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WO2008058399A1 - Procédés pour le diagnostic, le pronostic ou le traitement de la migraine et de troubles associés - Google Patents

Procédés pour le diagnostic, le pronostic ou le traitement de la migraine et de troubles associés Download PDF

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Publication number
WO2008058399A1
WO2008058399A1 PCT/CA2007/002084 CA2007002084W WO2008058399A1 WO 2008058399 A1 WO2008058399 A1 WO 2008058399A1 CA 2007002084 W CA2007002084 W CA 2007002084W WO 2008058399 A1 WO2008058399 A1 WO 2008058399A1
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Prior art keywords
kcnk18
migraine
polymorphism
gene
seq
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Ronald G. Lafrenière
Guy Rouleau
Jean-François POULIN
Maryse Simoneau
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EMERILLON THERAPEUTICS Inc
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EMERILLON THERAPEUTICS Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • 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
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • 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
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/035—Animal model for multifactorial diseases
    • A01K2267/0356—Animal model for processes and diseases of the central nervous system, e.g. stress, learning, schizophrenia, pain, epilepsy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/172—Haplotypes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2807—Headache; Migraine

Definitions

  • the present invention relates to methods of utilizing a gene marker and expression products thereof for the screening and identification of agents, including small organic compounds, useful in the diagnosis and treatment of migraine and related disorders in human patients, as well as methods of using these agents to diagnose, prognose, treat or otherwise ameliorate such disorders.
  • Migraine is one of the most prevalent neurological disorders.
  • the reason for the most prevalent neurological disorders is the most prevalent neurological disorders.
  • migraine migraine without aura
  • MA migraine with aura
  • Migraine is known to run in families, indicating a significant genetic component for the disease. Familial aggregation studies have shown an increased risk for migraine in relatives of probands with different types of migraine. Heritability is estimated to be between 40% and 60%, indicating that the disease is partly explained by non-genetic determinants. It is likely that both migraine subtypes (MO and MA) have some genetic determinants in common although different modifying factors (including genetic and lifestyle triggers) may contribute to the variable expression of the clinical end-point (Lea et al. 2004 BMC Med 2:3).
  • FHM familial hemiplegic migraine
  • CACNA1A voltage-dependent, P/Q type calcium channel gene located on chromosome 19p13
  • ATP1A2 alpha-2 subunit of the Na+/K+ transporting ATPase gene located on chromosome 1q21 were also found to cause a second form of FHM (FHM2) (De Fusco et al. 2003 Nat Genet 33:192).
  • chromosomes 4q24 (Wessman et al. 2002 Am J Hum Genet 70:652), Xq24-28 (MGR2; (Nyholt et al. 2000 Hum Genet 107:18)), 6p21.1-p12.2 (MGR3; (Carlsson et al.
  • a total of seven mutations have been found in the ATP1A2 gene in patients with FHM2. Each of these mutations is a missense, or amino acid substitution. Several of these mutations have been demonstrated to inhibit Na+/K+ pump activity, but not to affect assembly or translocation of the channel to the cell membrane. Similarly, a total of nine missense mutations have been found in the CACNA1A gene in patients with FHM1. These mutations can lead to increased calcium current density in cerebellar neurons, enhanced neurotransmission at the neuromuscular junction, and a reduced threshold and increased velocity of cortical spreading depression, which is the likely mechanism for the migraine aura (van den Maagdenberg et al. 2004 Neuron 41 :701).
  • migraine genes identified to date account for a very small proportion of all the migraine or headache syndromes. In addition, they have been identified in rare syndromes where the pattern of inheritance was clearly Mendelian. This is not the case for the vast majority of migraine patients, however, where the pattern of inheritance is not compatible with a simple Mendelian model. In fact, most authors consider migraine to be the result of a combination of many different genetic and environmental factors, features of a complex trait. Thus, to date, migraine related disorders are still not very well understood and only a very small proportion of migraine causes have been elucidated.
  • the present invention describes for the first time the KCNK18 gene (Potassium Channel, Subfamily K, Member 18 gene) as a therapeutic and diagnostic target for migraine or migraine-related disorders.
  • the present invention relates to the discovery that the human KCNK18 gene, when mutated, results in an increased susceptibility to develop migraine or migraine-related disorders. This constitutes an important step for developing more potent agents for treating migraine and migraine-related disorders.
  • the identification of the underlying genetic mutation or polymorphism provides a new therapeutic target for novel therapeutic agents.
  • KCNK18 can be used to identify and discover more effective therapeutic agents.
  • the present invention relates to the identification of mutations in exonic as well as intronic sequences of the KCNK18 gene in subjects affected by migraine or migraine-related disorders.
  • the present invention relates to the discovery of the KCNK18 gene as a theranostic, diagnostic and therapeutic target for migraine or migraine-related disorders. Having identified mutations in exonic as well as intronic KCNK18 sequences, it should be clear that mutations other than those particularly disclosed herein could easily be identified in the KCNK18 nucleic acid sequences and are thus, also encompassed by the present invention.
  • the present invention also relates to new methods and compositions for the diagnosis of migraine as well as related syndromes and for distinguishing between types of migraine disorders.
  • the present inventions relates to a method for determining an individual's predisposition to suffer from migraine or a migraine-raited disorder and/or responsiveness to therapy for migraine or a migraine-related disorder.
  • the KCNK18 gene can be used as a theranostic tool in order, for example, to identify and choose the correct treatment regime (particular drug, dose, mode and moment of administration) and monitor the patient's response to therapy.
  • the correct treatment regime particular drug, dose, mode and moment of administration
  • the present invention relates to the nucleic acid sequence of KCNK18, including the genomic sequence, mRNA or cDNA, polymorphic, allelic, isoforms and mutant forms thereof, and constructs of this nucleic acid sequence, including vectors, plasmids, recombinant cells and KCNK18 transgenic and knockout organisms.
  • Non-limiting examples of KCNK18 nucleic acid sequences of the present invention are set forth in SEQ ID NOs: 1 and 3.
  • the polymorphism is a substitution, and insertion or a deletion.
  • the mutation is a frameshift mutation.
  • KCNK18 gene or transgenic animals engineered to have perturbed KCNK18 function (eg. knock-in of a dominant negative mutation or knock out of the wild type gene), could be used as animal models for migraine and migraine-related disorders. These animals could also be used as tools for screening for KCNK18 modulating agents or for agents that would compensate for a malfunctioning KCNK18 gene or polypeptide.
  • the present invention relates to the gene product of KCNK18, including KCNK18 polypeptide, protein, and amino acid sequence, as well as polymorphic and allelic isoforms, splice variants and mutant forms thereof.
  • Recombinant cells and transgenic organisms wherein KCNK18 polypeptide or a variant thereof is expressed are also encompassed by the present invention.
  • Non-limiting examples of KCNK18 amino acid sequences of the present invention are set forth in SEQ ID NOs: 2 and 4.
  • KCNK18 gene or protein is used in a screening assay whereby compounds (including potential therapeutic agents) are tested to determine if they modulate KCNK18 gene expression or activity, thereby identifying potential therapeutic agents.
  • the present invention relates to a method to identify an agent which modulates the level of a polynucleotide (e.g. KCNK18) or the polypeptide encoded therefrom, whose expression contributes to the symptoms of migraine or migraine-related disorders.
  • a polynucleotide e.g. KCNK18
  • the polypeptide encoded therefrom whose expression contributes to the symptoms of migraine or migraine-related disorders.
  • the present invention relates to a method to identify an agent which modulates the level of KCNK18 polynucleotide comprising: (a) contacting an agent with a polynucleotide corresponding to a promoter sequence of KCNK18, comprising a sequence of SEQ ID NO: 5, or variants thereof; and (b) assessing the expression level of the polynucleotide in the presence of the agent as compared to in the absence thereof; wherein the detection of an increase or decrease in polynucleotide expression level results in the identification of an agent that modulates the polynucleotide expression.
  • the contacting between the agent and the polynucleotide is done under physiological conditions.
  • Such modulation may constitute a decrease or an increase in polynucleotide expression.
  • such expression is assessed by measuring the amount of an expression product encoded by the polynucleotide, most preferably an RNA or a polypeptide.
  • the KCNK18 promoter sequence is operably linked to a reporter gene (e.g. luciferase, ⁇ -galactosidase, chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), etc.) and the assay measures the relative expression of the reporter gene or its gene product.
  • a reporter gene e.g. luciferase, ⁇ -galactosidase, chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), etc.
  • the polynucleotide whose expression is to be measured or monitored is present in an intact cell, preferably a mammalian cell, most preferably a peripheral neuron, and may include a recombinant cell.
  • an intact cell is a cell that has been engineered to comprise the polynucleotide, (e.g. by genetic engineering) preferably wherein the cell does not normally express the subject gene or polynucleotide absent having been engineered to do so.
  • the present invention relates to a method for identifying an agent that modulates an activity of a KCNK18 polypeptide encoded by a polynucleotide as disclosed herein, comprising: (a) contacting an agent with a polypeptide encoded by a KCNK18 polynucleotide, such as a polynucleotide comprising SEQ ID NO: 2 or variants thereof; and (b) assessing the activity of the KCNK18 polypeptide in the presence of the agent as compared to in the absence thereof, wherein the detection of a change in the activity of a KCNK18 polypeptide in the presence of the agent results in the identification of an agent that modulates a KCNK18 polypeptide activity.
  • the observed change in activity in step (b) may be a decrease or an increase in KCNK18 activity.
  • the change in activity is the result of binding or interaction of the agent in step (b) with the KCNK18 polypeptide.
  • the agent inhibits or decreases the
  • the agent increases the KCNK18 activity.
  • the polypeptide is part of an intact cell, preferably a mammalian cell, such as a neuronal cell, which can all be of recombinant origin.
  • the cell e.g. a host cell
  • the cell has been engineered to comprise the polypeptide, (e.g. by genetic engineering).
  • the cell does not normally express the KCNK18 polypeptide of the present invention when not engineered to do so.
  • the KCNK18 polypeptide is a polypeptide that reacts with an antibody that is specific for a polypeptide having the amino acid sequence of SEQ ID NO: 2 or variants thereof. More particularly, and as well known in the art, the KCNK18 polypeptide sequence of SEQ ID NO: 2 may be modified by conservative amino acid substitutions.
  • the present invention relates to a method for identifying an agent for the treatment of migraine or migraine-related disorders, comprising: (a) administering to an animal a KCNK18 modulating agent of the present invention, and detecting in the animal a decrease (e.g. total or partial relief) in migraine (or related neurological conditions) symptoms or marker associated with same, or a decrease in the frequency of migraine episodes (or related neurological conditions) following the administration of the KCNK18 modulating agent, thereby identifying an agent for the treatment of migraine or migraine-related disorders.
  • a decrease e.g. total or partial relief
  • the animal which is treated or diagnosed is a mammal, including a human being.
  • the compound identified which modulates KCNK18 is more selective for KCNK18 as compared to related genes or proteins.
  • the present invention relates to a method for treating an animal afflicted with migraine or a related disorder comprising administering to the animal an effective amount of an agent which targets KCNK18, such as an agent identified by a screening assay of the present invention.
  • the animal is a human patient, (e.g. a patient afflicted with migraine or a related disorder).
  • KCNK18 modulating compounds of the present invention may act directly or indirectly on the expression or activity of KCNK18 mRNA or protein.
  • the KCNK18 modulating compounds act on KCNK18 interacting proteins thereby indirectly modulating KCNK18 activity.
  • the KCNK18 modulating agents of the present invention modulate the expression or activity of proteins (enhancers, transcription factors, etc) that in turn modulate the expression of the KCNK18 gene, thereby indirectly modifying the expression of the KCNK18 gene.
  • the compounds identified by the assays disclosed herein, and the methods of treating patients with such compounds are used in alternative or additional indications beyond migraine or migraine-related disorders, which are found to be treatable by modulating KCNK18 activity.
  • KCNK18 modulating compounds or agents of the present invention include antibodies, antisense compounds, gene therapy vectors and proteins, as well as small molecules and organic compounds.
  • the present invention relates to diagnostic, theranostic and pharmacogenomic compositions, kits and methods which allow the identification in a patient of the presence or absence of one or more mutations in a KCNK18 polynucleotide or a polypeptide (e.g. SEQ ID NOs: 1 or 2) including those specific mutations as set forth in SEQ ID NOs: 7 and 8.
  • a KCNK18 polynucleotide or a polypeptide e.g. SEQ ID NOs: 1 or 2
  • the identification of a mutation in KCNK18 gene or protein is most useful in diagnosing possible risk for developing migraine or migraine-related disorders as well as to determine response to therapy and to identify the appropriate treatment regimen.
  • the identification of a mutation also opens the way to gene therapy methods.
  • Figure 1A shows a cDNA sequence encoding human KCNK18
  • Figure 1B shows the corresponding amino acid sequence (SEQ ID NO: 2), wherein the start ATG codon (underlined) in Figure 1A is between positions 434-436; the stop TCA codon (underlined) is between positions 1586- 1588 and the capital letters in Figure 1A correspond to the open reading frame;
  • Figure 2A shows a cDNA sequence encoding mouse KCNK18
  • Figure 2B shows the corresponding amino acid sequence (SEQ ID NO: 4), wherein the start ATG codon (underlined) in Figure 2A is between positions 521-523; the stop TAA codon (underlined) is between positions 1703- 1705and the capital letters in Figure 2A correspond to the open reading frame;
  • Figure 3 shows the promoter region of the human KCNK18 gene (SEQ ID NO: 5), wherein the the predicted transcription start site is underlined and the start ATG codon (underlined) is located between positions 1337-1339;
  • Figure 4 shows the predicted exon/intron structure of the human
  • KCNK18 gene (SEQ ID NO: 6); wherein the exons are shown as black boxes and are numbered 1 through 3. Black boxes represent the protein coding regions of each exon;
  • Figure 5 shows the DNA sequence traces of the KCNK18 variations found in various samples, wherein Figure 5A shows a DNA sequence trace from a normal individual (left) compared to a migraine individual (right) heterozygous for the frameshift mutation; and Figure 5B shows a DNA sequence trace from a normal individual (left) compared to a migraine individual (right) heterozygous for the Trp101Ser substitution;
  • Figure 7 shows the alignment of the KCNK18 homologous protein sequences surrounding the Trp101 conserved residue (indicated by an asterisk (*)), wherein Figure 7A shows KCNK18 orthologs identified from various species and Figure 7B represents various human paralogs;
  • Figure 8 shows the alignment of the KCNK18 homologous protein sequences surrounding the Ala34 conserved residue (indicated by an asterisk (*)), wherein the location of the predicted first transmembrane domain (TMD1) is indicated above the sequences;
  • Figure 9 shows the expression pattern of KCNK18 in newborn
  • Figure 10 shows the expression pattern of KCNK18 in human tissues using TaqMan quantitative RT-PCR assay normalized to the expression level of the human Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene.
  • GPDH Glyceraldehyde 3-phosphate dehydrogenase
  • Migraine shows a clinical pattern similar to other known paroxysmal disorders that are caused by mutations in ion channel genes, the so-called “channelopathies”.
  • Migraine can also be treated with a number of agents, including beta-blockers, anti-depressants, calcium channel blockers, and a number of cerebral vasoconstrictor abortive agents such as sumatriptan and dihydroergotamine. Given that many of these agents function through modulation of ion channel function, and that CACNA1A and ATP1A2 function in ion homeostasis, it is possible that additional migraine susceptibility genes are ion channels.
  • dHPLC analysis enables the detection of mutations as small as single-base substitutions. Indeed, such substitutions, by altering the elution profiles of DNA molecules, allow one to identify different alleles of a given locus.
  • the identification of single base substitutions of genes using dHPLC is well known in the art, and numerous protocols are available therefor.
  • DNA variants were then characterized as to their predicted effect upon the gene or protein, and those variants deemed to have such an effect are further characterized by genotyping an extended collection of affected and control samples to determine allelic frequencies and/or clinical co-segregation within a given pedigree. Statistical tests were used to determine whether the DNA variation is associated with a given disease. Evolutionary conservation of DNA or protein sequences was also used to evaluate the functional significance of DNA variations. Such an analysis identifies genes or their proteins that could function as susceptibility factors in inherited forms of epilepsy, migraine, Tourette syndrome, bipolar disorder and/or essential tremor. The gene(s) is/are then used as relevant targets for the development of diagnostic and theranostic methods, therapies and therapeutics, and to develop relevant animal models of the disease.
  • An additional area of relevant background relates to the potential for discovery and development of therapeutic agents based on the discovery of the underlying genetic basis of migraine.
  • the identification of migraine related genes will lead to a new understanding of the disease and to the identification of possible new therapies for migraine and related disorders in humans and animals.
  • Therapeutic agents which modulate the biological activity of a given migraine-related gene target or its corresponding protein, constitute novel therapeutic agents for the treatment of migraine, pain, and related disorders.
  • a migraine susceptibility gene can be used in screening assays to identify therapeutic agents which would not have been known previously to be useful to modulate the target and/or treat migraine related disorders.
  • These screening assays are designed to select from a large library of compounds potential therapeutic agents that may interact with, bind to or somehow modulate the activity of the target gene/protein.
  • Combinatorial library methods known in the art including: biological libraries, spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring decon volution, the "one bead one compound” library method and synthetic library methods using affinity chromatography selection may be used in order to identify modulators of KCNK18 biological activity.
  • the biological approach comprises peptide libraries while the other 4 approaches are applicable to peptide, non- peptide, oligomer or small molecule libraries of compounds.
  • the choice of the particular combinatorial library depends on the specific KCNK18 activity that is to be modulated.
  • the present invention discloses the KCN K18 gene and protein as a novel therapeutic target involved in migraine and migraine-related disorders, and methods of using this target to identify therapeutic agents as well as compositions comprising these therapeutic agents and methods for treating such conditions in human patients.
  • the present invention discloses diagnostic and theranostic methods to identify patients that are at risk of suffering from migraine and migraine-related disorders and to identify, choose or adapt the appropriate treatment regimen.
  • the invention describes methods to produce animal models of migraine by modulating KCNK18 expression and/or modulating expression of factors that modulate KCNK18 expression. Definitions
  • nucleotide sequences are presented herein by single strand, in the 5' to 3' direction, from left to right, using the one-letter nucleotide symbols as commonly used in the art and in accordance with the recommendations of the IUPAC IUB Biochemical Nomenclature Commission. When presenting one such strand it should be recognized that a complementary sequence normally exists. Thus, it should be understood that in numerous embodiments, the present invention relates to complementary sequences.
  • KCNK18 nucleic acid or "KCNK18 polynucleotide” refers to a native KCNK18 nucleic acid sequence.
  • the human KCNK18 nucleic acid sequence is as set forth in SEQ ID NO: 1 or 6.
  • the human KCNK18 nucleic acid sequence encodes a KCNK18 protein as set forth in SEQ ID NO: 2.
  • the KCNK18 nucleic acid is a mouse nucleic acid encoding a mouse KCNK18 protein as set forth in SEQ ID NOs: 3 and 4 respectively.
  • the KCNK18 nucleic acid encodes a splice variant or allelic variant of KCNK18 (e.g.
  • the KCNK18 nucleic acid encodes a functional derivative or fragment of the KCNK18 protein.
  • the KCNK18 polynucleotides of the present invention include processed or unprocessed mRNA, including naturally occurring splice variants and alleles as well as functional derivatives.
  • the KCNK18 polynucleotide of the present invention are at least 40%, 50%, 60%, 65, 70%, 75%, 80%, 85%, 90%, 95%, or 98% identical to the KCNK18 sequence as set forth in SEQ ID NO: 1 or its complement thereof.
  • KCNK18 nucleic acid sequences encoding the same polypeptides and proteins as any of the nucleic acid sequences corresponding to KCNK18, regardless of the percent identity of such sequences, are also specifically contemplated by any of the methods of the present invention that rely on any or all of the sequences, regardless of how they are otherwise described or limited. Thus, any such sequences are available for use in carrying out any of the methods disclosed according to the invention.
  • KCNK18 sequences also include any open reading frames, as defined herein, present within a KCNK18 polynucleotide.
  • the sequences disclosed herein may represent less than the full genomic sequence. They may also represent sequences derived from alternate splicing of exons. Consequently, the genes present in the cell (and representing the genomic sequences) and the sequences disclosed herein, which are mostly cDNA sequences, may be identical or may be such that the cDNAs contain less than the full genomic sequence. Such genes and cDNA sequences are still considered KCNK18 nucleic acid sequences of the present invention because they encode similar RNA sequences.
  • RNA transcript which is then processed into a shorter mRNA
  • sequences disclosed herein correspond to genes contained in the cells and are used to determine relative levels of expression because they represent the same sequences or are complementary to RNAs encoded by these genes.
  • an "isolated nucleic acid molecule” refers to a polymer of nucleotides, and includes but should not be limited to DNA and RNA.
  • the "isolated” nucleic acid molecule is purified from its natural in vivo state.
  • a “nucleic acid” may comprise only conventional sugars, bases and linkages, as found in RNA and DNA, or may include both conventional components and substitutions (e.g., conventional bases linked via a methoxy backbone, or a nucleic acid including conventional bases and one or more base analogs).
  • Percent identity or “percent identical,” when referring to a sequence, means that a sequence is compared to a claimed or described sequence after alignment of the sequence to be compared (the "Compared Sequence") with the described or claimed sequence (the “Reference Sequence”).
  • C is the number of differences between the Reference Sequence and the Compared Sequence over the length of alignment between the Reference Sequence and the Compared Sequence wherein (i) each base or amino acid in the Reference Sequence that does not have a corresponding aligned base or amino acid in the Compared Sequence; (ii) each gap in the Reference Sequence; and (iii) each aligned base or amino acid in the Reference Sequence that is different from an aligned base or amino acid in the Compared Sequence, constitutes a difference. "R" is the number of bases or amino acids in the Reference Sequence over the length of the alignment with the Compared Sequence; with any gap created in the Reference Sequence also being counted as a base or amino acid.
  • the Compared Sequence has the specified minimum percent identity to the Reference Sequence even though alignments may exist in which the hereinabove calculated percent identity is less than the specified percent identity.
  • portion when used in relation to polynucleotides, refer to a continuous sequence of nucleotide residues which forms a subset of a larger sequence. Such terms include the sequences produced by treatment of the polynucleotides with any of the common endonucleases (e.g. restriction enzymes), or any stretch of polynucleotides that could be synthesized. These may include exonic and intronic sequences of the corresponding genes.
  • common endonucleases e.g. restriction enzymes
  • RNA molecules of the instant invention can also comprise non-standard nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally occurring RNA.
  • vector is commonly known in the art and defines a plasmid DNA, phage DNA, viral DNA and the like, which can serve as a DNA vehicle into which nucleic acid of the present invention can be cloned.
  • a targeting vector which may be used for homologous recombination with an endogenous target gene in a cell. Homologous recombination occurs between two sequences (i.e. the targeting vector and endogenous gene sequences) that are partially or fully complementary. Homologous recombination may be used to alter a gene sequence in a cell (e.g. embryonic stem cells, (ES cells)) in order to completely shut down protein expression or to introduce point mutations, substitutions or deletions in the target gene sequence. Such method is used for example to generate transgenic or knockout animals and is well known in the art.
  • ES cells embryonic stem cells
  • An expression vector is a vector or vehicle similar to a cloning vector but which is capable of expressing a gene which has been cloned into it, after transformation into a host.
  • the cloned gene (or nucleic acid sequence) is usually placed under the control of (i.e., operably linked to) certain control sequences such as promoter sequences which may be cell or tissue specific (e.g. neuronal or any KCNK18 expressing cells).
  • Expression control sequences will vary depending on whether the vector is designed to express the operably linked gene (or nucleic acid sequence) in a prokaryotic and/or eukaryotic host and can additionally contain transcriptional elements such as enhancer elements, termination sequences, tissue-specificity elements, and/or translational initiation and termination sites.
  • Vectors which can be used both in prokaryotic and eukaryotic cells are often called shuttle vectors.
  • the control sequences may allow general expression (i.e. expression in a large number of cell types) or tissue specific or cell specific expression of a particular nucleic acid sequence (e.g. neuronal cells or any other KCNK18 expressing cells).
  • Operably linked sequences may also include two segments that are transcribed onto the same RNA transcript.
  • two sequences such as a promoter and a "reporter sequence” are operably linked if transcription commencing in the promoter will produce an RNA transcript of the reporter sequence.
  • a promoter and a reporter sequence are operably linked if transcription commencing in the promoter will produce an RNA transcript of the reporter sequence.
  • a DNA construct can be a vector comprising a promoter that is operably linked to an oligonucleotide sequence of the present invention, which is in turn, operably linked to a heterologous gene, such as the gene for the luciferase reporter molecule.
  • Promoter refers to a DNA regulatory region capable of binding directly or indirectly to RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence.
  • the promoter is bound at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.
  • RNA polymerase a transcription initiation site (conveniently defined by mapping with S1 nuclease), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
  • Eukaryotic promoters will often, but not always, contain "TATA” boxes and "CCAT” boxes.
  • Prokaryotic promoters contain Shine Dalgarno sequences in addition to the -10 and -35 consensus sequences.
  • an expression vector can be constructed to assess the functionality of specific alleles of the KCNK18 polypeptide.
  • Non-limiting examples of such expression vectors include a vector comprising the KCNK18 nucleic acid sequence encoding the KCNK18 polypeptide (or part thereof) according to the present invention. These vectors can be transfected into cells. The sequences of the KCNK18 polypeptide in accordance with the present invention and their structure-function relationship could be assessed by a number of methods known to the skilled artisan.
  • One non- limiting example includes the use of cells co-expressing the KCNK18 polypeptide having a mutation which is linked to migraine or other related neurological disorders, and the KCNK18 polypeptide devoid of that mutation, as a control.
  • the functionality of the potassium channel could be tested as known to the skilled artisan and these cells could be used to screen for agents which could modulate the activity of the potassium channel.
  • agents could be tested and selected, which would increase the open state of the potassium channel.
  • Agents known to the person of ordinary skill as affecting potassium channels could be tested, for example, separately or in batches.
  • the KCNK18 gene expressed by these cells can be modified at will (e.g. by in vitro mutagenesis or the like).
  • the term "gene therapy” relates to the introduction and expression in an animal (preferably a human) of an exogenous sequence (e.g., a KCNK18 gene or cDNA sequence, KCNK18 siRNA or antisense nucleic acid) to supplement, replace or inhibit a target gene (i.e., KCNK18 gene), or to enable target cells to produce a protein (e.g., a KCNK18 chimeric protein to target a specific molecule in neuronal cells) having a prophylactic or therapeutic effect on migraine and/or migraine-related disorders.
  • the exogenous sequence is of the same origin as that of the animal (human sequence).
  • the exogenous sequence is of a different origin (e.g. human exogenous sequence in mice (e.g. knock-in)).
  • Nucleic acid sequences may be detected by using hybridization with a complementary sequence (e.g., oligonucleotide probes).
  • Hybridization detection methods may use an array of probes (e.g., on a DNA chip) to provide sequence information about the target nucleic acid which selectively hybridizes to an exactly complementary probe sequence in a set of four related probe sequences that differ by one nucleotide.
  • any other well-known hybridization technique may be used in accordance with the present invention.
  • Nucleic acid hybridization depends on the principle that two single-stranded nucleic acid molecules that have complementary base sequences will reform the thermodynamically favored double-stranded structure if they are mixed under the proper conditions.
  • the double-stranded structure will be formed between two complementary single-stranded nucleic acids even if one is immobilized on a nitrocellulose or nylon filter. In the Southern or Northern hybridization procedures, the latter situation occurs.
  • the DNA/RNA of the individual to be tested may be digested with a restriction endonuclease if applicable, prior to its fractionation by agarose gel electrophoresis, conversion to the single-stranded form, and transfer to nitrocellulose or nylon support, making it available for reannealing to the hybridization probe.
  • Non-limiting examples of hybridization conditions can be found in Ausubel, F. M. et al., Current protocols in Molecular Biology, John Wiley & Sons, Inc., New York, NY (1994).
  • an example of moderately stringent conditions for testing the hybridization of a polynucleotide of the present invention with other polynucleotides include prewashing, in a solution of 5X SSC, 0.5% SDS, 1mM EDTA (pH 8.0); hybridizing at 50°C-60°C, 5X SSC and 100 ⁇ g/ml denatured salmon sperm DNA overnight (12-16 hours); followed by washing twice at 6O 0 C for 15 minutes with each of 2X SSC, 0.5X SSC and 0.2X SSC containing 0.1% SDS.
  • the hybridization temperature is changed to 62, 63, 64, 65, 66, 67 or 68 0 C.
  • stringency of hybridization can be readily manipulated, such as by altering the salt and SDS concentration of the hybridizing and washing solutions and/or temperature at which the hybridization is performed.
  • the temperature and salt concentration selected is determined based on the melting temperature (Tm) of the DNA hybrid.
  • Tm melting temperature
  • Other protocols or commercially available hybridization kits using different annealing and washing solutions can also be used as well known in the art.
  • the use of formamide in different mixtures to lower the melting temperature may also be used and is well known in the art.
  • a "probe” is meant to include a nucleic acid oligomer that hybridizes specifically to a target sequence in a nucleic acid or its complement, under conditions that promote hybridization, thereby allowing detection of the target sequence or its amplified nucleic acid. Detection may either be direct (i.e., resulting from a probe hybridizing directly to the target or amplified sequence) or indirect (i.e., resulting from a probe hybridizing to an intermediate molecular structure that links the probe to the target or amplified sequence).
  • a probe's "target” generally refers to a sequence within an amplified nucleic acid sequence (i.e., a subset of the amplified sequence) that hybridizes specifically to at least a portion of the probe sequence by standard hydrogen bonding or "base pairing.”
  • sufficiently complementary is meant a contiguous nucleic acid base sequence that is capable of hybridizing to another sequence by hydrogen bonding between a series of complementary bases.
  • Complementary base sequences may be complementary at each position in sequence by using standard base pairing (e.g., G:C, A:T or A:U pairing) non standard base pairing (e.g., I:C) or may contain one or more residues (including a basic residues) that are not complementary by using standard base pairing, but which allow the entire sequence to specifically hybridize with another base sequence in appropriate hybridization conditions.
  • Contiguous bases of an oligomer are preferably at least about 80% (81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100%), more preferably at least about 90% complementary to the sequence to which the oligomer specifically hybridizes.
  • the binding free energy for a nucleic acid molecule with its complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed (e.g., RNAi activity).
  • the degree of complementarity between the sense and antisense region (or strand) of the siRNA construct can be the same or can be different from the degree of complementarity between the antisense region of the siRNA and the target RNA sequence (e.g., KCNK18 RNA sequence).
  • the target RNA sequence e.g., KCNK18 RNA sequence.
  • Complementarity to the target sequence of less than 100% in the antisense strand of the siRNA duplex is tolerated when these differences are located between the 5'-end and the middle of the antisense siRNA. Determination of binding free energies for nucleic acid molecules is well known in the art.
  • Perfectly complementary means that all the contiguous residues of a nucleic acid molecule will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence.
  • Appropriate hybridization conditions are well known to those skilled in the art, can be predicted readily based on sequence composition and conditions, or can be determined empirically by using routine testing (see Sambrook et al., cf. Molecular Cloning: A Laboratory Manual, Third Edition, edited by Cold Spring Harbor Laboratory, 2000) . Sequences that are "sufficiently complementary” allow stable hybridization of a probe sequence to a target sequence, even if the two sequences are not completely identical.
  • a detection step may use any of a variety of known methods to detect the presence of nucleic acid by hybridization to a probe oligonucleotide.
  • One specific example of a detection step uses a homogeneous detection method such as described in detail previously in Arnold et al. Clinical Chemistry 35:1588- 1594 (1989), and U.S. Patent Nos. 5,658,737 (Nelson et al.), and 5,118,801 and 5,312,728 (Lizardi et al.).
  • probes can be used include Southern blots (DNA detection), dot or slot blots (DNA, RNA), and Northern blots (RNA detection). Labeled proteins could also be used to detect a particular nucleic acid sequence to which it binds (e.g. protein detection by far western technology).
  • Other detection methods include kits containing reagents of the present invention on a dipstick setup and the like. Of course, it might be preferable to use a detection method which is amenable to automation.
  • a non-limiting example thereof includes a chip or other support comprising one or more (e.g. an array) different probes.
  • a "label” refers to a molecular moiety or compound that can be detected or can lead to a detectable signal.
  • a label is joined, directly or indirectly, to a nucleic acid probe or the nucleic acid to be detected (e.g., an amplified sequence).
  • Direct labeling can occur through bonds or interactions that link the label to the nucleic acid (e.g., covalent bonds or non-covalent interactions), whereas indirect labeling can occur through the use of a "linker” or bridging moiety, such as additional oligonucleotide(s), which is either directly or indirectly labeled.
  • Bridging moieties may amplify a detectable signal.
  • Labels can include any detectable moiety (e.g., a radionuclide, ligand such as biotin or avidin, enzyme or enzyme substrate, reactive group, chromophore such as a dye or colored particle, luminescent compound including a bioluminescent, phosphorescent or chemiluminescent compound, and fluorescent compound).
  • a detectable moiety e.g., a radionuclide, ligand such as biotin or avidin, enzyme or enzyme substrate, reactive group, chromophore such as a dye or colored particle, luminescent compound including a bioluminescent, phosphorescent or chemiluminescent compound, and fluorescent compound.
  • the label on a labeled probe is detectable in a homogeneous assay system, i.e., in a mixture, the bound label exhibits a detectable change compared to an unbound label.
  • oligonucleotides or “oligos” define a molecule having two or more nucleotides (ribo or deoxyribonucleotides). The size of the oligo will be dictated by the particular situation and ultimately on the particular use thereof and adapted accordingly by the person of ordinary skill.
  • An oligonucleotide can be synthesized chemically or derived by cloning according to well-known methods. While they are usually in a single-stranded form, they can be in a double- stranded form and even contain a "regulatory region". They can contain natural, rare or synthetic nucleotides. They can be designed to enhance a chosen criterion like stability, for example. Chimeras of deoxyribonucleotides and ribonucleotides may also be within the scope of the present invention.
  • Amplification refers to any known in vitro procedure for obtaining multiple copies ("amplicons") of a target nucleic acid sequence or its complement or fragments thereof.
  • In vitro amplification refers to the production of an amplified nucleic acid that may contain less than the complete target region sequence or its complement.
  • Known in vitro amplification methods include, e.g., transcription-mediated amplification, replicase-mediated amplification, polymerase chain reaction (PCR) amplification, ligase chain reaction (LCR) amplification, nucleic acid sequence-based amplification (NASBA), and strand-displacement amplification (SDA).
  • Replicase-mediated amplification uses self-replicating RNA molecules, and a replicase such as Q ⁇ -replicase.
  • PCR amplification is well known and uses DNA polymerase, primers and thermal cycling to synthesize multiple copies of the two complementary strands of DNA or cDNA.
  • LCR amplification uses at least four separate oligonucleotides to amplify a target and its complementary strand by using multiple cycles of hybridization, ligation, and denaturation.
  • SDA is a method in which a primer contains a recognition site for a restriction endonuclease that permits the endonuclease to nick one strand of a hemimodified DNA duplex that includes the target sequence, followed by amplification in a series of primer extension and strand displacement steps.
  • Other known strand- displacement amplification methods do not require endonuclease nicking.
  • Transcription-mediated amplification can also be used in the present invention.
  • TMA and NASBA isothermic methods of nucleic acid amplification are used.
  • oligonucleotide primer sequences of the present invention may be readily used in any in vitro amplification method based on primer extension by a polymerase.
  • the oligos are designed to bind to a complementary sequence under selected conditions.
  • a "primer" defines an oligonucleotide which is capable of annealing to a target sequence such as describe for probes, thereby creating a double stranded region.
  • the double stranded region can serve as an initiation point for nucleic acid synthesis under suitable conditions.
  • Primers can be, for example, designed to be specific for certain alleles so as to be used in an allele-specific amplification system.
  • Primers of the present invention may be derived from intronic sequences, exonic sequences, intron/exon junctions, promoter sequences etc. They may be designed to amplify all or portions (e.g. 1 , 2, 3, 4,, 5, 6, 7, 8, 9, 10, or more exons) of a KCNK18 nucleic acids.
  • the primer's 5 1 region may be non-complementary to the target nucleic acid sequence and include additional bases, such as a promoter sequence (which is referred to as a "promoter primer").
  • any oligomer that can function as a primer can be modified to include a 5' promoter sequence, and thus function as a promoter primer.
  • any promoter primer can serve as a primer, independent of its functional promoter sequence.
  • the design of a primer from a known nucleic acid sequence is well known in the art.
  • the oligos it can comprise a number of types of different nucleotides.
  • stereoisomers e.g., D-amino acids
  • ⁇ ,oc-disubstituted amino acids N-alkyl amino acids
  • lactic acid and other unconventional amino acids may also be suitable components for the polypeptides of the present invention.
  • Examples of unconventional amino acids include but are not limited to selenocysteine, citrulline, ornithine, norvaline, 4-(E)-butenyl-4(R) - methyl-N-methylthreonine (MeBmt), N-methyl-leucine (MeLeu), aminoisobutyric acid, statine, N-methyl-alanine (MeAIa).
  • protein or “polypeptide” means any peptide- linked chain of amino acids, regardless of post-translational modifications (e.g. acetylation, phosphorylation, glycosyjation, sulfatation, sumoylation, prenylation, ubiquitination, etc).
  • a "KCNK18 protein” or a “KCNK18 polypeptide” is an expression product of KCNK18 nucleic acid (e.g.
  • KCNK18 gene of SEQ ID NO: 6) such as native human KCNK18 protein (SEQ ID NO: 2), a KCNK18 natural splice variant, a KCNK18 allelic variant (SEQ ID NO: 7) or a KCNK18 protein homolog (e.g. mouse KCNK18: SEQ ID NO: 4) that shares at least 60%, 65, 70, 75, 80, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100% amino acid sequence identity with KCNK18 and displays at least one functional activity of native KCNK18 protein.
  • the units e.g. 66, 67...81 , 82%
  • a "KCNK18 interacting protein” refers to a protein which binds directly or indirectly (e.g. with another bridging protein or molecule) to the KCNK18 polypeptide in order to modulate or participate in a functional activity of the KCNK18 polypeptide.
  • These proteins may include kinases, phosphatases, other potassium channels of the KCNK subfamily, and any other proteins known to interact with KCNK18.
  • KCNK18 activity or “KCNK18 protein activity” especially, but not exclusively when relating to screening assays, is to be interpreted broadly and contemplates all directly or indirectly measurable and identifiable biological activities of the KCNK18 gene, gene products and KCNK18 protein.
  • Non-limiting examples of KCNK18 protein activity include all biological processes, interactions, binding behavior, binding-activity relationships, pKa, pKd, enzyme kinetics, stability, and functional assessments of the protein.
  • Non-limiting examples of KCNK18 protein activity in cell fractions, reconstituted cell fractions or whole cells include the rate at which KCNK18 protein performs any measurable biological characteristic and all measurable consequences of these effects, including cell growth, development or behavior and other direct or indirect effects of KCNK18 protein activity.
  • KCNK18 activity includes the rate, scale or scope of transcription of genomic DNA to generate RNA; the effect of regulatory proteins on such transcription, the effect of modulators of such regulatory proteins on such transcription; plus the stability and behavior of mRNA transcripts, post-transcriptional processing, mRNA amounts and turnover, and all measurements of expression and translation of the mRNA into polypeptide sequences.
  • Relating to KCNK18 activity in organisms this includes but is not limited to biological activities which are identified by their absence or deficiency in disease processes or disorders caused by aberrant KCNK18 biological activity in those organisms. Broadly speaking, KCNK18 biological activity can be determined by all these and other means for analyzing biological properties of proteins and genes that are known in the art.
  • KCNK18 protein includes activation by calcineurin in response to increased intracellular calcium concentration. Oligomerization of KCNK18 with specific proteins such as two pore potassium channels of the KCNK subfamily, as well as with itself is also considered a biological activity of KCNK18. Such interaction may be stable or transient. Thus, in accordance with the present invention, oligomerization and enzymatic activity are also considered as functional or biological activities of KCNK18. Interaction of KCNK18 with other known ligands not explicitly listed in the present disclosure may also be considered functional activities of KCNK18.
  • KCNK18 biological activity also includes any biochemical measurement of the protein, conformational changes, posttranslational modification status (e.g.
  • KCNK18 biological activities include generation of a background potassium current, establishment of the resting membrane potential of an excitable cell, repolarizing the resting membrane potential after an action potential, and other potassium channel properties as measured by electrophysiological experiments.
  • migraine refers to a primary headache disorder characterized by recurrent attacks of moderate to severe headaches that last 4-72 hours, are usually unilateral, pulsating and accompanied by nausea, vomiting, photophobia and/or phonophobia.
  • Migraine is clinically diagnosed based on criteria specified by the International Headache Society (IHS), which has defined two major classes of migraine: 1) migraine without aura (MO), which accounts for -70% of all migraine in the population, and 2) migraine with aura (MA), which comprises -25% of all migraine, and in which sufferers experience distinguishing neurological disturbances (the aura) that usually precede the headache phase of an attack.
  • IHS International Headache Society
  • Migraine-related disorders comprise other types of primary headache syndromes, including episodic and chronic tension-type headache, cluster headache, familial and sporadic hemiplegic migraine, basilar-type migraine, and aura without headache, as defined by the International Headache Society.
  • migraine-related disorders include, but are not limited to, headache pain, inflammatory and/or rheumatic pain, neuralgia, nerve entrapment syndrome, pain associated with a musculoskeletal disorder, neuropathies, seizure, tremor, anxiety, depression, Alzheimer's Disease, ALS, pain associated with multiple sclerosis, spinal cord injury, sciatica, failed back surgery syndrome, traumatic brain injury, epilepsy, Parkinson's disease, post-stroke, and vascularlesions in the brain and spinal cord, neuropathic pain associated with post mastectomy pain, phantom feeling, reflex sympathetic dystrophy, visceral pain such as pancreatits, intestinal cystitis, dysmenorrhea, irritable Bowel syndrome, Crohn's disease, biliary colic, ureteral colic, myocardial infarction, pain syndromes of the pelvic cavity, and acute pain.
  • epitope refers to a molecular region on the surface of an antigen capable of eliciting an immune response and of combining with the specific antibody produced by such a response.
  • KCNK18 antibody refers to an antibody that specifically binds to (interacts with) a KCNK18 protein and displays no substantial binding to other naturally occurring proteins other than the ones sharing the same antigenic determinants as the KCNK18 protein.
  • KCNK18 antibodies include polyclonal, monoclonal, humanized as well as chimeric antibodies.
  • animal as used herein in the context of screening assays, diagnostic and theranostic methods as well as in methods of treatments comprises all animals including mammals and particularly humans.
  • transgenic animal is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene.
  • rodent such as a rat or mouse
  • transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, and the like.
  • a "transgene” is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal.
  • a "homologous recombinant animal” is a non- human animal, preferably a mammal, more preferably a mouse, in which an endogenous KCNK18 gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal (e. g., an embryonic cell of the animal) prior to development of the animal.
  • the designation "functional derivative” denotes, in the context of a functional derivative of an amino acid sequence, a molecule that retains a biological activity (either function or structural) that is substantially similar to that of the original sequence.
  • This functional derivative or equivalent may be a natural derivative or may be prepared synthetically.
  • Such derivatives include amino acid sequences having substitutions, deletions, or additions of one or more amino acids, provided that the biological activity of the protein is conserved.
  • the substituting amino acid generally has chemico-physical properties, which are similar to that of the substituted amino acid.
  • the similar chemico-physical properties include, similarities in charge, bulkiness, hydrophobicity, hydrophylicity and the like.
  • the term “functional derivatives” is intended to include “segments", “variants”, “analogs” or “chemical derivatives” of the subject matter of the present invention.
  • chemical derivatives is meant to cover additional chemical moieties not normally part of the subject matter of the invention. Such moieties could affect the physico-chemical characteristic of the derivative (i.e. solubility, absorption, half life and the like, decrease of toxicity). Such moieties are exemplified in Remington: The Science and Practice of Pharmacy by Alfonso R. Gennaro, 2003, 21th edition, Mack Publishing Company. Methods of coupling these chemical physical moieties to a polypeptide are well known in the art.
  • a “mutation” is a detectable change in the genetic material which can be transmitted to a daughter cell.
  • a mutation can be, for example, a detectable change in one or more deoxyribonucleotides.
  • nucleotides can be added, deleted, substituted for, inverted, or transposed to a new position.
  • Spontaneous mutations and experimentally induced mutations exist.
  • the result of a mutation of nucleic acid molecule is a mutant nucleic acid molecule.
  • a mutant polypeptide can be encoded by this mutant nucleic acid molecule.
  • variant refers herein to a protein, which is substantially similar in structure and biological activity to the protein, or nucleic acid of the present invention to maintain at least one of its biological activities.
  • two molecules possess a common activity and can substitute for each other they are considered variants as that term is used herein, even if the composition, or secondary, tertiary or quaternary structure of one molecule is not identical to that found in the other, or if the amino acid sequence or nucleotide sequence is not identical.
  • Two gene or protein sequences are said to be homolgous if they share a certain level of sequence identity.
  • Two genes or proteins that are said to be “orthologous to” or “orthologs of one another are two genes or proteins that are the most homologous to one another but are isolated from two different species (eg. human and mouse KCNK18 proteins).
  • Two genes or proteins that are said to be “paralogous to” or “paralogs of one another are two genes or proteins that are homologous to one another but are isolated from the same species (eg. human KCNK3, KCNK9, KCNK18, etc proteins).
  • Orthologs represent identical genes or proteins from different species, whereas paralogs represent duplicated gene or protein families within a given species.
  • subject or "patient” as used herein refers to an animal, preferably a mammal, most preferably a human who is the object of treatment, observation or experiment.
  • purified refers to a molecule (e.g.
  • KCNK18 polypeptides, antisense or RNAi molecule, etc having been separated from a component of the composition in which it was originally present.
  • a "purified KCNK18 polypeptide or polynucleotide” has been purified to a level not found in nature.
  • a “substantially pure” molecule is a molecule that is lacking in most other components (e.g., 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 100% free of contaminants).
  • the term “crude” means molecules that have not been separated from the components of the original composition in which it was present.
  • sample components include nucleic acids such as, but not limited to, DNA or RNA, in a generally aqueous solution that may include other components, such as proteins, carbohydrates, or lipids.
  • a separating or purifying step preferably removes at least about 70% (e.g., 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 100%), more preferably at least about 90% (e.g., 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100%) and, even more preferably, at least about 95% (e.g., 95, 96, 97, 98, 99, 100%) of the other components present in the sample from the desired component.
  • the units e.g. 66, 67...81 , 82,...91 , 92%.
  • inhibitors when used in the claims and/or the specification includes any measurable decrease or complete inhibition of at least one biological activity of KCNK18 to achieve a desired result.
  • a compound is said to be inhibiting KCNK18 activity when a decrease in KCNK18 activity is measured following a treatment with the compounds of the present invention as compared to in the absence thereof.
  • Other non-limiting examples include a reduction in the K+ current or other elctrophysiological properties of the KCNK18 channel.
  • ligand are used interchangeably and broadly to refer to natural, synthetic or semisynthetic molecules or compounds.
  • compound and related terms, therefore denotes for example chemicals, macromolecules, cell or tissue extracts (from plants or animals) and the like.
  • Non-limiting examples of compounds include peptides, antibodies, carbohydrates, nucleic acid molecules and pharmaceutical agents.
  • the compound can be selected and screened by a variety of means including random screening, rational selection and by rational design using for example protein or ligand modeling methods such as computer modeling.
  • the terms “rationally selected” or “rationally designed” are meant to define compounds which have been chosen based on the configuration of interacting domains of the present invention.
  • the modulating compounds of the present invention are modified to enhance their stability and their bioavailability.
  • the compounds or molecules identified in accordance with the teachings of the present invention have a therapeutic value in diseases or conditions in which the physiology or homeostasis of the cell and/or tissue is compromised by KCNK18 production or response.
  • compounds of the present invention by acting on a biological activity of KCNK18 may modulate the function/activity of neuronal cells and therefore be used to treat migraine and/or related KCNK18 disorders.
  • KCNK18 inhibitors refer to any molecule or compound capable of inhibiting (completely or partially) a biological activity of KCNK18.
  • agonists refer to any molecule or compound capable of enhancing or stimulating (completely or partially) a biological activity of KCNK18.
  • the term “pharmaceutically acceptable” refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to human.
  • pharmaceutically acceptable means approved by a regulatory agency of the federal or state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the compounds of the present invention may be administered. Sterile water or aqueous saline solutions and aqueous dextrose and glycerol solutions may be employed as carrier, particularly for injectable solutions.
  • RFLP restriction fragment length polymorphism
  • polymorphism DNA polymorphism
  • linkage disequilibrium refers to any degree of non- random genetic association between one or more allele(s) of two different polymorphic DNA sequences, which is due to the physical proximity of the two loci. Linkage disequilibrium is present when two DNA segments are close enough on a given chromosome that they will tend to remain unseparated for several generations with the consequence that alleles of a DNA polymorphism (or marker) in one segment will show a non-random association with the alleles of a different DNA polymorphism (or marker) located in the other DNA segment nearby.
  • testing of a marker in linkage disequilibrium with the polymorphisms of the present invention at the KCNK18, gene (indirect testing), will give almost the same information as testing for the KCNK18 polymorphisms directly.
  • This situation is encountered throughout a given genome (e.g. human) when two DNA polymorphisms that are close to each other are studied.
  • Linkage disequilibrium is well known in the art and various degrees of linkage disequilibrium can be encountered between two genetic markers so that some are more closely associated than others.
  • the present invention should not be limited to the identification of the polymorphisms/mutations at the DNA level (whether on genomic DNA, amplified DNA, cDNA, or the like). Indeed, the herein-identified polymorphisms and/or mutations could be detected at the mRNA or protein level.
  • detections of polymorphism identification on mRNA or protein are known in the art. Non-limiting examples include detection based on oligos designed to hybridize to mRNA or ligands such as antibodies which are specific to the encoded polymorphism (i.e. specific to the protein fragment encoded by the distinct polymorphisms).
  • heterologous i.e. a heterologous gene region of a DNA molecule is a subsegment of DNA within a larger segment that is not found in association therewith in nature.
  • heterologous can be similarly used to define two polypeptidic segments not joined together in nature.
  • Non-limiting examples of heterologous genes include reporter genes such as luciferase, chloramphenicol acetyl transferase, B-galactosidase, and the like which can be juxtaposed or joined to heterologous control regions or to heterologous polypeptides.
  • sample biological sample
  • clinical sample is meant to include any tissue or material derived from a living or dead human (or from another animal) which may contain the KCNK18 target nucleic acid or protein.
  • samples include any tissue or material that may contain cells expressing the KCNK18 target or contain KCNK18 nucleic acid or protein such as blood or fraction thereof, lung biopsies, bronchial aspiration, feces, cerebrospinal fluid, skin, sputum, saliva, urine, or coughing samples from test patients (suspected cancer patients and control patients) or other body fluids or tissue that might be tested for KCNK18 expression.
  • the biological sample of the present invention is a crude sample (i.e.
  • the biological sample is semi-purified or substantially purified (e.g. a nucleic acid extract or protein extract).
  • the biological sample may be treated to physically disrupt tissue or cell structure, thus releasing intracellular components into a solution which may further contain enzymes, buffers, salts, detergents, and the like which are used to prepare the sample for analysis.
  • Biological samples to be tested include but should not be limited to samples from mammalian (e.g. human) or any other sources. Human samples are preferred biological samples in accordance with the present invention.
  • the clinical sample from the patient is not obtained through an invasive method.
  • the supernatant fluid or cell pellet of the present invention can be used in the diagnosis assay. Numerous clinical textbook and articles exist and are well known in the art concerning means of obtaining clinical samples and treatment thereof (in some conditions and for some applications) prior to use in the molecular diagnosis or for other uses.
  • KCNK18 gene and KCNK18 Polypeptides are intended to mean that therapeutic intervention for migraine or migraine-related disorders is achieved with therapeutic agents that modulate the activity of the gene or protein.
  • Module means to increase, to decrease, or to otherwise change a biological activity of the KCNK18 gene or protein.
  • the target may mean increasing the activity of the protein, i.e., with an agonist, or it may mean decreasing the activity of the protein, i.e. with an antagonist.
  • modulators or “to modulate” also means to increase, to decrease or to otherwise change a level of KCNK18 nucleic acid or protein.
  • the present invention identifies for the first time the KCNK18 gene and its corresponding protein as important in the etiology of migraine and migraine-related disorders.
  • the gene is named herein as KCNK18 (in human) or Kcnk18 (in other species, including mouse).
  • KCNK18 is predominantly used herein to designate the gene.
  • the polypeptide is alternately named KCNK18 polypeptide, KCNK18 protein, or TRESK. This naming is tentative and may be changed by the scientific community or otherwise without affecting the invention described and claimed herein.
  • Potassium channels are the most common type of ion channel.
  • Potassium channels form potassium-selective pores that span cell membranes.
  • Potassium channels are found in most cells and play an important role in control of cell function.
  • Potassium channels comprise a vast family of proteins which can be subdivided in different subtypes, each subtype having unique properties: voltage- gated potassium channels (voltage-gated ion channels that open or close in response to changes in the transmembrane voltage); calcium-activated potassium channels (open in response to the presence of calcium ions or other signalling molecules); inwardly rectifying potassium channels; and tandem pore domain potassium channels (also known as two-pore channels; these channels are constitutively open or possess high basal activation, such as the "resting potassium channels” or "leak channels” that set the negative membrane potential of neurons).
  • voltage- gated potassium channels voltage-gated ion channels that open or close in response to changes in the transmembrane voltage
  • calcium-activated potassium channels open in response to the presence of calcium ions or other signalling molecules
  • the mammalian family of two-pore domain K+ channels comprises 15 members and is subdivided into six subfamilies on the basis of sequence similarity (tandem of P domains in weak inward rectifier K+ channel (TWIK); TWIK-related K+ channel (TREK); TWIK-related acid sensitive K+ channel (TASK); TWIK-related alkaline pH activated K+ channel (TALK); tandem-pore domain halothane inhibited K+ channel (THIK); and TWIK-related spinal cord K+ channel (TRESK)).
  • sequence similarity tandem of P domains in weak inward rectifier K+ channel (TWIK); TWIK-related K+ channel (TREK); TWIK-related acid sensitive K+ channel (TASK); TWIK-related alkaline pH activated K+ channel (TALK); tandem-pore domain halothane inhibited K+ channel (THIK); and TWIK-related spinal cord K+ channel (TRESK)).
  • K2P channels form what is known as “leak channels” which possess Goldman-Hodgkin-Katz (open) rectification and can be regulated by several mechanisms including oxygen tension, pH, mechanical stretch, and G- proteins. Their name is derived from the fact that the ⁇ subunits consist of four transmembrane segments, each containing two pore loops. As such, they structurally correspond to two inward-rectifier ⁇ subunits and thus form dimers in the membrane. K2P channels are expressed in cells throughout the body and have been implicated in diverse cellular functions including maintenance of the resting potential and regulation of excitability, sensory transduction, ion transport, and cell volume regulation, as well as metabolic regulation and apoptosis.
  • K2P channel isoforms have been identified as important targets of several widely employed drugs, including general anesthetics, local anesthetics, neuroprotectants, and antidepressants.
  • general anesthetics including general anesthetics, local anesthetics, neuroprotectants, and antidepressants.
  • the KCNK18 gene encodes a K2P channel expressed at low levels in the brain and spinal cord, where it helps establish the resting membrane potential of excitable cells (Sano et al, 2003 JBC 278:27406).
  • KCNK18 also known in the art as TRESK, TWIK5 or WIK5, has been described in International patent application publication No WO 01/32872 and in U.S. Patent No. 6,670,149 (also US 6,664,373).
  • the wild type nucleic acid sequence of human KCNK18 is provided in SEQ ID NO: 1
  • the wild type amino acid sequence of human KCNK18 is provided in SEQ ID NO: 2.
  • the wild type nucleic acid sequence of mouse KCNK18 gene is provided in SEQ ID NO: 3
  • the wild type amino acid sequence of the mouse KCNK18 protein is provided in SEQ ID NO: 4.
  • the gene encodes a full-length conserved peptide 384 amino acids in length.
  • the gene is split into 3 exons that span 14 kb. Mutations in the KCNK18 gene in migraine patients
  • Trp101Ser missense substitution that is predicted to adversely affect KCNK18 function.
  • the Trp101 residue is very highly conserved, being found in all orthologs of KCNK18 found in different species (from human to fruitfly and worm), and in all other members of the KCNK family of two-pore K+ channels (see Figure 7). Additional variations in the KCNK18 gene have also been associated with migraine, including a missense Ala34Val variant that is found specifically in another migraine patient and is highly conserved in KCNK18 orthologs, and two variants (rs363344 and Ser231 Pro) that are significantly under-represented in migraine patient populations compared to controls, and thus may represent protective alleles.
  • the present invention discloses, for the first time, that variations in the KCNK18 gene are directly responsible for development of migraine in certain human populations and thus establishes the human clinical relevance of the KCNK18 gene, mRNA and KCNK18 protein in the etiology of migraine. Further, this discovery suggests that compounds which modulate the activity of KCNK18 may have application beyond the small groups of families with migraine with aura, and may have applicability for treating many or all forms of migraine and migraine- related disorders. Thus, the present invention relates to screening assays using KCNK18 gene or polypeptides to identify compounds having therapeutic benefits for migraine and migraine-related disorders. The present invention also concerns those compounds and their uses in the treatment of migraine and migraine-related disorders.
  • screening assays may utilize KCNK18 gene or KCNK18 protein or a fragment thereof from a different organism, preferably a vertebrate, and most preferably from a mammalian species.
  • the shared technical features of these forms of KCNK18 gene or KCNK18 protein, are that, when expressed, they have similar biological activity, and that they share functional similarity with KCNK18 or KCNK18 protein, as the case may be, such as may be determined by those skilled in the art. This approach is supported by the significant conservation of KCNK18 throughout evolution.
  • the invention encompasses the use of, including but not limited to, sheep, dog, rat, mouse or horse KCNK18 or KCNK18 protein, for the same purposes as set out more specifically herein for human KCNK18 or KCNK18 protein.
  • the KCNK18 gene and/or polypeptide used according to the invention may also be obtained from other mammalian species, other vertebrates, or invertebrates.
  • KCNK18 functional fragments as well as variants thereof may be used in accordance with the present invention.
  • screening assays of the present invention may use a particular allelic variant of KCNK18 (e.g. SEQ ID NO: 7) in order to screen for agents that will compensate for the mutation and thus, treat migraine.
  • Specific functional domains of KCNK18 may also be used in particular screening assays in order to screen for particular agents modulating a particular KCNK18 function.
  • the invention therefore relates to a method for treating migraine or migraine-related disorders comprising administering to a person in need thereof an effective amount of a selective KCNK18 agonist or antagonist, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing either entity.
  • the disorder is migraine or a migraine-related disorder.
  • the administering is by oral or intravenous means.
  • KCNK18 nucleic acid sequences and/or KCNK18 amino acid sequences for identifying therapeutic agents
  • KCNK18 activity in cells e.g. neuronal cells
  • several screening assays aiming at reducing or stimulating a functional activity of KCNK18 in cells can be designed in accordance with the present invention.
  • the preferred embodiment would be to increase KCNK18 activity to treat migraine and migraine-related disorders, although modulators that decrease KCNK18 activity are also contemplated in the invention.
  • KCNK18 activity e.g. compounds which increase K+ background current, increase plasma membrane localization, stabilize the dimerization of two KCNK18 subunits, increase the affinity of KCNK18 protein for calcineurin, reduce the affinity of KCNK18 protein for the kinase that targets Ser264, etc.
  • Inhibitors of other functional KCNK18 activities may also be identified in accordance with the present invention, as long as such functional activities are related to KCNK18 function in cells (e.g. neuronal cells).
  • libraries of candidate compounds may also be screened for inhibitors of KCNK18 activity (e.g.
  • the following methods and assays of the present invention may be developed for low-throughput, high-throughput, or ultra-high throughput screening formats.
  • methods and assays of the present invention are amenable to automation. Automation and low-throughput, high-throughput, or ultra-high throughput screening formats are possible for the screening of agents which modulate the level and/or activity of KCNK18.
  • KCNK18 modulators i.e. candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules, antisense RNA, ribozyme, or other drugs) may be based on assays which measure a biological activity of KCNK18.
  • the invention therefore provides a method (also referred to herein as a "screening assay") for identifying modulators, which have an inhibitory or stimulatory effect on, for example, a KCNK18 biological activity or expression thereof, or which bind to or interact with KCNK18 proteins, or which have a stimulatory or inhibitory effect on, for example the flux of ions through the channel.
  • Assays to measure such KCNK18 biological activity could consist of the any of the following: rubidium efflux from cell monolayers; electrophysiological studies or fluorescence measurement of cell membrane potential or ion subcellular concentrations.
  • the KCNK18 protein may be expressed in a cell, and functional, e.g., physical and chemical or phenotypic, changes can be assayed to identify potassium channel modulators.
  • these cell lines are loaded with rubidium (Rb) ions which permeate readily through potassium channels. The loaded cells can then be cultured in the presence or absence of extrinsic materials and KCNK18 channel modulators are identified by their ability to modulate Rb-efflux.
  • the Rb ion concentrations inside and outside the said cells can be measured directly using a scintillation counting device if radioactive 86Rb is used, or by atomic absorption spectroscopy if non-radioactive Rb is used. Both methods are well know to those skilled in the art.
  • molecular rearrangements of the membrane-spanning core of the KCNK18 protein as described herein can be determined using FRET combined with patch clamp (Du et a/., 2006, JBC, 280: 8633-8636) or by patch fluorometry (Zheng, Physiology, 2006, 21 : 6-12).
  • changes in ion flux may be assessed by determining changes in polarization (i.e., electrical potential) of the cell or membrane expressing the KCNK18 channel comprising a KCNK18 polypeptide.
  • changes in polarization i.e., electrical potential
  • a preferred means to determine changes in cellular polarization is by measuring changes in current (thereby measuring changes in polarization) with voltage-clamp and patch-clamp techniques, e.g., the "cell-attached" mode, the "inside-out” mode, and the "whole cell” mode. Whole cell currents are conveniently determined using standard methodology.
  • fluorescence assays using voltage- sensitive dyes or ion sensitive dyes are used to measure changes in ion flux or membrane polarization.
  • Assays for compounds capable of inhibiting or increasing ion flux through the KCNK18 potassium channel can be performed by application of the compounds to a bath solution in contact with and comprising cells having a channel of the present invention.
  • the compounds to be tested are present in the range from 1 pM to 100 mM.
  • this invention also includes secondary KCNK18 screens which may involve assays utilizing mammalian cell lines or host cells expressing KCNK18, a KCNK18 ortholog or a KCNK18 paralog.
  • a host cell is intended to mean a cell transformed with the
  • the host cell can be prokaryotic or eukaryotic.
  • prokaryotic cells are bacterial microorganisms such as E. coli, B. subtilis, Pseudomonas, and B. stearothermophilus.
  • eukaryotic cells are yeast, such as Saccharomyces cerevisiae (baker's yeast) or higher animal cells such as cells of insect or mammalian origin. Mammalian cell systems often will be in the form of monolayers of cells although mammalian cell suspensions may also be used.
  • Non- limiting examples of mammalian cell lines include VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, HEK 293, WI38, BHK, COS-7 or MDCK cell lines.
  • Host cells are used in lab techniques such as DNA cloning to receive, maintain, and allow the reproduction of recombinant DNA cloning vectors. The DNA introduced with the vector is replicated whenever the cell divides and the recombinant proteins encoded for by the plasmid are reproduced in large quantities.
  • Tertiary screens may involve the study of the identified modulators in the appropriate rat and mouse models. Accordingly, it is within the scope of this invention to further use an agent identified as described herein in an appropriate animal model.
  • a test compound identified as described herein e.g., a KCNK18 stimulating or inhibiting agent, an antisense KCNK18 nucleic acid molecule, a KCNK18 siRNA, a KCNK18 antibody, etc.
  • a test compound identified as described herein e.g., a KCNK18 stimulating or inhibiting agent, an antisense KCNK18 nucleic acid molecule, a KCNK18 siRNA, a KCNK18 antibody, etc.
  • this invention pertains to uses of novel agents identified by the above-described screening assays for treatment of migraine and migraine- related disorders, as described herein.
  • the present invention also readily affords different means for the identification of agents for treating migraine and migraine-related disorders according to their ability to modulate the activity of a KCNK18 nucleic acid sequence or KCNK18 protein.
  • Such means involve testing libraries of chemical compounds, either one at a time or in combinations, in an assay format which is designed to measure a biological activity related to a KCNK18 nucleic acid sequence or KCNK18 protein. Those compounds which modulate the biological activity in the desired fashion are thereby identified as modulating/therapeutic agents of the invention.
  • exemplary methods useful for the identification of such compounds are detailed herein, although those skilled in the art will be aware of alternative means.
  • compounds are sequentially tested to determine whether they influence a measurable biological activity of KCNK18 nucleic acid sequence or protein.
  • Assays may be based on one or more of the diverse measurable biological activities of the KCNK18 nucleic acid sequence or KCNK18 protein.
  • assays and other uses with the KCNK18 nucleic acid or amino acid sequence need not necessarily be based on a use of a full length KCNK18 sequence.
  • Particular sequences, fragments, variants, functional domains and the like can be used in methods and assays of the present invention.
  • the invention therefore provides screening assays which measure an activity of KCNK18 and are useful for the identification of compounds which modulate a KCNK18 activity.
  • the invention also invites those skilled in the art to develop further KCNK18 screening assays which go beyond those disclosed herein, for use in screening compound libraries.
  • a non-limiting list of typical assays available to those skilled in the art which may be adapted to screen for KCNK18 modulating agents include: 1) protein based assays; 2) gene expression assays; and 3) computational assays.
  • the present invention relates to a method for identifying an agent that modulates the activity of a polypeptide encoded by a polynucleotide as disclosed herein, comprising: (a) contacting an agent with a polypeptide encoded by a polynucleotide corresponding to a KCNK18 nucleic acid (e.g. SEQ ID NO: 1 or part thereof); and (b) detecting a change in the activity of the polypeptide as a result of this contacting, thereby identifying an agent that modulates the polypeptide activity.
  • a KCNK18 nucleic acid e.g. SEQ ID NO: 1 or part thereof
  • the observed change in activity in step (b) is a decrease or an increase in activity, most preferably wherein the change in activity is the result of binding to the polypeptide by the agent of step (b).
  • agents are useful for treating migraine or a migraine-related disorder.
  • the polypeptide is part of an intact cell, preferably a mammalian cell, such as a neuronal cell, most preferably a recombinant cell.
  • the cell has been engineered to comprise the polypeptide (e.g. by genetic engineering), especially where the cell does not possess the polypeptide if not modified to do so.
  • the present invention also contemplates embodiments in which the cell is engineered by means other than genetic engineering, such as where the activity of a polypeptide is to be enhanced and the cell has been engineered to contain, or have on its surface, the polypeptide but wherein the polypeptide is present due to physical insertion of the polypeptide into the membrane or cytoplasm of the cell and not through expression of a gene contained within the cell.
  • Methods well known in the art such as use of polyethylene glycol, viruses, and the like, are available to effect such insertions and the details of such procedures need not be further described herein.
  • the polypeptide is a polypeptide that reacts with an antibody that reacts with, or is specific for, a polypeptide having an amino acid sequence at least 95% identical to, more preferably at least 98% identical to the sequence of SEQ ID NO: 2 or a subsequence thereof, and where any difference in amino acid sequence is due only to conservative amino acid substitutions.
  • the polypeptide comprises the amino acid sequence of SEQ ID NO: 2.
  • the polypeptide has the full- length amino acid sequence of SEQ ID NO: 2.
  • the KCNK18 protein assays of the invention may employ compound screening technology such as (but not limited to) the ability of various dyes to change color in response to variations in assay conditions resulting from the activity of the polypeptides.
  • Compound screening assays can also be based upon the ability of test compounds to modulate the interaction of the target peptide (KCNK18 protein) and known interacting proteins.
  • interacting proteins can be identified by a variety of methods known in the art, including, for example, radioimmunoprecipitation, co-immunoprecipitation, co-purification, protein fragmentation assays and yeast two-hybrid screening. Such interactions can be further assayed by means including but not limited to fluorescence polarization or scintillation proximity methods.
  • KCNK18 protein in cells would also act to control migraine or migraine-related disorders.
  • cells expressing a wild-type KCNK18 protein are transiently metabolically labeled during translation, contacted with a candidate compound, and the half-life of the polypeptide is determined using standard techniques (generally known as pulse chase techniques).
  • Compounds that modulate the half-life of the polypeptide are useful compounds in the present invention.
  • the polypeptide (or a polypeptide fragment thereof or an epitope-tagged form or fragment thereof) is bound to a suitable support (e.g., nitrocellulose or an antibody or a metal agarose column in the case of, for example, a His-tagged form of the polypeptide). Binding to the support is preferably done under conditions that allow proteins associated with the polypeptide to remain associated with it. Such conditions may include use of buffers that minimize interference with protein-protein interactions. If desired, other proteins (e.g., a cell lysate) are added, and allowed time to associate with the polypeptide.
  • a suitable support e.g., nitrocellulose or an antibody or a metal agarose column in the case of, for example, a His-tagged form of the polypeptide. Binding to the support is preferably done under conditions that allow proteins associated with the polypeptide to remain associated with it. Such conditions may include use of buffers that minimize interference with protein-protein interactions. If desired, other proteins (e.g., a cell
  • the immobilized polypeptide is then washed to remove proteins or other cell constituents that may be non-specifically associated with the polypeptide or the support.
  • the immobilized polypeptide can then be used for multiple purposes.
  • compounds can be tested for their ability to interfere with interactions between KCNK18 protein and other bound molecules (which are presumably KCNK18 protein interacting proteins). Compounds which can successfully displace interacting proteins are thereby identified as KCNK18 protein modulating agents of the present invention.
  • the immobilized polypeptide is dissociated from its support, and proteins bound to it are released (for example, by heating), or, alternatively, associated proteins are released from the polypeptide without releasing the latter polypeptide from the support.
  • the released proteins and other cell constituents can be analyzed, for example, by SDS-PAGE gel electrophoresis, Western blotting and detection with specific antibodies, phospho-amino acid analysis, protease digestion, protein sequencing, or isoelectric focusing. Normal and mutant forms of such polypeptide can be employed in these assays to gain additional information about which part of the polypeptide a given factor is binding to.
  • an assay can be performed using a purified or semi-purified protein or other molecule that is known to interact with a polypeptide encoded by a KCNK18 polynucleotide.
  • the assay may also include the following steps: (1) harvest the encoded polypeptide and couple a suitable fluorescent label to it; (2) label an interacting protein (or other molecule) with a second, different fluorescent label.
  • Fluorescent Resonance Energy Transfer This assay may comprise the following steps: (1) Provide the encoded protein or a suitable polypeptide fragment thereof and couple a suitable FRET donor (e.g. nitro-benzoxadiazole (NBD)) to it; (2) label an interacting protein (or other molecule) with a FRET acceptor (e.g. rhodamine); (3) expose the acceptor-labeled interacting molecule to the donor-labeled polypeptide in the presence or absence of a compound being tested for its ability to interfere with an interaction between the two; and (4) measure fluorescence resonance energy transfer.
  • a suitable FRET donor e.g. nitro-benzoxadiazole (NBD)
  • NBD nitro-benzoxadiazole
  • FRET acceptor e.g. rhodamine
  • Quenching and FRET assays are related. Either one can be applied in a given case, depending on which pair of fluorophores is used in the assay.
  • BRET bioluminescence resonance energy transfer
  • PCA protein fragment complementation assay
  • the methodology basically comprises: generating at least a first fragment of a reporter molecule linked to a first interacting domain and at least a second fragment of a reporter molecule linked to a second interacting domain and subsequently allowing interaction of said domains; and detecting the reconstituted reporter molecule activity.
  • drug-screening assays can also be based on polypeptide functions deduced upon antisense interference with the gene function.
  • Intracellular localization of migraine-related polypeptides, or effects which occur upon a change in intracellular localization of such proteins or upon a change in the level of such protein, can also be used in screening assays of the present invention.
  • the present invention provides the amino acid sequence of a protein, designated KCNK18 protein or KCNK18 polypeptide, that is found in neuronal cells (e.g. SEQ ID NO: 2) and which is associated with migraine or migraine-related disorders.
  • KCNK18 protein or KCNK18 polypeptide that is found in neuronal cells (e.g. SEQ ID NO: 2) and which is associated with migraine or migraine-related disorders.
  • SEQ ID NO: 2 neuronal cells
  • SEQ ID NO: 7 several mutations have been found in this sequence derived from individuals found to have migraine or migraine-related disorders.
  • agents that counteract the phenotypic effects of these mutations such as aberrant protein structure and decreased, or absent, function represent candidate compounds for evaluation as therapeutic agents of the present invention.
  • the present invention relates to a method for identifying an agent that modulates the activity of a polynucleotide whose expression contributes to migraine or migraine-related disorders, comprising: contacting under physiological conditions a chemical agent with a polynucleotide corresponding to the promoter region of KCNK18 (SEQ ID NO: 5); and detecting a change in the expression of the polynucleotide as a result of this contacting, thereby identifying an agent that modulates the polynucleotide or gene activity.
  • Such modulation corresponds to a decrease or an increase in expression.
  • the modulation in expression is assessed by measuring the amount of an expression product (e.g. KCNK18).
  • the promoter region of KCNK18 is operably linked to a reporter gene, i.e. a gene whose expression is conveniently measured.
  • reporter genes such as Green Fluorescent Protein, luciferase, chloramphenicol acetyl-transferase (CAT), ⁇ -galactosidase, etc.
  • the polynucleotide whose expression is to be measured or monitored is present in an intact cell, preferably a mammalian cell, most preferably a neuronal cell.
  • an intact cell is a cell that has been engineered to comprise the polynucleotide, (e.g. by genetic engineering) most preferably wherein the cell does not express the subject gene or polynucleotide absent having been engineered to do so.
  • upstream untranslated regions and promoter regions of KCNK18 gene are readily obtained from SEQ ID NO: 5 as well as from publicly retrievable sequence databases.
  • genomic or untranslated regions may be included in plasmids comprising the identified gene, such as in assays to identify compounds which modulate expression thereof.
  • the upstream genomic region is ligated to a reporter gene, and incorporated into an expression plasmid.
  • the plasmid is transfected into a cell, and the recombinant cell is exposed to test compound(s).
  • Those compounds which increase or decrease the expression of the reporter gene are identified as modulators of the gene/protein, and are considered therapeutic agents of the present invention.
  • the invention also relates to recombinant cells engineered to express a KCNK18 polynucleotide or a KCNK18 polypeptide as disclosed herein.
  • the gene disclosed herein (or a fragment thereof) as being involved in migraine or migraine-related disorders in an animal can be used to express a protein in an appropriate cell in vitro, or can be cloned into expression vectors which can be used to produce large enough amounts of protein to use in in vitro assays for drug screening.
  • the expression construct may employ the genomic promoter region of KCNK18 and link it to a gene, such as a reporter gene, whose expression level is easily measured.
  • Expression systems which may be employed include but are not limited to baculovirus, herpes virus, adenovirus, adeno- associated virus, bacterial systems, and eukaryotic systems such as CHO cells. Naked DNA and DNA-liposome complexes can also be used.
  • the invention thus concerns recombinant cell lines containing a heterologous KCNK18 gene or variants thereof.
  • Such recombinant cells may be used in expression assays for analyzing the levels of expression of KCNK18 gene or a suitable reporter gene after contacting the cells with agents that may have anti-migraine properties.
  • the levels of gene expression can be quantified by Northern blot analysis or RT-PCR, or, alternatively, by measuring the amount of protein produced, by one of a number of methods known in the art, or by measuring the levels of biological activity of polypeptides encoded thereby or other genes.
  • the gene expression can serve as a marker, indicative of the physiological response of the cells to the agent. Accordingly, this response state may be determined before, and at various time points during treatment of the subject with the agent.
  • Recombinant cell lines are also preferred for the preparation of purified protein, if a purified protein assay is desired. Those skilled in the art are capable of producing recombinant cell lines and extracting protein fractions containing highly purified proteins. These samples can be used in a variety of binding assays to identify compounds which interact with KCNK18 proteins. Compounds that interact with a KCNK18 are therapeutic agents of the invention, or analogs thereof.
  • Target selectivity is an important aspect of the development of therapeutic agents.
  • the present invention specifically contemplates the identification of chemical agents, such as small organic molecules, that modulate the expression of KCNK18 gene, as defined herein, or the activity of the KCNK18 polypeptide (SEQ ID NO: 2) encoded thereby, with high specificity and that have little or no effect on other genes and/or polypeptides.
  • the methods disclosed herein for identifying an agent that modulates the expression of a KCNK18 gene comprises first identifying such agent and then testing such agent for effects on expression or activity of at least one other related gene or polypeptide, preferably a gene or polypeptide with important physiological consequences that are preferably not disturbed by a therapeutic intervention, and demonstrating little or no effect.
  • the invention provides a method for computationally identifying a compound of the invention.
  • the method involves (a) determining the active site of the KCNK18 protein (i.e. through X-Ray crystallography or other techniques); and (b) through computational modeling, identifying a compound which interacts with the active site, thereby identifying a compound, or its analog, as a compound which is useful for modulating the activity of such a polypeptide. This process is sometimes referred to as in silico screening. Sophisticated software for testing the probability of test compounds to interact with the target protein, which can test tens of millions of computer generated compounds, is available to those skilled in the art.
  • the present invention relates to a method for identifying an anti-migraine agent, comprising: (a) administering to an animal a KCNK18 modulating agent identified by using an assay or screening method of the present invention, and (b) detecting in the animal a decrease in migraine-related symptoms following the administration, thereby identifying an antimigraine agent.
  • the animal is a mammal, such as a human being.
  • the decrease in migraine symptoms could be, for example, a decrease in susceptibility to cortical spreading depression (CSD), the phenomenon that underlies the migraine aura, decreased calcium channel activation by stronger stimuli with consequent decreased calcium entry into neurons, or decreased release of excitatory neurotransmitters.
  • CSD cortical spreading depression
  • an electrical stimulus may be used.
  • the stimulus may be represented as a sharp or dull sensation.
  • the animal may otherwise react normally to such stimulus so that a decrease in normal response due to the test agent is being measured whereas in other cases the animal may initially possess a heightened sensitivity to the stimulus prior to administering the test agent.
  • observation of an antimigraine effect need not necessarily involve a reduced sensitivity or response to migraine but may involve simply a reduced sensation to a particular stimulus.
  • the anti-migraine agents identified by the methods of the present invention may induce general analgesia in an animal or may have more localized analgesic or anesthetic effects.
  • the present invention relates to a method for treating a condition in an animal afflicted with a source of chronic migraine due to another disease or condition (e.g. cancer) comprising administering to the animal an effective amount of an anti-migraine agent first identified by an assay method of the invention.
  • the animal is a human patient.
  • the screening assays of the invention thus simplify the evaluation, identification and development of therapeutic agents for the treatment of migraine and migraine-related disorders.
  • the invention also includes antibodies and immuno-reactive substances which target, interact with or bind to a KCNK18 protein, fragment thereof or epitopes thereof (including allelic variants and mutants associated with increase migraine risks).
  • Polypeptides encoded by the polynucleotides disclosed herein can be used as an antigen to raise antibodies, including monoclonal antibodies. Such antibodies will be useful for a wide variety of purposes, including, but not limited to, functional studies and drug screening assays and diagnostics.
  • Monitoring the influence of agents (e.g., small organic compounds) on the expression or biological activity of the migraine-related polypeptides identified in accordance with the present invention can be applied not only in basic drug screening, but also in clinical trials.
  • the effectiveness of an agent determined by a screening assay as described herein to increase or decrease gene expression, protein levels, or biological activity can be monitored in clinical trials of subjects exhibiting symptoms of chronic or persistent migraine due to inadequate gene expression, protein levels, or biological activity (for example, the individuals studied herein).
  • the effectiveness of an agent determined by a screening assay to modulate expression of KCNK18 gene, as well as structurally and functionally related genes, including genes with high homology thereto, and including protein levels, or biological activity can be monitored in clinical trials of subjects exhibiting decreased altered gene expression, protein levels, or biological activity.
  • the expression or activity of the genes or polypeptides disclosed herein can be used to ascertain the effectiveness of a particular anti-migraine drug.
  • the present invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate identified by the screening assays described herein) including the steps of (i) determining that a patient exhibits discomfort due to a disease or disorder that causes some type of migraine or headache syndrome; (ii) administering an effective amount of an agent identified using one of the screening assays of the present invention; (iii) ascertaining a reduction of migraine or other symptoms or measurable traits following the administration and (iv) altering the administration of the agent to the subject accordingly.
  • an agent e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate identified by the screening assays described herein
  • an agent e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid,
  • biopsy samples can be taken to show a change in gene expression, such as by measuring levels of protein, mRNA, or genomic DNA post-administration samples and comparing the level of expression or activity of the protein, mRNA, or genomic DNA in the pre- administration sample with that of the corresponding post-administration sample or samples, thereby showing the effects of drug administration on one or more of the genes disclosed herein and concomitant reduction in migraine symptoms and/or sensitivity.
  • Candidate modulators may be purified (or substantially purified) molecules or may be one component of a mixture of compounds (e.g., an extract or supernatant obtained from cells).
  • a mixed compound assay gene expression is tested against progressively smaller subsets of the candidate compound pool (e.g., produced by standard purification techniques until a single compound or minimal compound mixture is demonstrated to modulate gene or protein activity or expression in a manner having anti-migraine effects.
  • Specific compounds which modulate the gene expression or gene transcript levels of KCNK18 in a cell include antisense nucleic acids, ribozymes, siRNAs and other nucleic acid compositions which specifically hybridize with KCNK18 or a KCNK18-modulating protein (including exons or introns of such genes, promoters, 5' and 3' untranslated regions, etc.). These specific compounds are compounds of the invention, and are useful for treating the diseases such as migraine and migraine-related disorders. Design and manufacturing of such compounds are well known to those skilled in the art.
  • KCNK18 protein and polypeptides include antibodies (polyclonal or monoclonal) which specifically bind to an epitope of the polypeptide.
  • antibodies polyclonal or monoclonal
  • These specific compounds are compounds of the invention, and are useful for treating the diseases previously discussed. Design and manufacturing of such compounds are well known to those skilled in the art.
  • KCNK18 in the body include gene therapy vectors comprising all or a part of the KCNK18 gene sequence or mutant KCNK18 sequence.
  • gene therapy allows the delivery of KCNK18 to cells in an organism where it is taken up and expressed, thus changing the level or amount of KCNK18 protein in such cell.
  • These vectors thereby modulate the activity of KCNK18 in the body and are useful for the therapeutic indications disclosed herein.
  • Specific compounds which modulate the activity of KCNK18 in the body include small organic molecules. Such compounds may be naturally occurring, or they may be synthetic. Collections and combinatorial libraries of such compounds are widely available from commercial sources. As known to those skilled in the art, a screening assay, such as the assays disclosed in the instant specification, can be easily adapted to identify therapeutic agents which have the desired KCNK18 modulating ability. Agonists, antagonists, or mimetics found to be effective at reducing migraine symptoms may be confirmed as useful in animal models (for example, mice, chimpanzees, etc.). In other embodiments, treatment with a compound of the invention may be combined with other anti-migraine agents to achieve a combined, possibly even synergistic, effect.
  • novel drugs having anti-migraine properties are identified from large libraries of both natural product or synthetic (or semi-synthetic) extracts or chemical libraries according to methods known in the art.
  • test extracts or compounds are not critical to the screening procedure(s) of the invention. Accordingly, virtually any number of chemical extracts or compounds can be screened using the exemplary methods described herein. Examples of such extracts or compounds include, but are not limited to, plant-, fungal-, prokaryotic- or animal-based extracts, fermentation broths, and synthetic compounds, as well as modification of existing compounds.
  • Synthetic compound libraries are commercially available from Brandon Associates (Merrimack, NH) and Aldrich Chemical (Milwaukee, Wl).
  • libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are commercially available from a number of sources, including Biotics (Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceangraphics Institute (Ft. Pierce, FL), and PharmaMar, U.S.A. (Cambridge, MA).
  • natural and synthetically produced libraries are produced, if desired, according to methods known in the art, e.g., by standard extraction and fractionation methods.
  • any library or compound is readily modified using standard chemical, physical, or biochemical methods.
  • combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring decon volution; the One-bead one-compound 1 library method; and synthetic library methods using affinity chromatography selection may be used in order to identify modulators of KCNK18 biological activity.
  • the biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds. Methods for the synthesis of molecular libraries are known to those skilled in the art. The choice of a particular combinatorial library depends on the specific KCNK18 activity that needs to be modulated.
  • these screening methods provide a ready means for selecting either natural product extracts or synthetic compounds of interest from a large population (i.e. a chemical library, for example, one produced by combinatorial means) which are further evaluated and condensed to a few active core structures. Multiple analogs of such core structures may be developed and tested to identify those preferred analogs which have improved characteristics as therapeutic agents.
  • Improved analogs may also include compounds with improved stability, biodistribution, pharmacokinetics or other desirable features for therapeutic agents which are not directly related to modulation of the therapeutic target.
  • the improved analog of the invention is effectively delivered, either by physiological means or assisted means, to cells of the body expressing the KCNK18 protein.
  • Analog compounds are systematically screened to evaluate whether they modulate the identified biological activity and those that effectively do so are then therapeutic agents, or further analogs thereof, according to the invention.
  • the present invention has identified KCNK18 as a target for the treatment of migraine or migraine-related disorders.
  • the present invention generally relates to KCNK18 expression modulation and the use thereof to treat or prevent migraine and related KCNK18 disorders.
  • RNA interference refers to the process of sequence specific suppression of gene expression mediated by small interfering RNA (siRNA) without generalized suppression of protein synthesis. While the invention is not limited to a particular mode of action, RNAi may involve degradation of messenger RNA (e.g., KCNK18 mRNA) by an RNA induced silencing complex (RISC), preventing translation of the transcribed targeted mRNA. Alternatively, it may involve methylation of genomic DNA 1 which shuts down transcription of a targeted gene. The suppression of gene expression caused by RNAi may be transient or it may be more stable, even permanent.
  • messenger RNA e.g., KCNK18 mRNA
  • RISC RNA induced silencing complex
  • RNA interference is triggered by the presence of short interfering RNAs of about 20-25 nucleotides in length which comprise about 19 base pair duplexes.
  • siRNAs can be of synthetic origin or they can be derived from a ribonuclease III activity (e.g., dicer ribonuclease) found in cells.
  • the RNAi response also features an endonuclease complex containing siRNA, commonly referred to as an RNA-induced silencing complex (RISC), which mediates the cleavage of single stranded RNA having a sequence complementary to the antisense region of the siRNA duplex.
  • RISC RNA-induced silencing complex
  • siRNA of the present invention refers to any nucleic acid molecule capable of mediating RNA interference "RNAi" or gene silencing.
  • siRNA of the present invention are double stranded RNA molecules from about ten to about 30 nucleotides long that are named for their ability to specifically interfere with protein expression.
  • siRNA of the present invention are 12-28 nucleotides long, more preferably 15-25 nucleotides long, even more preferably 19-23 nucleotides long and most preferably 21-23 nucleotides long. Therefore preferred siRNA of the present invention are 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28 nucleotides in length.
  • siRNA molecules need not to be limited to those molecules containing only RNA, but further encompass chemically modified nucleotides and non-nucleotides.
  • the length of one strand designates the length of a siRNA molecule.
  • a siRNA that is described as a 23 ribonucleotides long could comprise two opposite strands of RNA that anneal together for 21 contiguous base pairing. The two remaining ribonucleotides on each strand would form what is called an "overhang".
  • the siRNA of the present invention contains two strands of different lengths. In this case, the longer strand designates the length of the siRNA. For example, a dsRNA containing one strand that is 20 nucleotides long and a second strand that is 19 nucleotides long is considered a 20 mer.
  • siRNAs that comprise an overhang are desirable.
  • the overhang may be at the 3' or 5' end.
  • the overhangs are at the 3' end of an RNA strand.
  • the length of an overhang may vary but preferably is about 1 to 5 nucleotides long. Generally, 21 nucleotides siRNA with two nucleotides 3'- overhang are the most active siRNAs.
  • siRNA of the present invention are designed to decrease
  • siRNA of the present invention comprise a sense region and an antisense region wherein the antisense region comprises a sequence complementary to a KCNK18 mRNA sequence and the sense region comprises a sequence complementary to the antisense sequence of KCNK18 mRNA.
  • a siRNA molecule can be assembled from two nucleic acid fragments wherein one fragment comprises the sense region and the second fragment comprises the antisense region of siRNA molecule.
  • the sense region and antisense region can also be covalently connected via a linker molecule.
  • the linker molecule can be a polynucleotide linker or a non- polynucleotide linker.
  • the present invention features a siRNA molecule having RNAi activity against KCNK18 RNA, wherein the siRNA molecule comprises a sequence complementary to any RNA having a KCNK18 encoding sequence.
  • a siRNA molecule of the present invention can comprise any contiguous KCNK18 sequence (e.g. 19-23 contiguous nucleotides present in a KCNK18 sequence).
  • the present invention can be used to inhibit gene expression of a particular gene family member through the targeting of the appropriate exon(s) (e.g., to specifically knock down the expression of a KCNK18 particular transcript) or of the full length transcript.
  • siRNAs of the present invention comprise a ribonucleotide sequence that is at least 80% identical to a KCNK18 ribonucleotide sequence.
  • the siRNA molecule is at least 90%, at least 95% (e.g., 95, 96, 97, 99, 99, 100%), at least 98% (e.g., 98, 99, 100%) or at least 99% (e.g., 99, 100%) identical to the ribonucleotide sequence of the target gene (e.g., KCNK18 RNA).
  • siRNA molecule with insertion, deletions, or single point mutations relative to the target may also be effective. Mutations that are not in the center of the siRNA molecule are more tolerated.
  • Tools to assist siRNA design are well known in the art and readily available to the public. For example, a computer-based siRNA design tool is rendered accessible by Dharmacon (Thermo Fisher Scientific).
  • the siRNA molecules of the present invention are chemically modified to confer increased stability against nuclease degradation but retain the ability to bind to the target nucleic acid that is present in a cell.
  • Modified siRNAs of the present invention comprise modified ribonucleotides, and are resistant to enzymatic degradation such as RNAse degradation, yet they retain their ability to reduce KCNK18 expression in a target cell.
  • the siRNA may be modified at any position of the molecule so long as the modified siRNA is still capable of binding to the target sequence and is more resistant to enzymatic degradation. Modifications in the siRNA may be in the nucleotide base (i.e., purine or pyrimidine), the ribose or phosphate.
  • the siRNA may be modified in at least one purine, in at least one pyrimidine or a combination thereof.
  • all purines adenosine or guanine
  • all pyrimidines cytosine or uracyl
  • Ribonucleotides on either one or both strands of the siRNA may be modified.
  • Non-limiting examples of chemical modification that can be included in an siRNA molecule include phosphorothioate intemucleotide linkages, 2'-O-methyl ribonucleotides, 2'-O-methyl modified ribonucleotides, 2'-deoxy-2'- fluoro ribonucleotides, 2'-deoxy-2'-fluoro modified pyrimidines nucleotides, 5-C- methyl nucleotides and deoxyabasic residue incorporation.
  • the ribonucleotides containing pyrimidine bases can be modified at the 2' position of the ribose residue.
  • a preferable modification is the addition of a molecule from the halide chemical group such as fluorine.
  • siRNAs of the present invention may also be modified by the attachment of at least one receptor binding ligand to the siRNA.
  • Receptor binding ligand can be any ligand or molecule that directs the siRNA of the present invention to a specific target cell (e.g., neuronal cells). Such ligands are useful to direct delivery of siRNA to a target cell in a body system, organ or tissue of a subject such as neuronal cells.
  • Receptor binding ligand may be attached to one or more siRNA ends, including any combination of 5' or 3' ends. The selection of an appropriate ligand for delivering siRNAs depends on the cells, tissues or organs that are targeted and is considered to be within the ordinary skill of the art.
  • cholesterol may be attached at one or more ends, including 3' and 5' ends.
  • Other conjugates such as other ligands for cellular receptors (e.g., peptides derived from naturally occurring protein ligands), protein localization sequences (e.g., ZIP code sequences), antibodies, nucleic acid aptamers, vitamins and other cofactors such as N-acetylgalactosamine and folate, polymers such as polyethyleneglycol (PEG), polyamines (e.g., spermine or spermidine) and phospholipids can be linked (directly or indirectly) to the siRNA molecule for improving its bioavailability.
  • PEG polyethyleneglycol
  • polyamines e.g., spermine or spermidine
  • phospholipids can be linked (directly or indirectly) to the siRNA molecule for improving its bioavailability.
  • siRNAs can be prepared in a number of ways well known in the art, such as by chemical synthesis, T7 polymerase transcription, or by treating long double stranded RNA (dsRNA) prepared by one of the two previous methods with Dicer enzyme.
  • Dicer enzyme create mixed population of dsRNA from about 21 to 23 base pairs in length from double stranded RNA that is about 500 base pairs to about 1000 base pairs in size.
  • Dicer can effectively cleave modified strands of dsRNA, such as 2'-fluoromodified dsRNA.
  • vectors are employed for producing siRNAs by recombinant techniques.
  • a DNA segment encoding a siRNA derived from a KCNK18 sequence may be included in anyone of a variety of expression vectors for expressing any DNA sequence derived from a KCNK18 sequence.
  • Such vectors include synthetic DNA sequences (e.g., derivatives of SV40, bacterial plasmids, baculovirus, yeast plamids, viral DNA such as vaccinia, fowl pox virus, adenovirus, lentivirus, retrovirus, adeno-associated virus, alphavirus etc), chromosomal, and non-chromosomal vectors.
  • any vector may be used in accordance with the present invention as long as it is replicable and viable in the desired host.
  • the DNA segment in the expression vector is operatebly linked to an appropriate expression control sequence(s) (e.g., promoter) to direct siRNA synthesis.
  • the promoters of the present invention are from the type III class of RNA polymerase III promoters (e.g., U6 and H1 promoters).
  • the promoters of the present invention may also be inducible, in that the expression may be turned on or turned off (e.g., tetracycline-regulatable system employing the U6 promoter to control the production of siRNA targeted to KCNK18).
  • the present invention utilizes a vector wherein a DNA segment encoding the sense strand of the RNA polynucleotide is operatebly linked to a first promoter and the antisense strand of the RNA polynucleotide is operably linked to a second promoter (i.e., each strand of the RNA polynucleotide is independently expressed).
  • the DNA segment encoding both strands of the RNA polynucleotide are under the control of a single promoter.
  • the DNA segment encoding each strand are arranged on the vector with a loop region connecting the two DNA segments (e.g., sense and antisense sequences), where the transcription of the DNA segments and loop region creates one RNA transcript.
  • the siRNA folds back on itself to form a short hairpin capable of inducing RNAi.
  • the loop of the hairpin structure is preferably from about 4 to 6 nucleotides in length. The short hairpin is processed in cells by endoribonucleases which removes the loop thus forming a siRNA molecule.
  • siRNAs of the present invention comprising a hairpin or circular structures are about 35 to about 65 nucleotides in length (e.g., 35, 36, 37, 38, 49, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 63, 64, 65 nucleotides in length), preferably between 40 and 64 nucleotides in length comprising for example about 18, 19, 20, 21 , 22, or 23, 24,25 base pairs.
  • the vector of the present invention comprises opposing promoters.
  • the vector may comprise two RNA polymerase III promoters on either side of the DNA segment (e.g., a specific KCNK18 DNA segment) encoding the sense strand of the RNA polynucleotide and placed in opposing orientations, with or without a transcription terminator placed between the two opposing promoters.
  • a specific KCNK18 DNA segment e.g., a specific KCNK18 DNA segment
  • the present invention also features antisense nucleic acid molecules which can be used for example to decrease or abrogate the expression of KCNK18.
  • An antisense nucleic acid molecule according to the present invention refers to a molecule capable of forming a stable duplex or triplex with a portion of its targeted nucleic acid sequence (DNA or RNA). The use of antisense nucleic acid molecules and the design and modification of such molecules is well known in the art.
  • Antisense nucleic acid molecules according to the present invention can be derived from the nucleic acid sequences and modified in accordance to well known methods.
  • some antisense molecules can be designed to be more resistant to degradation to increase their affinity to their targeted sequence, to affect their transport to chosen cell types or cell compartments, and/or to enhance their lipid solubility by using nucleotide analogs and/or substituting chosen chemical fragments thereof, as commonly known in the art.
  • antisense approach of the present invention involves the design of oligonucleotides (either DNA or RNA) that are complementary to KCNK18 mRNA.
  • the antisense oligonucleotides bind to KCNK18 mRNA and prevent its translation. Absolute complementarity, although preferred, is not a definite prerequisite.
  • Absolute complementarity although preferred, is not a definite prerequisite.
  • One skilled in the art can identify a certain tolerable degree of mismatch by use of standard methods to determine the melting point of the hybridized antisense complex.
  • oligonucleotides that are complementary to the 5'untranslated region (up to the first AUG initiator codon) of KCNK18 mRNA should work more efficiently at inhibiting translation and production of KCNK18 protein.
  • oligonucleotides that are targeted to a coding portion of the sequence may produce inactive truncated protein or diminish the efficiency of translation thereby lowering the overall expression of KCNK18 protein in a cell.
  • Antisense oligonucleotides targeted to the 3' untranslated region of messages have also proven to be efficient in inhibiting translation of targeted mRNAs.
  • the KCNK18 antisense oligonucleotides of the present invention are less than 100 nucleotides in length, particularly, less than 50 nucleotides in length and more particularly less than 30 nucleotides in length.
  • effective antisense oligonucleotides are at least 15 or more oligonucleotides in length.
  • antisense oligonucleotides of the present invention can be any antisense oligonucleotide.
  • antisense oligonucleotides of the present invention may include modified bases or sugar moiety.
  • modified bases include xanthine, hypoxanthine, 2-methyladenine, N6-isopentenyladenine, 2-methylguanine, 3- methylcytosine, 5-methylcytosine, N6-adenine, 7-methyguanine, 5-fluorouracil, 5- chlorouracil, 5-bromouracil, 5-iodouracyl, 5-carboxymethylaminomethyluracil, 5- methoxycarboxymethyluracil, queosine, 4-thiouracil and 2,6-diaminopurine.
  • modified sugar moieties include hexose, xylulose, arabinose and 2- fluoroarabinose.
  • the antisense oligonucleotides of the present invention may also include modified phosphate backbone such as methylphosphonate, phosphoramidate, phosphoramidothioates, phosphordiamidate and alkyl phosphotriesters.
  • modified phosphate backbone such as methylphosphonate, phosphoramidate, phosphoramidothioates, phosphordiamidate and alkyl phosphotriesters.
  • the synthesis of modified oligonucleotides can be done according to methods well known in the art.
  • an antisense oligonucleotide or siRNA is designed, its effectiveness can be appreciated by conducting in vitro studies that assess the ability of the antisense to inhibit gene expression (e.g., KCNK18 protein expression). Such studies ultimately compare the level of KCNK18 RNA or protein with the level of a control experiment (e.g., an oligonucleotide which is the same has that of antisense experiment but being a sense oligonucleotide or an oligonucleotide of the same size as the antisense oligonucleotide but that does not bind to a specific KCNK18 sequence).
  • a control experiment e.g., an oligonucleotide which is the same has that of antisense experiment but being a sense oligonucleotide or an oligonucleotide of the same size as the antisense oligonucleotide but that does not bind to a specific KCNK18 sequence.
  • KCNK18 in particular conditions it might be useful to stimulate or increase the expression of KCNK18 in cells such as to overcome the effect of a mutation (e.g. SEQ ID NO: 7) which reduces or changes the activity of KCNK18. This could decrease the ability of cells to modulate an inappropriate KCNK18 biological response useful for example in the treatment of diseases caused by insufficient KCNK18 expression or activity (e.g. migraine).
  • a mutation e.g. SEQ ID NO: 7
  • the present invention features gene therapy methods to increase KCNK18 expression in cells.
  • the KCNK18 sequences used in the gene therapy method of the present invention may be either a full-length KCNK18 nucleic acid sequence (e.g. SEQ ID NO: 1) or be limited to sequences encoding the biologically active domains of a KCNK18 protein.
  • any KCNK18 sequence having at least one conserved biological activity of native KCNK18 protein may be used in accordance with the present invention.
  • the KCNK18 sequence may be under the control of its natural promoter or under the control of other strong promoter allowing either general expression or cell-type or tissue specific expression.
  • an exogenous sequence e.g., a KCNK18 gene or cDNA sequence, a KCNK18 siRNA or antisense nucleic acid
  • an animal preferably a human
  • a target gene i.e., KCNK18 gene
  • target cells e.g., a KCNK18 chimeric protein to target a specific molecule to neuronal cells
  • a protein e.g., a KCNK18 chimeric protein to target a specific molecule to neuronal cells having a prophylactic or therapeutic effect toward migraine or migraine-related disorders.
  • Non virus-based and virus-based vectors for insertion of exogenous nucleic acid sequences into eukaryotic cells are well known in the art and may be used in accordance with the present invention.
  • Virus-based vectors (and their different variations) for use in gene therapy are well known in the art. In virus-based vectors, parts of a viral gene are replaced by the desired exogenous sequence so that a viral vector is produced. Viral vectors are very often designed to no longer be able to replicate due to DNA manipulations.
  • lentivirus derived vectors are used to target a KCNK18 sequence (e.g., siRNA, antisense, nucleic acid encoding a partial or complete KCNK18 protein) into specific target cells (e.g., neuronal cells).
  • KCNK18 sequence e.g., siRNA, antisense, nucleic acid encoding a partial or complete KCNK18 protein
  • target cells e.g., neuronal cells.
  • the vectors of the present invention may contain a gene that acts as a marker by encoding a detectable product.
  • One way of performing gene therapy is to extract cells from a patient, infect the extracted cells with a viral vector and reintroduce the cells back into the patient.
  • a selectable marker may or may not be included to provide a means for enriching the infected or transduced cells.
  • vectors for gene therapy that are specially formulated to reach and enter target cells may be directly administered to a patient (e.g., intravenously, orally etc.).
  • exogenous sequences e.g. antisense RNA, siRNA, a
  • KCNK18 sequence may be delivered into cells that express KCNK18 according to well-known methods.
  • methods to deliver nucleic acid into cells include DEAE dextran lipid formulations, liposome- mediated transfection, CaCI 2 -mediated transfection, electroporation or using a gene gun.
  • Synthetic cationic amphiphilic substances such as dioleoyloxypropylmethylammonium bromide (DOTMA) in a mixture with dioleoylphosphatidylethanolamine (DOPE), or lipopolyamine, have gained considerable importance in charged gene transfer.
  • DOTMA dioleoyloxypropylmethylammonium bromide
  • DOPE dioleoylphosphatidylethanolamine
  • the substance mixture complexes with negatively charged genes and binds to the anionic cell surface.
  • Other methods include linking the exogenous oligonucleotide sequence (e.g., siRNA, antisense, KCNK18 sequence encoding a KCNK18 protein, KCNK18 targeting vector for homologous recombination, etc.) to peptides or antibodies that especially binds to receptors or antigens at the surface of a target cell.
  • methods of targetting cell-specific non-viral vectors for inserting at least one gene into cells of an organism relate to the uses of non-viral carriers that are cationized to enable them to complex with the negatively charged DNA.
  • the method also includes the use of a ligand (e.g., a monoclonal antibody or fragment thereof that is specific in this particular case for membrane antigen present on the surface of neuronal cells or other KCNK18 expressing cells) can specifically bind to the desired target cell in order to enter it.
  • a ligand e.g., a monoclonal antibody or fragment thereof that is specific in this particular case for membrane antigen present on the surface of neuronal cells or other KCNK18 expressing cells
  • a ligand e.g., a monoclonal antibody or fragment thereof that is specific in this particular case for membrane antigen present on the surface of neuronal cells or other KCNK18 expressing cells
  • an effective method utilizes a recombinant DNA construct in which the nucleic acid sequence is placed under a strong promoter and the entire construct is targeted into the cell.
  • a strong promoter may constitutively or inducibly produce the KCNK18 sequence encoding KCNK18 protein (or portion thereof), antisense RNA or siRNA of the present invention.
  • the agents contemplated by the present invention are highly selective for the KCNK18 gene or the KCNK18 protein and administration of such agents to a human or other animal in need thereof provide a treatment for any of the disorders exemplified by, or later found to be related to, migraine.
  • an inhibitor mimics the effects of one or more of the mutated forms of the gene as disclosed herein.
  • an agonist would control or reverse effects of migraine or a migraine-related disorder.
  • Compounds first identified as useful in reducing susceptibility to migraine or migraine-related disorders using one or more of the assays of the present invention may be administered with a pharmaceutically-acceptable diluent, carrier, or excipient, in unit dosage form.
  • a pharmaceutically-acceptable diluent, carrier, or excipient in unit dosage form.
  • Conventional pharmaceutical practice may be employed to provide suitable formulations or compositions to administer such compositions to patients.
  • oral administration is preferred, any appropriate route of administration may be employed, for example, intravenous, perenteral, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intrathecal, epidural, intracisternal, intraperitoneal, intranasal, or aerosol administration.
  • Therapeutic formulations may be in the form of liquid solutions or suspension; for oral administration, formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal
  • Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes.
  • Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds.
  • parenteral delivery systems for agonists of the invention include ethylenevinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.
  • Formulations for inhalation may contain excipients, (e.g. lactose) or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel.
  • Combination therapies are also contemplated by the present inventors.
  • An analgesic agent identified by one of the screening methods disclosed herein may be administered along with another agent intended to treat a coincident condition, such as where analgesic and anti-tumor agents are given together or contemporaneously.
  • the present invention also relates to a process that comprises a method for producing a product comprising identifying an agent according to one of the disclosed processes for identifying such an agent (i.e., the therapeutic agents identified according to the assay procedures disclosed herein) wherein the product is the data collected with respect to the agent as a result of the identification process, or assay, and wherein the data is sufficient to convey the chemical character and/or structure and/or properties of the agent.
  • identifying an agent i.e., the therapeutic agents identified according to the assay procedures disclosed herein
  • the product is the data collected with respect to the agent as a result of the identification process, or assay, and wherein the data is sufficient to convey the chemical character and/or structure and/or properties of the agent.
  • the present invention specifically contemplates a situation whereby a user of an assay of the present invention may use the assay to screen for compounds having the desired enzyme modulating activity and, having identified the compound, then conveys that information (i.e., information as to structure, dosage, etc) to another user who then utilizes the information to reproduce the agent and administer it for therapeutic or research purposes according to the present invention.
  • information i.e., information as to structure, dosage, etc
  • the user of the assay may screen a number of test compounds without knowing the structure or identity of the compounds (such as where a number of code numbers are used the first user is simply given samples labeled with the code numbers) and, after performing the screening process, using one or more assay processes of the present invention, then imparts to a second user (user 2), verbally or in writing or some equivalent fashion, sufficient information to identify the compounds having a particular modulating activity (for example, the code number with the corresponding results).
  • This transmission of information from user 1 to user 2 is specifically contemplated by the present invention.
  • the present invention relates to diagnostic and theranostic methods and kits. More particularly, the present invention concerns the analysis of the KCNK18 gene for the diagnosis of migraine or migraine-related disorders as well as the selection of the appropriate therapeutic agent and treatment regimen for a particular patient (i.e. pharmacogenomics).
  • the term theranostics describes the use of diagnostic testing to diagnose the disease, choose the correct treatment regime and monitor the patient response to therapy.
  • Theranostics (therapy specific diagnostics) are being developed specifically for predicting and assessing drug response in individual patients rather than diagnosing disease.
  • Theranostic tests can be used to select patients for treatments that are particularly likely to benefit them and unlikely to produce side-effects. They can also provide an early and objective indication of treatment.
  • nucleic acid analysis can be used to identify
  • KCNK18 mutations e.g. SEQ ID NO: 7
  • Many nucleic acid diagnostic techniques are well known to those skilled in the art. Such techniques include DNA sequencing, hybridization probing, single stranded conformational analysis (SSCP), denaturing high performance liquid chromatography (dHPLC), PCR-based techniques such as mismatch amplification, and a myriad of other well known methods. All of the above analyses can be performed on a small sample of blood, saliva, urine or other tissue provided by a subject or patient (i.e. a person wishing to be diagnosed).
  • SSCP single stranded conformational analysis
  • dHPLC denaturing high performance liquid chromatography
  • PCR-based techniques such as mismatch amplification
  • All of the above analyses can be performed on a small sample of blood, saliva, urine or other tissue provided by a subject or patient (i.e. a person wishing to be diagnosed).
  • protein based analyses such as antibody based assays (Elisa, Radioimmunoassay and the like) can be employed to identify the expression, amount, presence or absence of a KCNK18 protein (e.g. SEQ ID NO: 1 , SEQ ID NO: 7).
  • a KCNK18 protein e.g. SEQ ID NO: 1 , SEQ ID NO: 7
  • Gene expression as well as biological activity, and mutational analysis can each serve as a diagnostic and tool theranostic for migraine and migraine-related disorders.
  • determination of the amount of KCNK18 mRNA can be used to diagnose the presence or absence of a mutation correlated with migraine or a migraine-related disorder and to determine the appropriate treatment regimen.
  • Pharmacogenomics and theranostic methods deal with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons.
  • two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drugs act on the body (altered drug action) or genetic conditions transmitted as single factors altering the way the body acts on drugs (altered drug metabolism). Altered drug action may occur in a patient having a polymorphism (e.g., an single nucleotide polymorphism or SNP) in promoter, intronic, or exonic sequences of KCNK18 gene.
  • a polymorphism e.g., an single nucleotide polymorphism or SNP
  • determining the presence and prevalence of polymorphisms within the KCNK18 gene allows for prediction of a patient's response to a particular therapeutic agent.
  • This pharmacogenomic analysis can lead to the tailoring of drug treatments according to patient genotype, including prediction of side effects upon administration of therapeutic agents, particularly therapeutic agents for treating disorders disclosed herein.
  • Pharmacogenomic allows for the selection of agents (e.g., drugs) for therapeutic or prophylactic treatment of an individual based on the genotype of the individual (e.g., the genotype of the individual is examined to determine the ability of the individual to respond to a particular agent).
  • KCNK18 can also be useful in selecting patients for clinical trials of a potential therapeutic agent. Patients can be stratified according to the DNA or protein sequence of their KCNK18 gene and their response to drug treatment can be evaluated. Such stratification can greatly reduce the number of patients required to establish efficacy for a potential therapeutic agent.
  • kits for practicing the diagnostic, theranostic and pharmacogenomic methods described herein.
  • the kits may include a carrier for the various components of the kit.
  • the carrier can be a container or support, in the form of, e.g., bag, box, tube, rack, and is optionally compartmentalized.
  • the carrier may define an enclosed confinement for safety purposes during shipment and storage.
  • the kit also includes various components useful in detecting nucleotide or amino acid variants or mutations or polymorphisms discovered in accordance with the present invention using the detection techniques discussed herein.
  • the detection kit includes one or more oligonucleotides useful in detecting the genetic variants or mutations in KCNK18 gene sequence in accordance with the present invention.
  • the oligonucleotides are designed such that they are specific to a KCNK18 nucleic acid variant of the present invention under stringent conditions. That is, the oligonucleotides should be designed such that they can be used in distinguishing one genetic variant or polymorphism from another at a particular locus under predetermined stringent hybridization conditions as described herein. Examples of such oligonucleotides include nucleic acids having a sequence selected from SEQ ID NOs: 2, 7 and 8.
  • the oligonucleotides can be used in mutation-detecting techniques such as allele-specific oligonucleotides (ASO), allele-specific PCR, TaqMan-based quantitative PCR, chemiluminescence-based techniques, molecular beacons, and improvements or derivatives thereof, e.g., microchip technologies.
  • the kit includes one or more oligonucleotides suitable for use in detecting techniques such as ARMS, oligonucleotide ligation assay (OLA), and the like.
  • the oligonucleotides in the detection kit can be labeled with any suitable detection marker including but not limited to, radioactive isotopes, fluorophores, biotin, enzymes (e.g., alkaline phosphatase), enzyme substrates, ligands and antibodies, etc.
  • any suitable detection marker including but not limited to, radioactive isotopes, fluorophores, biotin, enzymes (e.g., alkaline phosphatase), enzyme substrates, ligands and antibodies, etc.
  • the oligonucleotides included in the kit are not labeled, and instead, one or more markers are provided in the kit so that users may label the oligonucleotides at the time of use.
  • the detection kit contains one or more antibodies selectively immunoreactive with a KCNK18 protein variant of the present invention. Methods for producing and using such antibodies have been described above in detail.
  • the detection kit of this invention Various other components useful in the detection techniques may also be included in the detection kit of this invention. Examples of such components include, but are not limited to, DNA polymerase, reverse transcriptase, deoxyribonucleotides, dideoxyribonucleotides, other primers suitable for the amplification of a target DNA or mRNA sequence, RNase A, mutS protein, and the like.
  • the detection kit preferably includes instructions on using the kit for detecting genetic variants in KCNK18 gene sequences, particularly the genetic variants of the present invention.
  • the invention provides a kit for detecting an individual's predisposition to suffer from migraine or a migraine-related disorder, said kit comprising at least one ligand binding to the KCNK18 gene containing a polymorphism or mutation and instructions for use.
  • the ligand may be e.g. an oligonucleotide having a sequence selected from SEQ ID NOs: 2, 7 and 8.
  • the polymorphism may be located in an exon or an intron and may be a substitution, an insertion or a deletion.
  • the polymorphism is a serine to proline substitution at position 231 of SEQ ID NO: 2, or a tryptophan to serine substitution at amino acid 101 of SEQ ID NO: 2, or as set forth in SEQ ID NO: 7, or comprised in the amino acid sequence as set forth in SEQ ID NO: 8.
  • a further embodiment of the present invention relates to animal models of migraine, in which the KCNK18 function has been perturbed.
  • This may include animals that harbor a naturally occuring mutation or variant in the KCNK18 gene that affects function of the gene, including but not limited to decreased activity of the KCNK18 gene or protein.
  • Transgenic animal models of migraine can also be engineered using standard methods in which exogenous KCNK18 sequences are stably introduced into animal cells, and the cells are used to produce animals.
  • These models may include: 1) KCNK18 knock-out animals in which the endogenous KCNK18 gene has been replaced by a sequence that disrupts KCNK18 expression or function, 2) KCNK18 knock-in animals, in which the endogenous KCNK18 gene is replaced with a copy of the gene that perturbs normal KCNK18 function (eg.
  • KCNK18 heterologous recombinant animals in which heterologous integration of KCNK18 sequences lead to peturbed KCNK18 function (eg. stable expression of a dominant negative KCNK18 mutation, such as in SEQ ID NO. 7, or of a siRNA that downregulates KCNK18 expression).
  • Both knock-out and knock-in animal models are considered homologous recombinant animals.
  • the transgene in these animal models may be either heterozygous (one or more copies of the transgene in the presence of the endogenous allele) or homozygous (eg. two identical copies of the transgene in the absence of the endogenous allele).
  • Animal models of migraine may also be produced to contain selected systems which allow for regulated expression of the transgene.
  • One example of such a system is the creloxP recombinase system of bacteriophage P1.
  • creloxP recombinase system is the creloxP recombinase system of Saccharomyces cerevisiae. If a creloxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein are required.
  • Such animals can be provided through the construction of "double" transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.
  • animal models of migraine may be used as part of screening strategies designed to identify compounds which are capable of ameliorating migraine, or modulating physiological phenomena associated with migraine (eg. cortical spreading depression, vascular constriction, or meningeal inflammation).
  • the animal models of migraine may be used to identify or improve drugs, pharmaceuticals, therapies and interventions which may be effective in treating migraine or migraine-related disorders.
  • LD50 half lethal dose
  • ED50 half effective dose
  • Cells from transgenic animals may be used for in vitro applications such as to determine KCNK18 function, or binding partners, or cellular responses to exogenous substances or stimuli (eg. electrophysiological characteristics of neuronal cells from a transgenic animal). These transgenic cellular systems can also be used to identify or improve drugs, pharmaceuticals, therapies and interventions which may be effective in treating migraine or migraine-related disorders.
  • the KCNK18 gene was identified as a potentially interesting candidate ion channel gene for migraine based on its expression in brain and spinal cord (Sano et al. 2003 J Biol Chem 278:27406).
  • the genomic sequence was downloaded from a publicly available website and the exon/intron structure of the gene determined by comparing all available human and mouse cDNA and EST (expressed sequence tag) sequences. It was determined that there was one major isoform expressed from the human gene, and that the gene was split into 3 exons (Figure 4) (SEQ ID NO: 6).
  • a total of 4 amplicons covering the Open Reading Frame (ORF) and 4 amplicons covering the 5' flanking region were used to screen the complete human KCNK18 gene in a panel of 368 patients affected with either migraine, bipolar disorder, Tourette syndrome, epilepsy or essential tremor. Amplicons were designed to minimize the amount of intronic sequence for each amplicon, and to avoid repetitive elements such as AIu repeats.
  • PCR primers were designed using PrimerSelectTM (DNASTAR, Madison Wl) and purchased from BioCorp (Montreal). The primer sequences, amplicon sizes and amplification conditions are given in Table 1.
  • the TD1 protocol for PCR amplification consisted of the following: initial denaturation for 4 min.
  • the TD8 protocol for PCR amplification consisted of the following: initial denaturation for 4 min.
  • Samples were amplified using Taq DNA polymerase (Qiagen) on a PE 9700 PCR thermocyclerTM (Perkin Elmer). Amplification conditions were determined by testing several annealing temperatures, and by addition of Q solution (Qiagen) for problematic amplicons on control genomic DNA samples. All amplified fragments were tested by agarose gel electrophoresis. The PCR fragments were then optimized for melting temperature on a model 3500HT WAVE (Transgenomic Inc., Omaha, NB with Wavemaker software version 4.1.44) dHPLC apparatus.
  • DNA samples from a panel of 368 patients with various forms of epilepsy, bipolar, Tourette's syndrome, essential tremor and migraine were amplified using the optimized conditions and the fragments were then tested on agarose gels.
  • Genomic DNA from patients and normal individuals was obtained by conventional methods.
  • Amplified fragments were pooled 4x, denatured at 95 0 C in a heating block for 5 min. and renatured slowly by cooling the block to 25 0 C over the course of 3 hours.
  • the pooled PCR fragments were run on the dHPLC apparatus at two different melting temperatures.
  • a total of twelve base-pair variations were identified: one variation mapped to the 5 1 flanking region of the gene, possibly in the promoter, three variations were predicted to be silent, seven variations were predicted to result in substitution of an amino acid residue, and one variation was predicted to cause a frameshift that would prematurely truncate the KCNK18 protein.
  • Each DNA variation is shown in Table 2.
  • Each variant was genotyped in the full panel of 368 patients, to establish allelic frequencies. Genotyping was done either by allele specific oligo (ASO) hybridization or by direct sequencing. For each ASO assay, two oligonucleotides were designed to differentiate the wild-type from the mutant alleles. For instance, the sequences of the oligonucleotides used in the ASO assay for the G458xO3Av1 (F139WfsX24) variant are shown in SEQ ID NOs: 9 and 10. The ASO assays was done essentially as described (Bourgeois & Labuda 2004, Anal Biochem 324:309).
  • each oligonucleotide was end-labeled with Y 32 P- dATP using T4 kinase in a 25ul total reaction as described. Then the probes were hybridized to nylon membranes to which was affixed the desired PCR amplified fragments, washed, and exposed to X-ray film. Films were scored and the genotypes for all successfully amplified samples was determined. The minor allele frequencies established are shown in Table 2 for each variant.
  • G458xO3Av1 (F139WfsX24) variant with migraine in the large pedigree was calculated using standard procedures.
  • the variant gave a LOD of 2.3, the maximum score obtainable for this pedgree.
  • the prematurely truncated KCNK18 protein which still retains its TMD1 and dimerization domain (containing the Cysteine reside that can form disulfide bonds with another KCNK18 protein), might bind to wild type KCNK18 channels and negatively affect their channel conductances, prevent wild type channels from reaching the plasma membrane, or lead to premature proteolysis of wild type channels.
  • TMD1 and dimerization domain containing the Cysteine reside that can form disulfide bonds with another KCNK18 protein
  • N-terminal fragment including the first transmembrane segment of the rat delayed rectifier K+ channel Kv1.1 coassembles with other K+ channels of the Kv1 subfamily in vitro, inhibits the currents encoded by Kv1.5 in a dominant-negative manner when coexpressed in Xenopus oocytes, and traps Kv1.5 polypeptide in the endoplasmic reticulum of GH3 cells.
  • This truncated N-terminal fragment thus acts as a dominant negative construct.
  • HIV-1 accessory protein Vpu which consists of 81 aa containing 1 TMD with homology to the K2P3 channel (TASK-1 , KCNK3) N-terminal end.
  • the frameshifted KCNK18 protein would be predicted to interact with wild type KCNK18 channels, and either keep them from reaching the plasma membrane, or negatively interact with them at the plasma membrane. Alternately, defective KCNK18 dimers may be degraded, reducing the effective number of functional KCNK18 channels that reach the plasma membrane, below a critical threshold required for normal KCNK18 activity, especially in relation to the control of hyperexcitability in KCNK18-expressing neurons. It is also possible that frameshifted KCNK18 channels interfere with the function of another K channel by heterodimerization and negative control.
  • Trp101 is perfectly conserved, being present in KCNK18 orthologs identified from human, chimpanzee, Rhesus monkey, lemur, dog, cow, rat, mouse, hedgehog, platypus, chicken, 3 species of fish (Medaka, Stickleback, and zebrafish), 2 species of fruitfly, and 1 species of worm (C. elegans) ( Figure 7A).
  • Trp101 residue is also perfectly conserved in each of the 15 human paralogs of KCNK18 protein, or members of the KCNK family of channels ( Figure 7B). This very high level of sequence conservation for the Trp101 residue strongly suggests that the residue is critical to the protein's structure and/or function.
  • Trp101 residue is predicted to lie very close to the P1 domain of the KCNK18 channel, and may therefore play a critical role in the function of that domain, its secondary or tertiary structure, or the exact position of the P1 domain in the context of the plasma membrane.
  • Trp residues have been shown to be critical for correct positioning of trans-membrane domains in lipid bilayers.
  • the Trp101 residue may also be critical for binding of K+ ions, other ions, or other ligands critical for KCNK18 function. In this respect, Gruss et al (2004, MoI Pharmacol.
  • Trp101 in KCNK18 protein is paralogous to Trp127 in TREK1
  • Trp101 in KCNK18 protein is paralogous to Trp127 in TREK1
  • the Trp101Ser missense in KCNK18 protein will affect channel activation by Cu++ or other ionic substance.
  • Trp101Ser missense mutation was found only in 1/110 migraine patients (s132, migraine without aura) and not in 711 other individuals.
  • the G458xO3Av6 variation consists of a C to T substitution at position c.718 resulting in a silent mutation (Leu240Leu). This silent variation was found only in one migraine patient (s296) out of 110 migraine patients, and not in 258 non-migraine individuals. Although the variation is not predicted to change the amino acid composition, analysis using ESE Finder 3.0 predicted that the variation would destroy at least two exon splicing enhancer elements, and could presumably interfere with the proper splicing or expression of the gene. Improper splicing of the KCNK18 transcript could lead to premature truncation of the KCNK18 protein, and loss of KCNK18 function.
  • the G458xO1v5 variation consists of a C to T substitution at position c.101 resulting in a missense mutation (Ala34Val).
  • This missense variation was found only in one migraine patient (s1900) out of 553 migraine patients, and not in 1782 non-migraine individuals.
  • the Ala34 residue is located within transmembrane domain 1 of the channel, and is conserved in orthologs of KCNK18 from mammals (11 species), chicken, fish (3 species), and worm (C. elegans) suggesting functional conservation (Figure 8).
  • the rs363344 variant was genotyped using the lllumina platform technology in 368 patient samples and 368 control samples.
  • the variant was significantly under-represented in migraine populations versus non-migraine populations, with a Fisher's exact test yielding a p value of 0.049.
  • haplotype block is characterized by precise allelic frequency differences between Causasian, Asian and African populations: minor allele frequency of 0.008 in Caucasian, 0.144 in Asian, and 0.133 in African populations.
  • the haplotype contains at least 34 single nucleotide variations that have been genotyped by the HapMap project, and spans at least 19 kb.
  • KCNK18 IS EXPRESSED IN TISSUES IMPORTANT IN MIGRAINE
  • ISH in situ hybridization
  • mKCNK18 mRNA was undetectable in embryonic stages e ⁇ .5; e4.5; e5.5; e6.5; e8.5; e9.5 and e12.5 but detectable at later intrauterine life stages starting on e15.5.
  • mKcnk18 mRNA expression was restricted to the embryonic, postnatal and adult mouse peripheral nervous system (PNS) including trigeminal ganglion (TG) and dorsal root ganglia (DRG).
  • mKCNK18 mRNA levels were relatively low in TG and DRG on day 15.5, increased through e18 stage to reach a peak in newborn mouse (p1) followed by a decline in adulthood.
  • Microanalysis performed under microscopy identified mKCNK18 expression in a subpopulation of adult mouse medium-size and small-size sensory neurons, suggesting expression in B or C fibres (or a combination of B and C fibres).
  • mKcnk18 mRNA was expressed exclusively in peripheral nervous system structures such as the TG, DRG, SG and PVG, and was not detected in any other mouse tissues.
  • KCNK18 Expression of KCNK18 in human tissues was tested using quantitative RT- PCR (Applied Biosystems, TaqMan Gene Expression Assay Hs00699272_m1) on a panel of human mRNAs isolated from various tissues. GAPDH expression was used as an endogenous control (Applied Biosystems, Cat#: 4326317E). PCR reagents were purchased from ABI and used according to the manufacturer's instructions. Optical 96-well plates and optical seals were used for the experiments. The experiment was run on a ABI7000 thermocycler and the data analysed using SDS v1.2.3 software.
  • KCNK18 expression could be detected specifically in dorsal root ganglion (DRG), and not in any other of the 21 tissues tested. Based on comparison with the expression of the human GAPDH gene, the KCNK18 gene was expressed at a level 9000 times higher in DRG than in any other tissue tested. Hence the expression pattern seen in human tissues is consistent with the specific PNS expression pattern seen in mouse.
  • DRG dorsal root ganglion
  • the trigeminal nerve and trigeminal ganglia are central to the pathogenesis of migraine.
  • the trigeminal nerve afferents become activated by a mechanism that may involve cortical spreading depression or other type of neuronal hyperexcitability.
  • Activated TG afferents innervating the meningeal blood vessels secrete pro-inflammatory peptides (such as CGRP and substance P) that causes local inflammation, and intensifies activation of the TG afferents. This leads to central desensitization and pain perception, symptoms of which include photophobia, phonophobia and headache.

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Abstract

La présente invention concerne la migraine et l'identification d'un marqueur de gène associé à la migraine. L'invention concerne en outre des séquences d'acide nucléique, et des séquences de protéine de ce locus ainsi que leur utilisation pour évaluer, diagnostiquer, pronostiquer ou traiter la migraine afin de prévoir la réponse au médicament chez un patient migraineux et d'identifier les agents qui modulent la fonction dudit marqueur ou de ladite protéine. L'invention concerne également des essais de criblage utilisant un tel gène ou une telle protéine ou des fragments de ceux-ci afin d'identifier des composés, qui sont bénéfiques pour les patients migraineux ou souffrant de troubles associés à la migraine. Enfin, l'invention concerne des procédés pour produire des modèles animaux de migraine par la modulation de l'expression de KCNK18 et/ou la modulation de l'expression de facteurs qui modulent l'expression de KCNK18.
PCT/CA2007/002084 2006-11-17 2007-11-16 Procédés pour le diagnostic, le pronostic ou le traitement de la migraine et de troubles associés Ceased WO2008058399A1 (fr)

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WO2018154111A1 (fr) * 2017-02-24 2018-08-30 Aalborg Universitet Méthode de diagnostic de migraine
CN109658980A (zh) * 2018-03-20 2019-04-19 上海交通大学医学院附属瑞金医院 一种粪便基因标志物的筛选及应用
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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018154111A1 (fr) * 2017-02-24 2018-08-30 Aalborg Universitet Méthode de diagnostic de migraine
CN109658980A (zh) * 2018-03-20 2019-04-19 上海交通大学医学院附属瑞金医院 一种粪便基因标志物的筛选及应用
CN109658980B (zh) * 2018-03-20 2023-05-09 上海交通大学医学院附属瑞金医院 一种粪便基因标志物的筛选及应用
US11154240B2 (en) 2019-04-02 2021-10-26 Kpn Innovations Llc Methods and systems for utilizing diagnostics for informed vibrant constitutional guidance
US11289206B2 (en) 2020-06-02 2022-03-29 Kpn Innovations, Llc. Artificial intelligence methods and systems for constitutional analysis using objective functions
US12176108B2 (en) 2020-06-02 2024-12-24 Kpn Innovations Llc Artificial intelligence methods and systems for constitutional analysis using objective functions
US11211158B1 (en) 2020-08-31 2021-12-28 Kpn Innovations, Llc. System and method for representing an arranged list of provider aliment possibilities
CN114317716A (zh) * 2021-12-31 2022-04-12 上海锐翌生物科技有限公司 痛风症标志基因及其应用

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