • Novel GPcR genes may be isolated using expression cloning, by synthesizing specific oligonucleotides based on the sequence of purified proteins, using low stringency hybridization (Ausubel FM et al, eds., 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc., and John Wiley & sons, Inc., New York) and by degenerate PCR using known receptor sequences.
  • GPcR genes may also be identified by large scale sequencing, as in the Human Genome Project, followed by analysis of expressed sequence tags (ESTs), or complete sequences present in databases. Known GPcR sequences or conserved regions thereof may be employed as query sequences to extract novel GPcR sequences from these databases.
  • ESTs expressed sequence tags
  • the present invention provides IGPcRl 7, a novel G protein-coupled receptor protein described for the first time herein, and characterized as having seven hydrophobic domains which span the plasma membrane and which are connected by alternating extracellular and intracellular hydrophilic loops.
  • the IGPcRl 7 sequences encode a protein of 342 amino acids. At the amino acid level IGPcRl 7 has 47% identity and 66% sequence homology with conserved substitutions to human KIAAOOOl sequences encoding a 338 amino acid protein (Genpept accession number NP055694, PID g7661848; Nomura et al, 1994, DNA
  • KIAAOOOl is related to proteinase-activated receptors 1, 2 and 3, and to platelet activating factor receptor. It is widely expressed, with expression being highest in placenta, adipose tissue, stomach and intestine, moderate in brain, spleen, lung and heart, and low in kidney (Chambers et al, 2000, J. Biol. Chem., 275:10767-10771). Recently, ligands to this orphan receptor were found: UDP-glucose and the closely related UDP-galactose,
  • KIAAOOOl UDP-glucuronic acid and UDP-N-acetylglucoseamine, but not uridine or uridine nucleotides were found to activate recombinant KIAAOOOl receptors.
  • KIAAOOOl was also shown to be coupled to G proteins of the Gi/o class (Chambers et al, 2000, J. Biol. Chem., 275:10767-10771).
  • the KIAAOOOl gene is localized within the chromosome 3q region linked to Usher syndrome type III, representing progressive hearing loss and retinal degradation. However, no mutations were detected in the coding region among affected patients, although they might have been present in the gene regulatory regions (Joensuu et al, 2000, Genomics, 63:409-416).
  • the mouse ortholog gene to the human IGPcRl 7 gene was identified by a search of public databases EMBL and Genbank using BLAST (Basic Local Alignment Search Tool, National Institutes of Health, Bethesda MD, U.S.A.) using the full-length coding sequence of the human gene as a query sequence. The highest score was assigned to an EMBL entry, EST/MM1179620: Soares mouse 3NbMs Mus musculus cDNA clone IMAGE 764455; length 542 bp. The EST open reading frame was used in primer design.
  • mouse IGPcRl 7 full-length cDNA SEQ ID NO:6
  • flanking genomic sequences were isolated and sequenced.
  • the predicted mouse IGPcRl 7 amino acid sequence SEQ ID NO: 7 revealed that the amino acid identity of human IGPcRl 7 to mouse IGPcRl 7 is 89 %.
  • the invention encompasses sequences coding for IGPcRl 7 polypeptides, or functional domains of the IGPcRl 7, mutated, truncated or deleted IGPcRl 7, and IGPcRl 7 fusion proteins.
  • the invention also encompasses nucleotide constructs that inhibit expression of the IGPcRl 7 gene, such as anti-sense and ribozyme constructs, or enhance expression of IGPcRl 7 in combination with regulatory sequences such as promoters and enhancers.
  • the cDNA sequence (SEQ ID NO:l) and deduced amino acid sequence (SEQ ID NO:2) of human IGPcRl 7 of this invention are shown in Fig. 1 and Fig. 2.
  • the IGPcRl 7 nucleotide sequences of the invention include the DNA sequence shown in
  • Fig. 1 nucleotide sequences that encode the amino acid sequence shown in Fig. 2 and any nucleotide sequence that hybridizes to the complement of the DNA sequence shown in Fig. 1 under highly stringent conditions (Ausubel FM et al, eds., 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc., and John Wiley & sons, Inc., New York).
  • Functional equivalents of the IGPcRl 7 gene product include naturally occurring IGPcRl 7, mutant and degenerate variants present in humans and other species.
  • Preferred IGPcRl 7 nucleic acids encode polypeptides that are at least 55% identical or similar to the amino acid sequence shown in Fig. 2. Nucleic acids which encode polypeptides which are at least 70%>, and even more preferably, in increasing order of preference, at least 80%, 85%, 90%,
  • the nucleic acid of the present invention encodes a polypeptide having an overall amino acid sequence homology or identity of, in increasing order of preference, at least 70%), 80%, 85%, 90%, 95%, 98%, or at least 99% with the amino acid sequence shown in Fig. 2.
  • the invention also provides DNA molecules that are the complements of the nucleotide sequences described above and which may act as IGPCR17 anti-sense molecules useful in IGPcRl 7 gene regulation.
  • Orthologs of the human IGPCR17 gene present in other species can be identified and readily isolated. They can be useful for developing cell and animal model systems for purposes of drug discovery.
  • cDNA or genomic DNA libraries derived from the organism of interest can be screened by hybridization using the nucleotides described above, or by performing PCR using degenerate oligonucleotide primers. (See Sambrook et al, 1989, "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Press, New
  • IGPcRl 7 gene sequences may additionally be used to isolate mutant IGPcRl 7 gene alleles, or to detect defects in the regulatory sequences of the IGPcRl 7 using DNA obtained from an individual suspected of or known to carry the mutant IGPcRl 7 allele. Mutant alleles may be isolated from individuals either known or proposed to have a genotype which contributes to the symptoms of disorders arising from the aberrant expression or activity of the IGPcRl 7 protein.
  • sequences derived from the human gene can be used to design primers for use in PCR assays to detect mutations for diagnostics.
  • the nucleotides of this invention are also preferred for use in mapping the location of the gene to the chromosome, in a process termed chromosomal mapping.
  • chromosomal mapping Various techniques known to those skilled in the art, including but not limited to in situ hybridization of labeled probes to flow-sorted chromosomes, fluorescence in situ hybridization (FISH) and PCR mapping of somatic cell hybrids may be employed. This allows the physical location of gene regions to be associated with genetic diseases, based on a genetic map. Genetic linkage analysis can then be used to identify the relationship between genes and diseases (see Egeland et al, 1987, Nature, 325:783-787). Preferred uses of this map include diagnostic tests and reagents, in pharmacogenetics studies and in monitoring patient responses to drugs in clinical trials.
  • Fig. 2 shows the amino acid sequence of the human IGPcRl 7 protein.
  • the amino acid sequence of IGPcRl 7 contains hydrophilic domains located between the transmembrane domains, arranging an alternating location of the hydrophilic domains inside and outside the cell membrane.
  • Polypeptides which are at least 70%, and even more preferably at least 80%, 85%, 90%, 95%, 98% or 99% identical or similar to the amino acid sequence represented by Fig. 2 are encompassed by this invention.
  • the invention encompasses IGPcRl 7 polypeptides, or functional domains of the IGPcRl 7, mutated, truncated or deleted IGPcRl 7, and host cell expression systems that can produce such IGPcRl 7 products.
  • IGPcRl 7 proteins, polypeptides and peptides can be prepared for the generation of antibodies, as reagents in diagnostic assays, in the identification of other cellular gene products involved in regulating IGPcRl 7, as reagents for screening for compounds that can be used in the treatment of conditions involving IGPcRl 7, and as pharmaceutical reagents useful in the treatment of related disorders.
  • the invention also encompasses proteins that are functionally equivalent to the
  • IGPcRl 7 encoded by the nucleotide sequences, as defined by the ability to bind natural IGPcRl 7 ligand, the resulting biological effect of natural IGPcRl 7 ligand binding, e.g., signal transduction, a change in cellular metabolism or change in phenotype.
  • Such functionally equivalent IGPcRl 7 proteins include but are not limited to additions or substitutions of amino acid residues, which result in a silent change.
  • mutant IGPcRl 7 proteins with increased function, and/or greater signaling capacity; or decreased function, and/or decreased signal transduction capacity which may be generated by random mutagenesis techniques and site-directed mutagenesis techniques well known to those skilled in the art.
  • the same strategy can also be used to design mutant forms of IGPcRl 7 based on the alignment of human IGPcRl 7 and IGPcRl 7 orthologs from other species. Highly preferred are other mutations to the IGPcRl 7 coding sequence that can be made to generate IGPcRl 7 constructs that are better suited for expression, scale up, etc. in the host cells chosen. Host cells may be chosen depending on their varying capacity to modify synthesized proteins.
  • IGPcRl 7 Peptides corresponding to one or more domains of the IGPcRl 7 (e.g., ECD, TM or CD), truncated or deleted IGPcRl 7s, as well as fusion proteins are also within the scope of the invention and can be designed on the basis of the IGPcRl 7 nucleotide and IGPcRl 7 amino acid sequences disclosed above.
  • IGPcRl 7 polypeptides, peptides and fusion proteins can be produced using techniques well known in the art for expressing protein encoding IGPcRl 7 sequences. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.
  • IGPcRl 7 nucleotide sequences of the invention may be utilized to express the IGPcRl 7 nucleotide sequences of the invention.
  • the IGPcRl 7 peptide or polypeptide may be anchored in the cell membrane and purified or enriched from such expression systems using appropriate detergents and lipid micelles, and methods well known to those skilled in the art. Or, where the IGPcRl 7 peptide or polypeptide is secreted by the cells, it may be isolated from the culture media.
  • host cells themselves may be used to assess biological activity, e.g., in drug screening assays.
  • the expression systems that may be used for purposes of the invention include, but are not limited to microorganisms such as bacteria (e.g., E. coli, B. subtilis); yeast (e.g., Saccharomyces sp., Pichia sp.); insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus); plant cell systems infected with recombinant viral or plasmid expression vectors; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3) harboring recombinant expression constructs containing mammalian promoters. Lower amounts of functional protein are expressible in E. coli and yeast, particularly as E.
  • microorganisms such as bacteria (e.g., E. coli, B. subtilis); yeast (e.g., Saccharomyces sp., Pichia sp.); insect cell systems infected with recombinant virus expression vectors (e
  • G proteins may be added to E.coli expressing G protein-coupled receptors in cell membrane, in the cell-based assays.
  • Yeast cells may be humanized by co- transfixing human G proteins. The yeast Pichia pas tor is is preferred over
  • Saccharomyces cerevisiae for purification of G protein-coupled receptors for structural studies.
  • the most preferred systems for expression are the baculovirus/insect cell and mammalian cell systems, as they can produce the largest quantities of G protein-coupled receptors in functional form for analysis.
  • Mammalian cells are preferred because they express the necessary G proteins, and vaccinia and Semliki Forest virus are preferred as vectors. (See Tate et al, 1996,
  • the invention encompasses antibodies directed against IGPcRl 7 proteins or peptides, or IGPcRl 7 fusion proteins, as described above.
  • Such antibodies include but are not limited to polyclonal antibodies, monoclonal antibodies (mAbs), humanized or chimeric antibodies, single chain antibodies, anti-idiotypic (anti-Id) antibodies, including Fab fragments.
  • the antibodies may be generated and purified, or conjugated according to methods well known in the art. See for example Harlow E and Lane D, 1988, “Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, which is incorporated herein by reference in its entirety.
  • the antibodies of the invention may be used, for example, as part of a diagnostic or a prognostic, and as a part of compound screening schemes, for the evaluation of the effect of test compounds on expression and/or activity of the IGPcRl 7 gene product.
  • antibodies may be used in therapeutic regimes as a method for the inhibition of abnormal IGPcRl 7 activity.
  • the antibodies may be employed as part of an enzyme immunoassay (EIA), a radioimmunoassay, or as an antibody labeled with a chemiluminescent or a fluorescent compound.
  • EIA enzyme immunoassay
  • the IGPcRl 7 proteins or peptides, IGPcRl 7 fusion proteins, IGPcRl 7 nucleotide sequences, antibodies, antagonists and agonists can be useful for the detection of mutant forms of IGPcRl 7 or inappropriately expressed forms of IGPcRl 7, for the diagnosis of disorders including but not limited to central nervous system disorders, neurologic, psychiatric and behavioral disorders, metabolic disorders, visual and olfactory disorders, immune, neuroimmune, neuroendocrine and inflammatory disorders and diseases.
  • DNA encoding IGPcRl 7 or parts thereof may be used in hybridization or amplification assays of biological samples to detect abnormalities involving IGPcRl 7 gene structure, including point mutations, insertions, deletions and chromosomal rearrangements.
  • genotyping assays may include, but are not limited to Southern analyses, single stranded conformational polymorphism analyses (SSCP), and PCR analyses (See Mullis KB, U.S. Pat. No. 4,683,202), the use of restriction fragment length polymorphisms (RFLPs), of variable numbers of short, tandemly repeated DNA sequences between the restriction enzyme sites (see Weber, U.S. Pat.
  • IGPcRl 7 proteins or peptides are also within the scope of the invention.
  • IGPcRl 7 fusion proteins are also within the scope of the invention. These can be used for screening for drugs effective in the treatment of disorders.
  • the use of engineered host cells and/or animals may offer an advantage in that both compounds that bind to the ECD of the IGPcRl 7 and compounds that affect the signal transduced by the activated IGPcRl 7 may be identified.
  • the invention encompasses the pharmacological testing wherein the cloned IGPcRl 7 genes are expressed in yeast, insect or mammalian cells and screened for a response to cognate or surrogate agonists.
  • the agonists may be present in, but are not limited to, biological extracts, peptide libraries and/or complex compound collections.
  • the invention provides for screening which may utilize libraries of known compounds, including natural products or synthetic chemicals, and biologically active materials, including proteins, for compounds which are inhibitors or activators.
  • Candidate test compounds include all kinds of combinatorial chemistry derived molecular libraries of amino acids, peptides, soluble peptides, modified peptides, antibodies, small organic and inorganic molecules.
  • a labeled test compound can be incubated with the receptor to determine whether one binds to the other.
  • Functional assays including fibroblast and BM transformation assays, cell cycle analysis can be performed; as well as responses using signal transduction assays, including protein phosphorylation, guanylate cyclase activity, ion fluxes (e.g. calcium) and pH changes can be measured.
  • High throughput drug screening systems are most preferred and may use assays including, but not limited to, the production of intracellular second messengers, such as cAMP, diacylglycerol and inositol phosphates; the activation of reporter gene transcription, such as luciferase and beta-galactosidase under for example the cAMP-responsive element; receptor-mediated actions on adenylyl cyclase and phospholipase C leading also for example to dispersion or aggregation of frog melanophores. (Reviewed in Tate et al, 1996, Tibtech 14:426-430; included in entirety herein).
  • a functional genomics approach for protein- protein interaction screening may be employed wherein the GPcR is produced in "humanized yeast cells": expression in yeast along with endogenous or promiscuous mammalian or human G-alpha proteins.
  • Transient expression of cDNA can also be carried out using mammalian CHO, HEK-293 cells or COS-7 cells and receptors can be analyzed for ligand binding and drug interactions (for example as described in Fraser et al, 1995, J. Nucl. Med., 36:17S-21S).
  • site-directed mutagenesis to define regions of IGPcRl 7 that have functional importance.
  • Site- directed mutagenesis may be used to map ligand-binding pockets and to identify residues important for receptor interaction and activation.
  • Compounds that can be generated using modeling methods to bind these residues are also within the scope of this invention.
  • receptor down-regulation and the development of drug tolerance such as seen in asthma patients who use bronchial dilators which are beta- adrenergic agonists leading to tachyphylaxis, can be studied in these cell systems.
  • the expression of both intact and hybrid receptors is preferred.
  • the number of receptors, as well as mRNA levels can be measured.
  • Agents for radionuclide imaging to monitor level changes can be developed.
  • the invention encompasses antagonists and agonists of IGPcRl 7, as well as compounds or nucleotide constructs that inhibit expression of the IGPcRl 7 gene (anti-sense and ribozyme molecules), or promote expression of IGPcRl 7 (wherein IGPcRl 7 coding sequences are operatively associated with promoters, enhancers, etc.).
  • IGPcRl 7 protein products especially soluble derivatives of IGPcRl 7, or truncated polypeptides lacking the TM or CD domains
  • fusion protein products antibodies and anti-idiotypic antibodies, antagonists or agonists (including compounds that modulate signal transduction which may act on downstream targets in the IGPcRl 7 signal transduction pathway) that can be used for therapy of such diseases, by inhibiting receptor activity.
  • Nucleotide constructs encoding functional forms of IGPcRl 7 and mutant forms of IGPcRl 7 are preferred embodiments of the invention, as their uses include employment in the genetic engineering of host cells.
  • Other preferred embodiments of the invention are anti-sense and ribozyme molecules, preferred for use in "gene therapy” approaches in the treatment of disorders or diseases arising from the aberrant or altered activity of IGPcRl 7.
  • the gene therapy vector alone or when incorporated into recombinant cells may be administered in a suitable formulation for intravenous, intra-muscular, intra-peritoneal delivery, or may be incorporated into a timed release delivery matrix.
  • the animal-based and cell-based models can be used to identify drugs, biologicals, therapies and interventions which can be effective in treating disorders with aberrant expression or activity.
  • IGPcRl 7 sequences can be introduced into, and over- expressed and/or can be disrupted in order to under-express or inactivate IGPcRl 7 gene expression.
  • the IGPcRl gene products can also be expressed in transgenic animals.
  • Non-human animals of any species including, but not limited to, mice, rats, rabbits, guinea pigs, sheep, cows, goats, may be used to generate IGPcRl 7 transgenic animals.
  • the present invention provides for transgenic animals that carry the IGPcRl 7 transgene in all their cells, as well as animals which carry the transgene in some, but not all their cells, i.e., mosaic animals.
  • the transgene may be expressed in all tissues of the animal, or may be limited to specific tissues. Any technique known in the art may be used to introduce the IGPcRl 7 transgene into animals to produce the founder lines of transgenic animals.
  • Such techniques include, but are not limited to pronuclear microinjection (Hoppe PC and Wagner TE, U.S. Patent No. 4,873,191); retrovirus mediated gene transfer into germ lines (Van der Putten et al, 1985, Proc. Natl. Acad. Sci., USA 82:6148-6152); gene targeting in embryonic stem cells (Thompson et al, 1989, Cell 56:313-321); electroporation of embryos (Lo, 1983, Mol. Cell. Biol., 3:1803-1814); and sperm- mediated gene transfer (Lavitrano et al, 1989, Cell 57:717-723); etc.
  • pronuclear microinjection Hoppe PC and Wagner TE, U.S. Patent No. 4,873,191
  • retrovirus mediated gene transfer into germ lines Van der Putten et al, 1985, Proc. Natl. Acad. Sci., USA 82:6148-6152
  • gene targeting in embryonic stem cells Tho
  • the present invention relates to knock-out animals engineered by homologous recombination to be deficient in the production of the IGPcRl 7.
  • the present invention is directed to a knock-out animal having a phenotype characterized by the substantial absence of IGPcRl 7, otherwise naturally occurring in the animal.
  • the invention encompasses the DNA constructs and embryonic stem cells used to develop the knock-out animals and assays which utilize either the animals or tissues derived from the animals.
  • these cells, tissues and cell lines are characterized by the substantial absence of IGPcRl 7 that would otherwise be naturally occurring in their normal counterparts.
  • Gene targeting is a procedure in which foreign DNA sequences are introduced into a specific locus within the genome of a host cell.
  • endogenous IGPcRl 7 gene expression can be reduced by inactivating or knocking out the IGPcRl 7 gene or its promoter using targeted homologous recombination, (e.g., see Smithies et al, 1985, Nature 317:230-234; Thomas & Capecchi, 1987, Cell 51 :503-512; Thompson et al, 1989, Cell 5:313-321 ; each of which is incorporated by reference herein in its entirety).
  • targeted homologous recombination e.g., see Smithies et al, 1985, Nature 317:230-234; Thomas & Capecchi, 1987, Cell 51 :503-512; Thompson et al, 1989, Cell 5:313-321 ; each of which is incorporated by reference herein in its entirety).
  • a mutant, non-functional IGPcRl 7 flanked by DNA homologous to the endogenous IGPcRl 7 gene (either the coding regions or regulatory regions of the IGPcRl 7 gene) can be used, with or without a selectable marker and/or a negative selectable marker, to transfect cells that express IGPcRl 7 in vivo. Insertion of the DNA construct, via targeted homologous recombination into the genome, results in abolishing IGPcRl 7 gene function.
  • One preferred technique for targeted mutagenesis in this invention is based on homologous recombination.
  • the general methodologies of targeting mutations into the genome of cells, and the process of generating mouse lines from genetically altered embryonic stem (ES) cells with specific genetic lesions are well known (Bradley, 1991, Cur. Opin. Biotech. 2: 823-829). See also U.S. patents 5,557,032 by
  • Preferred in this invention is a synthetic recombination vector which contains the genetic information of the targeted chromosomal locus recombines with the genomic DNA after introduction into a cell.
  • a strategy of "positive/negative selection” can be used to enrich the cell population for cells in which targeting vectors have integrated into the host cell genome, and recombination has occurred at the desired gene locus (Mansour, et al, 1988, Nature 336:348).
  • the vector usually contains a positive selection cassette which is flanked by the genetic information of the target locus to enrich for cells where the vector successfully recombines with the chromosomal DNA against the pool of non-recombinant cells.
  • the likelihood of obtaining an homologous recombination event increases with the size of the chromosomal vector DNA and is further dependent on the isogenicity between the genomic DNA of the vector and the target cell (See te Reile et al, 1992, P.N.A.S. USA 89:5128-5132; Deng et al, 1991, Mol. Cell. Biol., 12, 3365-3371).
  • Also preferred in this invention are large stretches of genomic DNA flanking the
  • IGPcRl 7 gene ortholog in the target animal species The cloning of large chromosomal fragments of the target gene, the sub-cloning of this DNA into a bacterial plasmid vector, the mapping of the gene structure, the integration of the positive selection cassette into the vector and finally, the flanking of one or both homologous vector arms by a negative selection marker are well described in the literature. Also preferred are replacement-type targeting vectors using yeast host cells are described by Storck et al, 1996, Nuc. Acids Res. 24:4594-4596. The use of other vectors such as bacteriophage ⁇ and vectors for phage-plasmid recombination have been described by Tsuzuki et al, 1998, Nuc. Acids Res 26:988-993; transposon-generated gene targeting constructs have also been described by
  • the vector includes a linear lambda vector (lambda-KO- St?) that comprises a stuffer fragment; an E. coli origin of replication; an antibiotic resistance gene for bacterial selection, two negative selection markers suitable for use in mammalian cells; LoxP sequences for cre-recombinase mediated conversion of linear Lambda phages into high copy plasmids.
  • a linear lambda vector (lambda-KO- St?) that comprises a stuffer fragment; an E. coli origin of replication; an antibiotic resistance gene for bacterial selection, two negative selection markers suitable for use in mammalian cells; LoxP sequences for cre-recombinase mediated conversion of linear Lambda phages into high copy plasmids.
  • the stuffer fragment is replaced by nucleotide sequences representing a left arm of homology, an ES cell selection cassette, and a right arm of homology.
  • the transformation of mouse 129 ES cells with the final vector construct is done according to standard procedures.
  • the targeting vector is linearized and then introduced by electroporation into ES cells.
  • Cell clones are positively selected with G418 and negatively selected with GANC (ganciclovir, 0.2 ⁇ M).
  • GANC ganciclovir, 0.2 ⁇ M
  • the invention also encompasses embryonic stem (ES) cells derived from a developing mouse embryo at the blastocyst stage, that are modified by homologous recombination to contain a mutant IGPcRl 7 gene allele.
  • ES embryonic stem
  • the modified ES cells are reintroduced into a blastocyst by microinjection, where they contribute to the formation of all tissues of the resultant chimeric animal, including the germ line (Capecchi, 1989, Trends Genet., 5:70; Bradley, et al, 1984, Nature, 309:255). Modified ES cells may also be stored before reimplantation into blastocysts.
  • the chimeric blastocysts are implanted into the uterus of a pseudopregnant animal, prepared by mating females with vasectomized males of the same species.
  • chimeras typically have genes coding for a coat color or another phenotypic marker that is different from the corresponding marker encoded by the stem cell genes.
  • chimeric male non-human animals and their heterozygous offspring carrying the IGPcRl 7 gene mutation which are bred to obtain progeny which are homozygous for the mutation, preferred animals being mice.
  • a phenotype selection strategy may be employed, or chromosomal DNA may be obtained from the tissue of offspring, screened using Southern blots and/or PCR amplification for the presence of a modified nucleotide sequence at the IGPcRl 7 gene locus, liked described in the above section of identifying positivlly targeted ES cells.
  • Other means for identifying and characterizing transgenic knock-out animals are also available.
  • Northern blots can be used to probe mRNA obtained from tissues of offspring animals for the presence or absence of transcripts coding for either the IGPcRl 7, the marker gene, or both.
  • Western blots might be used to assess IGPcRl 7 expression by probing with antibody specific for the receptor.
  • These animals are characterized by including, but not limited to, a loss in the ability to bind ligands specific for IGPcRl 7 and/or by a loss in expression from the IGPcRl 7 gene locus.
  • the animals Preferably, the animals produce no functional forms of
  • IGPcRl 7 at all. Once homozygous transgenic animals have been identified, they may preferably be interbred to provide a continual supply of animals that can be used in identifying pathologies dependent upon the absence of a functional IGPcRl 7 and in evaluating drugs in the assays described above. Also highly preferred in this invention, are these animals as providing a source of cells, tissues and cell lines that differ from the corresponding cells, tissues and cell lines from normal animals by the absence of fully functional forms of IGPcRl 7.
  • the methodology needed to make such animals can be adapted to any non-human animal, preferably rodents such as hamsters, rats or mice, and most preferably, mice.
  • clones of the non-human transgenic animals can be produced according to methods described in Wilmut et al, 1997, Nature, 385:810- 813.
  • Example 1 Identification of a full-length human cDNA coding for a novel GPcR, IGPcR17.
  • a coding sequence of 1026 bp was identified as follows from the EMBL alert HTGH (High Throughput Genome) database (see Fig.l, SEQ ID NOT).
  • a search was performed using the nucleotide sequences of known GPcRs.
  • a sequence with a statistically significant score was returned and searched for open reading frames. Subsequently a putative coding region was assigned and used in primer design.
  • the tracked human genomic IGPcRl 7 sequence contains the full-length cDNA sequence, identified as a single exon coding GPcR.
  • IGPcRl 7 encodes a protein of 343 amino acids, (see Fig.2, SEQ ID NO:2).
  • a BLASTP search (Basic Local Alignment Search Tool for Proteins, National Institutes of Health, Bethesda MD, U.S.A.) revealed that the human protein most closely related to human IGPcRl 7 is KIAAOOOl, a human G protein-coupled receptor of 338 amino acids (Genpept. accession number NP055694; Nomura et al, 1994, DNA Res. 1 :27-35; Nomura et al, 1994, DNA Res. 1 :47-56).
  • IGPcR17 has 47% identity and 66%> sequence homology with conserved substitutions to KIAAOOOl.
  • Example 2 Tissue-specific expression of human IGPcRlJ, analysis by RT- PCR.
  • RNA from 29 human tissues was tested in a reverse transcription-polymerase chain reaction (RT-PCR) assay.
  • the sequence of the primers used to amplify a 466 bp product is as follows: 5'- CAC TGC TCT ACA CTG TCC TG (coding sequence position 86 to 105; SEQ ID NO: 3) 5'- GAC CGA ACT CTG ATT TAA GG (coding sequence position 501 to 486;
  • the conditions for the PCR were: denaturation at 94°C for 45 seconds, annealing at 56°C for 1 minute, and extension at 72°C for 30 seconds, for a total of 35 cycles, in a Thermocycler (MJ Research, Watertown MA, USA; type PTC-225).
  • the PCR products were analyzed on an 1.8% agarose gel and stained with ethidium bromide to visualize DNA by ultraviolet imaging.
  • the tissues analyzed were: skin, whole brain, fetal brain, cerebellum, thymus, esophagus, trachea, lung, breast, mammary gland, heart, liver, fetal liver, kidney, spleen, adrenal gland, pancreas, stomach, small intestine, skeletal muscle, adipose tissue, uterus, placenta, bladder, prostate, testis, colon, rectum and cervix. Positive (human genomic DNA) and negative (water) controls were included.
  • Example 3 Tissue-specific expression of human IGPcR17 transcript, analysis by Northern hybridization.
  • Example 2 The probe spans sequences coding for transmembrane regions 1 to 4 and is 466 bp in length.
  • Commercially available Multiple Tissue Northern Blots (Clontech Laboratories, Palo Alto CA, USA) containing approximately 2 micrograms of poly A + RNA per lane, adjusted to provide a consistent beta-actin signal in each lane, were hybridized, following the manufacturer's instructions.
  • blots are optimized to give best resolution in the 1.0-4.0 kb range, and marker RNAs of 9.5, 7.5, 4.4, 2.4, 1.35 and 0.24 kb are run for reference.
  • marker RNAs 9.5, 7.5, 4.4, 2.4, 1.35 and 0.24 kb are run for reference.
  • a different set of commercially available Multiple Tissue Northern Blots (BioChain Institute, Hayward CA, USA) containing 3 micrograms of human tissue poly A + RNA per lane, was hybridized. Membranes were pre-hybridized for 30 minutes and hybridized overnight at 68°C in ExpressHyb hybridization solution (Clontech Laboratories, Palo Alto CA, USA) as per the manufacturer's instructions.
  • the cDNA probe used was labeled with [ ⁇ P] dCTP using a random primer labeling kit (Megaprime DNA labeling system, Amersham Pharmacia Biotech, Piscataway NJ, USA) and had a specific activity of 1 x 10 9 dpm/ ⁇ g.
  • the blots were washed several times in 2X SSC, 0.05% SDS for 30-40 min at room temperature, and were then washed in 0.1X SSC, 0.1% SDS for 40 min at 50°C (see Sambrook et al, 1989, "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Press, New York, USA).
  • the blots were covered with standard domestic plastic wrap and exposed to X-ray film at -70°C with two intensifying screens for 36 hours.
  • IGPcRl 7 is expressed as an approximately 2.4 kb transcript in human brain & spinal cord (see Fig. 3a).
  • a less intense signal was obtained at a lower molecular weight of about 1.8 kb in the trachea. This band is most probably a cross-reactive RNA species, as it is also observed upon hybridization with probes detecting a variety of GPcRs.
  • IGPcRl 7 is expressed most strongly in a predominant 2.4 kb transcript in human brain. A fainter transcript species of about 4.4 kb in size is also detected in brain. Additional tissues expressing low levels of the IGPcR17 2.4 kb transcript are skeletal muscle, lung, intestine, placenta, uterus, cervix and prostate (see Fig. 3b).
  • Example 4 Tissue-specific expression and relative abundance of human IGPcRl 7 transcript, dot blot analysis.
  • IGPcRl 7-specific DNA probe was generated by radiolabeling the purified and sequenced PCR product generated using primers as described in Example 2. The probe spans sequences coding for transmembrane domains 1 to 4 and is 466 bp in length.
  • a commercially available Multiple Tissue Expression Array membrane (Clontech Laboratories, Inc., Palo Alto CA, USA, cat. no. 7775-1) containing polyA + RNA in each 1mm dot, normalized to the mRNA levels of eight housekeeping genes, was hybridized (see Fig. 4a and 4b). Thus changes seen can be attributed to actual differences in mRNA abundance.
  • the membrane was pre-hybridized for 30 minutes and hybridized overnight, at 65°C in ExpressHyb Hybridization Solution as per the manufacturer's instructions (Clontech Laboratories, see above).
  • the cDNA probe used was labeled with [ ⁇ 32 P] dCTP using a random primer labeling kit (Megaprime DNA labeling system, Amersham Pharmacia Biotech, Piscataway NJ, USA) and had a specific activity of 1 xlO 9 dpm/ ⁇ g.
  • the blot was washed several times in 2X SSC, 0.05% sodium dodecyl sulfate (SDS) for 30-40 minutes at room temperature, then washed in 0.1X SSC, 0.1% SDS for 40 min at 50°C (see Sambrook et al, 1989, "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Press, New York, USA).
  • the blot was covered with plastic wrap and an X-ray film exposure made for two days at -70°C using two intensifying screens.
  • IGPcRl 7 is expressed in all regions of the human brain and CNS examined, such as whole brain, cerebellum, substantia nigra, cerebral cortex, accumbens nucleus, frontal lobe, corpus callosum, thalamus, parietal lobe, amygdala, occipital lobe, caudate nucleus, spinal cord, temporal lobe, paracentral gyrus of cerebral cortex, medulla oblongata, pons and putamen, as well as, at a lower level in fetal brain (see Figure 4a).
  • Human IGPcRl 7 is a protein of 342 amino acid residues. The encoded protein was compared to amino acid sequences present in public databases EMBL and Genbank. IGPcRl 7 has 47% identity and 66% similarity to amino acid sequences of KIAAOOOl, a G protein- coupled receptor of 338 amino acids in size (Genpept accession number NP055694; Nomura et al, 1994, DNA Res., 1:27-35; Nomura et al, 1994, DNA Res., 1 :47-56). Recently a rat ortholog of human KIAAOOOl, NTR 15-20 (Genpept accesion number
  • Fig. 7a shows the amino acid sequence of IGPcR17 ('query') compared to the amino acid sequence of the mouse ortholog of IGPcRl 7 ('sbjct'), and Fig. 7b shows the amino acid sequence of IGPcRl 7 ('query') compared to the amino acid sequence of human KIAAOOOl ('sbjct'); as abstracted from the SWISSPROT database and analyzed using a BLASTP alignment program.
  • the predicted transmembrane domains of IGPcR17 are flanked by amino acids 29-50 (TM1), 61-80 (TM2), 100- 124 (TM3), 142-160 (TM4), 190-212 (TM5), 238-257 (TM6), 282-306 (TM7), as underlined.
  • Human IGPcRl 7 protein and the mouse ortholog IGPcRl 7 protein exhibit an amino acid identity of 88.7% in an overlap of 337 residues.
  • Fig. 8 shows a hydropathy plot for the predicted amino acid sequence of the human IGPcRl 7 protein compared to the sequences predicted for human KIAAOOOl and the mouse ortholog IGPcRl 7.
  • the analysis was performed using the method of Kyte and DooLittle (1982, J. Mol. Biol., 157:105-32), with the DAMBE program (Data Analysis in Molecular Biology and Evolution), University of Hong Kong, version 4.0.41.
  • the mouse ortholog gene to the human IGPcRl 7 gene was identified by a BLAST search of public databases EMBL and Genbank using the full-length coding sequence of the human gene as a query sequence. The highest score was assigned to an EMBL EST/MM 1179620, Soares mouse 3NbMs Mus musculus cDNA clone
  • IMAGE 764455 length 542 bp.
  • the open reading frame was used in primer design.
  • genomic clones containing the full-length cDNA (SEQ ID NO:6, Fig. 5) and flanking genomic sequences were isolated and were utilized in the construction of targeting vectors.
  • the cDNAs of the human and mouse IGPcRl 7 were found to be 90 %> identical.
  • Several clones were isolated from a mouse 129 genomic library by hybridization of a probe corresponding to a 356 bp PCR product, as described in Example 7.
  • Example 7. Tissue-specific expression of mouse orthologue gene, analysis by RT-PCR.
  • a panel of cDNAs derived from total RNA isolated from 22 mouse tissues was tested in a reverse transcription-polymerase chain reaction (RT-PCR) assay.
  • the sequence of the primers used to amplify a 356 bp product (SEQ ID NO: 10), which includes predicted transmembrane domains 3 to 5, is as follows: 5'- CGT TCC TGG GGT TGA TAA CC (coding sequence position 355- 374; SEQ ID NO:8) 5'- CTT GGG AAC TTT GGC TGA AC (coding sequence position 710-691 ; SEQ ID NO: 10
  • PCR polymerase chain reaction
  • Example 8 Tissue-specific expression of mouse IGPcR17, analysis by Northern hybridization.
  • RNA originating from several tissues was isolated, separated by agarose gel electrophoresis and blotted onto a Hybond N+ Nylon membrane (Amersham Pharmacia Biotech, Piscataway NJ, USA). After covalent fixation to the membrane by UV crosslinking, the Northern blot was hybridized with a mouse IGPcR-17 probe, as indicated in Example 7. Tissues analyzed are: total brain, cerebrum, cerebellum, heart, spleen, testis, epididymis and uterus.
  • a single transcript of about 2.4kb in size was detected, being strongly expressed in total brain, cerebrum and cerebellum, and expressed in testis and epididymis to a weaker extent.
  • a single faint transcript species of about 2.8 kb in size was detected, as depicted in Figure 9.
  • the mouse IGPcRl 7 protein (Fig.6, SEQ ID NO:7) is a protein of 347 amino acid residues.
  • the encoded protein has 88.7% identity to the amino acid sequence of the human IGPcRl 7 protein.
  • Figure 7a shows the amino acid sequence of human IGPcRl 7 ('query') compared to that of the mouse ortholog ('sbjct'), as abstracted from the SWISSPROT database and analyzed using the BLASTP alignment program.
  • the predicted transmembrane domains (TM 1 to 7) of mouse IGPcR17 are flanked by amino acids 34-55 (TM1),
  • TM2 69-86
  • TM3 99-121
  • TM4 144-167
  • TM5 192-214
  • TM6 240-262
  • TM7 279-301
  • Figure 8 shows a hydropathy plot for the predicted sequence of the mouse IGPcRl 7 protein compared to the human IGPcRl 7 protein and that of human KIAAOOOl .
  • the analysis was performed using the method of Kyte and DooLittle (1982, J. Mol. Biol, 157:105-32), with the DAMBE program (Data Analysis in Molecular Biology and Evolution), University of Hong Kong, version 3J.49.
  • Example 10 Generation of ES cells with a modified IGPcR17 allele, produced by homologous recombination.
  • the most preferred method in this invention is described in Wattler S & Nehls M, German patent application DE 100 16 523.0, "Klonticianssystem Kunststoff Konstruktion von homologen Rekombiationsvektoren", filed April 03, 2000.
  • This method reduces the time required for the construction of such a vector from 3-6 months to about 14 days.
  • the vector includes a linear lambda vector (lambda-KO-Sfi) that comprises a stuffer fragment; an E. coli origin of replication; an antibiotic resistance gene for bacteria selection, two negative selection markers suitable for use in mammalian cells; LoxP sequences for cre-recombinase mediated conversion of linear lambda phages into high copy plasmids.
  • the stuffer fragment is replaced by nucleotide sequences representing a left arm of homology, an ES cell selection cassette, and a right arm of homology.
  • mice IGPcRl 7 To abolish the gene function of mouse IGPcRl 7 (mIGPcR17) a deletion of approximately 880 bp of the coding region starting approximately 10 bp upstream of the ATG was performed (see Fig. 10).
  • the left arm of homology (hereafter referred to as A/C) is PCR amplified with the primers C and A.
  • the primers contain Sfi I restriction sites A and C in their 5 '-ends, respectively. Sfi recognizes and cuts the nucleotide sequence 5-GGCCNNNNNGGCC-3'. By changing the nucleotides designated N, unique and non-compatible Sfi restriction sites are generated.
  • primer A is homologous to 25 bp of mouse IGPcRl 7, ending with the 10 bp downstream of the ATG.
  • the 3 '-end (25 bp) of primer C is homologous to a position approximately 2500 basepairs upstream of the ATG.
  • the right arm of homology (hereafter referred to as B/D) is PCR amplified with primers B and D: B is located approximately 880 bp downstream of the ATG, and D approximately 2000 bp downstream of the stop codon. Both primers contain S ⁇ z-restriction sites B or D in their 5'-ends, respectively.
  • PCR-System Boehringer Mannheim / Roche Diagnostics, Basel CH
  • a ligation of A/C with B/D and a selection cassette leads to an approximately 880 bp deletion of the mIGPcR17 coding region, thereby creating a null allelle.
  • Both PCR-products A/C and B/D are purified using Qiaquick PCR Purification Kit according to the manufacturer (Quiagen, Nenlo, ⁇ L). The PCR-products are cleaved
  • the ES-cell selection cassette (IRES- ⁇ - lactamase-MCSneo) contains Sfi-sites A and B 5'- and 3'-, respectively (Wattler S, et al, 1999, Biotechniques, 26:1150-1159).
  • a typical ligation is 50 ng lambda-KO-Sfi- arm (S z-cleaved), 10 ng selection cassette, 1 ng A/C, 1 ng B/D, 1 x ligation buffer and 1U T4 ligase (Boehringer Mannheim / Roche Diagnostics, Basel CH).
  • the ligation is carried out for 2 hours at room temperature. Two ⁇ l of the ligation are used for in vitro packaging ('Gigapack plus' from Stratagene, La Jolla CA, USA) for 1.5 hours at room temperature according to the manufacturer's instructions. Aliquots of 10 ⁇ l and 50 ⁇ l are used to infect C600 bacteria (Stratagene, La Jolla CA, USA) and infection is performed overnight. Single plaques in SM-buffer (Ausubel FM et al, 1994, "Current Protocols in Molecular Biology", John Wiley & Sons, New York) are taken to infect BNN 132 bacteria (30 min at 30°C) for plasmid conversion and infection. Bacteria are cultured over 16 hours at 30°C in TB media (Ausubel FM et al, 1994, "Current Protocols in Molecular Biology", John Wiley & Sons, New
  • Plasmids are harvested using the Qiagen plasmid kit (Qiagen cat. no. 12143) according to the manufacturer's instructions. To verify plasmid integrity, Sfi and EeoR -digests are performed.
  • Electroporated 129 mouse ES cells are double-selected with G418 (400 ⁇ g/ml) for 7 days and GANC (ganciclovir, 0.2 ⁇ M) for 3 days, starting on day 3 after electroporation, for positive and negative selection, respectively, thereby enriching for transformants having the neomycin resistance gene integrated into an endogenous IGPcRl 7 allele.
  • G418 400 ⁇ g/ml
  • GANC ganciclovir, 0.2 ⁇ M
  • Single cell clones are propagated, frozen down and expanded for DNA isolation.
  • ES cell DNA is isolated from selected clones, incubated with an appropriate restriction enzyme, and the digestion products separated on an agarose gel.
  • Southern blots are hybridized with a 5' external probe and positive targeted candidates are verified by hybridization with a 3' external probe. A single integration is confirmed by hybridization with a probe derived from the neomycin gene. Positive ES cells are isolated and expanded in culture.
  • Example 11 Mice Deficient in the Expression of the IGPcR17 Gene.
  • mice Male chimeric mice are generated by micro-injection of ES cells carrying a recombined allele into 129/SvEv mouse blastocysts, using standard methodology.
  • the chimeric blastocyst is implanted into the uterus of a pseudopregnant mouse, prepared by mating females with vasectomized males of the same species.
  • the chimeras are bred to wild type animals.
  • Tail DNA is isolated from the offspring of these chimeric mice and analyzed by incubation with appropriate restriction enzymes followed by Southern analysis, using the same strategy as outlined above to determine germline transmission.
  • the blots demonstrate the transmission into the mouse genome of the mutation altering the IGPcRl 7 allele in transformant ES cells.
  • mice homozygous for the mutation (-/-).
  • Northern blots are used to probe mRNA obtained from tissues of offspring for the presence or absence of transcripts encoding either the IGPcR27, the marker gene, or both.
  • Western blots are used to assess IGPcR27 expression by probing with antibody specific for the receptor.

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Abstract

Selon l'invention, une nouvelle protéine de récepteur couplée aux protéines G humaines (GPcR), IGPcR17, a été identifiée et caractérisée. L'invention concerne également des nucléotides codant le IGPcR17, des protéines de IGPcR17 et des protéines de fusion, des anticorps dirigés contre le récepteur, des systèmes d'expression de cellules hôtes, des modèles animaux dans lesquels le gène IGPcR17 subit une mutation, des animaux 'knock-out' de recombinaison qui n'expriment pas le IGPcR17, et des animaux transgéniques qui expriment un transgène de IGPcR17, ainsi que des composés qui modulent l'expression génique ou l'activité de récepteur du IGPcR17 et leur utilisation pour la sélection de médicaments, et pour le diagnostic ou le traitement de maladies, en particulier de troubles du système nerveux central.
PCT/EP2001/007532 2000-06-30 2001-07-02 Recepteur couple aux proteines g humaines igpcr17, et son utilisation WO2002002599A2 (fr)

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Cited By (3)

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US6762029B2 (en) 1999-12-23 2004-07-13 Portola Pharmaceuticals, Inc. Methods of identifying agents that modulate P2Y12 receptor activity
US7115374B2 (en) 2002-10-16 2006-10-03 Gen-Probe Incorporated Compositions and methods for detecting West Nile virus
US7927840B2 (en) 2006-09-11 2011-04-19 Gen Probe Incorporated Method for detecting West Nile Virus nucleic acids in the 3′ non-coding region

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US6762029B2 (en) 1999-12-23 2004-07-13 Portola Pharmaceuticals, Inc. Methods of identifying agents that modulate P2Y12 receptor activity
US7115374B2 (en) 2002-10-16 2006-10-03 Gen-Probe Incorporated Compositions and methods for detecting West Nile virus
US7732169B2 (en) 2002-10-16 2010-06-08 Gen-Probe Incorporated Method for detecting West Nile virus nucleic acids in the 5′ non-coding/capsid region
US8759003B2 (en) 2002-10-16 2014-06-24 Gen-Probe Incorporated Detection of West Nile virus nucleic acids in the viral 3′ non-coding region
US9580762B2 (en) 2002-10-16 2017-02-28 Gen-Probe Incorporated Detection of west nile virus nucleic acids in the viral 3′ non-coding region
US10781495B2 (en) 2002-10-16 2020-09-22 Gen-Probe Incorporated Detection of West Nile virus nucleic acids in the viral 3′ non-coding region
US7927840B2 (en) 2006-09-11 2011-04-19 Gen Probe Incorporated Method for detecting West Nile Virus nucleic acids in the 3′ non-coding region

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