EP1280902A2 - 23949 et 32391, nouveaux canaux ioniques humains, et utilisation associee - Google Patents

23949 et 32391, nouveaux canaux ioniques humains, et utilisation associee

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Publication number
EP1280902A2
EP1280902A2 EP01930885A EP01930885A EP1280902A2 EP 1280902 A2 EP1280902 A2 EP 1280902A2 EP 01930885 A EP01930885 A EP 01930885A EP 01930885 A EP01930885 A EP 01930885A EP 1280902 A2 EP1280902 A2 EP 1280902A2
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Prior art keywords
nucleic acid
polypeptide
protein
seq
acid molecule
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EP01930885A
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German (de)
English (en)
Inventor
Maria A. Glucksmann
Rory A. J. Curtis
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Millennium Pharmaceuticals Inc
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Millennium Pharmaceuticals Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the ion channel family of proteins is a large family of membrane-bound proteins responsible for a wide range of important transport and signaling functions in cells.
  • the ion channel family includes at least three subfamilies: calcium ion channels (i.e., Ca channels), potassium channels (i.e., K channels) and sodium channels (Na channels).
  • Ca channels calcium ion channels
  • K channels potassium channels
  • Na channels sodium channels
  • Members of the ion channel family are characterized by the presence of six (6) transmembrane helices in which the last two helices flank a loop which determines ion selectivity.
  • the domain is repeated four times
  • others e.g., K channels
  • the protein forms as a tetramer in the membrane.
  • Calcium channel proteins are involved in the control of neurotransmitter release from neurons (Williams et al. (1992) Science 257:389-395), and play an important role in the regulation of a variety of cellular functions, including membrane excitability, muscle contraction and synaptic transmission (Mori et al. (1991) Nature 350:398-402).
  • the calcium channel proteins are composed of four (4) tightly-coupled subunits ( ⁇ l, ⁇ 2, ⁇ and ⁇ ), the ⁇ l subunit from each creating the pore for the import of extracellular calcium ions.
  • the ⁇ l subunit shares sequence characteristics with all voltage-dependent cation channels, and exploits the same 6-helix bundle structural motif.
  • Sodium channels are transmembrane (TM) voltage-dependent proteins responsible for the depolarising phase of the action potential in most electrically excitable cells (George et al. (1992) Proc. Natl. Acad. Sci. USA 89:4893-4897). They may exist in 3 states (Noda et al. (1984) Nature 312:121-127): the resting state, where the channel is closed; the activated state, where the channel is open; and the inactivated state, where the channel is closed.
  • Several different structurally and functionally distinct isoforms are found in mammals, coded for by a multigene family (Rogart et al. (1989) Proc. Natl. Acad. Sci.
  • the structure of sodium channels is based on 4 internal repeats of a 6- helix bundle (Noda et al. (1986) Nature 320:188-192) (in which 5 of the membrane- spanning segments are hydrophobic and the other is positively charged), forming a 24- helical bundle.
  • the charged segments are believed to be localized within clusters formed by their 5 hydrophobic neighbors. It is postulated that the charged domain may be the voltage sensor region, possibly moving outward on depolarization, causing a conformational change.
  • Potassium channels are the most diverse group of the ion channel family (possibly as a result of gene duplication and alternative splicing of the genes (Perney and Kaczmarek (1991) Curr. Opin. Cell. Biol. 3:663-670 and Luneau et al. (1991) FEBS Lett. 288:163-167). They are important in shaping the action potential, and in neuronal excitability and plasticity (Tempel et al. (1988) Nature 332:837-839).
  • the potassium channel family is composed of several functionally distinct isoforms, which can be broadly separated into 2 groups
  • Potassium channels are transmembrane (TM) proteins that contain 6 membrane- spanning ⁇ - helical segments, 5 of which are hydrophobic, the other being positively charged. The charged segment is believed to be localized within a cluster formed by the hydrophobic helices. As with Na channels, it is postulated that the charged segment may constitute the voltage sensor region, possibly moving outward on depolarisation, causing a conformational change.
  • the 6-helix bundle is a common structural motif in sodium channels (in which it is repeated 4 times within the sequence to form a 24-helix bundle), and in calcium channels (where it also forms a 24-helix bundle, which itself is tightly bound to 3 different subunits).
  • Ion channels play a role in regulating ion transport and signaling in virtually every cell in the human body.
  • the present invention is based, at least in part, on the discovery of novel ion channel family members, refened to herein as ion channels 23949 and 32391, or "IC" (e.g., IC23949 and IC32391) nucleic acid and protein molecules.
  • IC e.g., IC23949 and IC32391
  • the IC molecules of the present invention are useful as targets for developing modulating agents to regulate a variety of cellular processes, including ion transport (e.g., ion conductance); membrane excitability and/or polarization; synaptic transmission; signal transduction; cell activation, proliferation, growth, differentiation and/or migration; and muscle contraction.
  • this invention provides isolated nucleic acid molecules encoding IC proteins or biologically active portions thereof, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection of IC-encoding nucleic acids.
  • an IC nucleic acid molecule of the invention is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the nucleotide sequence (e.g., to the entire length of the nucleotide sequence) shown in SEQ ID NO: 1 , 3 , 4, or 6 or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number or , or a complement thereof.
  • the isolated nucleic acid molecules include the nucleotide sequences shown in SEQ ID NO: 1, 3, 4, or 6, or a complement thereof.
  • the IC23949 nucleic acid molecule includes SEQ ID NO:3 and nucleotides 1-244 of SEQ ID NO:l.
  • the IC23949 nucleic acid molecule includes SEQ ID NO:3 and nucleotides 5462-7007 of SEQ ID NO:l.
  • the IC32391 nucleic acid molecule includes SEQ ID NO:6 and nucleotides 1-206 of SEQ TD NO:4.
  • the IC32391 nucleic acid molecule includes SEQ ID NO:6 and nucleotides 3237-3534 of SEQ ID NO:4.
  • the nucleic acid molecule consists of the nucleotide sequence shown in SEQ ID NO:l, 3, 4, or 6.
  • the nucleic acid molecule includes a fragment of at least 50 nucleotides (e.g., 50 contiguous nucleotides) of the nucleotide sequence of SEQ ID NO:l, 3, 4, or 6 or a complement thereof.
  • an IC nucleic acid molecule includes a nucleotide sequence encoding a protein having an amino acid sequence sufficiently identical to the amino acid sequence of SEQ ID NO:2 or 5 or an amino acid sequence encoded by the DNA insert of the plasmid deposited with ATCC as Accession Number or .
  • an IC nucleic acid molecule includes a nucleotide sequence encoding a protein having an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the entire length of the amino acid sequence of SEQ ID NO: 2 or 5 or the amino acid sequence encoded by the DNA insert of the plasmid deposited with ATCC as Accession Number or .
  • an isolated nucleic acid molecule encodes the amino acid sequence of human IC23949 or human IC32391.
  • the nucleic acid molecule includes a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID NO: 2 or 5 or the amino acid sequence encoded by the DNA insert of the plasmid deposited with ATCC as Accession Number or .
  • the nucleic acid molecule is at least 50 nucleotides in length.
  • the nucleic acid molecule is at least 50 nucleotides in length and encodes a protein having IC23949 or IC32391 activity (as described herein).
  • Another embodiment of the invention features nucleic acid molecules, preferably
  • IC23949 nucleic acid molecules which specifically detect IC23949 and IC32391 nucleic acid molecules relative to nucleic acid molecules encoding non-IC23949 and non-IC32391 proteins.
  • a nucleic acid molecule is at least 50-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, 900-1000, 1000-1100, 1100-1200, 1200-1300, 1300-1400, 1400-1500, 1500-1600, 1600-1700, 1700- 1800, 1800-1900, 1900-2000, 2000-2100, 2100-2200, 2200-2300, 2300-2400, 2400-2500, 2500-2600, 2600-2700, 2700-2800, 2800-2900, 2900-3000, 3000-3100, 3100-3200, 3200- 3300, 3300-3400, 3400-3500, 3500-3600, 3600-3700, 3700-3800, 3800-3900, 3500-3600, 3600
  • the nucleic acid molecules are at least 15 (e.g., 15 contiguous) nucleotides in length and hybridize under stringent conditions to SEQ ID NO:l or 4.
  • the nucleic acid molecule encodes a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:2 or 5 or an amino acid sequence encoded by the DNA insert of the plasmid deposited with ATCC as Accession Number or , wherein the nucleic acid molecule hybridizes to a nucleic acid molecule comprising SEQ ID NO:l, 3, 4 or 6 under stringent conditions.
  • Another embodiment of the invention provides an isolated nucleic acid molecule which is antisense to an IC nucleic acid molecule, e.g., the coding strand of an IC nucleic acid molecule.
  • Another aspect of the invention provides a vector comprising an IC nucleic acid molecule.
  • the vector is a recombinant expression vector.
  • the invention provides a host cell containing a vector of the invention.
  • the invention provides a host cell containing a nucleic acid molecule of the invention.
  • the invention also provides a method for producing a protein, preferably an IC protein, by culturing in a suitable medium, a host cell, e.g., a mammalian host cell such as a non-human mammalian cell, of the invention containing a recombinant expression vector, such that the protein is produced.
  • Another aspect of this invention features isolated or recombinant IC proteins and polypeptides.
  • an isolated IC23949 protein has one or more of the following domains: a transmembrane domain and an ion transport protein domain.
  • an IC23949 protein includes at least one or more of the following domains: a transmembrane domain and an ion transport protein domain and has an amino acid sequence at least about 50%, 55%, 60%, 65%, 67%, 68%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%), 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:2, or the amino acid sequence encoded by the DNA insert of the plasmid deposited with ATCC as Accession Number .
  • an isolated IC32391 protein has one or more of the following domains: a transmembrane domain and a ligand-gated ion channel domain.
  • an IC32391 protein includes at least one or more of the following domains: a transmembrane domain and a ligand-gated ion channel domain, and has an amino acid sequence at least about 50%, 55%, 60%, 65%, 67%, 68%, 70%, 75%, 80%, 85%, 90%, 95%), 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:5, or the amino acid sequence encoded by the DNA insert of the plasmid deposited with ATCC as Accession Number .
  • an IC protein in another prefened embodiment, includes at least one transmembrane domain and has an IC activity (as described herein).
  • an IC23949 protein includes one or more of the following domains: a transmembrane domain and an ion transport protein domain and is encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:l or 3.
  • an IC32391 protein includes one or more of the following domains: a transmembrane domain and a ligand-gated ion channel domain, and is encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:4 or 6.
  • the invention features fragments of the protein having the amino acid sequence of SEQ ID NO:2 or 5, wherein the fragment comprises at least 15 amino acids (e.g., contiguous amino acids) of the amino acid sequence of SEQ ID NO:2 or
  • an IC protein has the amino acid sequence of SEQ ID NO:2 or 5.
  • the invention features an IC protein which is encoded by a nucleic acid molecule consisting of a nucleotide sequence at least about 50%, 55%, 60%,
  • This invention further features an IC protein, which is encoded by a nucleic acid molecule consisting of a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, 3, 4, or 6, or a complement thereof.
  • the proteins of the present invention or portions thereof, e.g., biologically active portions thereof, can be operatively linked to a non-IC polypeptide (e.g., heterologous amino acid sequences) to form fusion proteins.
  • the invention further features antibodies, such as monoclonal or polyclonal antibodies, that specifically bind proteins of the invention, preferably IC proteins.
  • the IC proteins or biologically active portions thereof can be incorporated into pharmaceutical compositions, which optionally include pharmaceutically acceptable carriers.
  • the present invention provides a method for detecting the presence of an IC nucleic acid molecule, protein, or polypeptide in a biological sample by contacting the biological sample with an agent capable of detecting an IC nucleic acid molecule, protein, or polypeptide such that the presence of an IC nucleic acid molecule, protein or polypeptide is detected in the biological sample.
  • the present invention provides a method for detecting the presence of IC activity in a biological sample by contacting the biological sample with an agent capable of detecting an indicator of IC activity such that the presence of IC activity is detected in the biological sample.
  • the invention provides a method for modulating IC activity comprising contacting a cell capable of expressing IC with an agent that modulates IC activity such that IC activity in the cell is modulated.
  • the agent inhibits IC activity.
  • the agent stimulates IC activity.
  • the agent is an antibody that specifically binds to an IC protein.
  • the agent modulates expression of IC by modulating transcription of an IC gene or translation of an IC mRNA.
  • the agent is a nucleic acid molecule having a nucleotide sequence that is antisense to the coding strand of an IC mRNA or an IC gene.
  • the methods of the present invention are used to treat a subject having a disorder characterized by abenant or unwanted IC protein or nucleic acid expression or activity by administering an agent which is an IC modulator to the subject.
  • the IC modulator is an IC protein.
  • the IC modulator is an IC nucleic acid molecule.
  • the IC modulator is a peptide, peptidomimetic, or other small molecule.
  • the disorder characterized by abenant or unwanted IC protein or nucleic acid expression is a CNS disorder, such as a cognitive or neurodegenerative disorder.
  • the disorder characterized by abenant or unwanted IC protein or nucleic acid expression is a cardiovascular disorder.
  • the disorder characterized by abenant or unwanted IC protein or nucleic acid expression is a muscular disorder.
  • the disorder characterized by abenant or unwanted IC activity is a pain disorder.
  • the disorder characterized by abenant or unwanted IC activity is a cell proliferation, growth, differentiation, or migration disorder.
  • the present invention also provides diagnostic assays for identifying the presence or absence of a genetic alteration characterized by at least one of (i) abenant modification or mutation of a gene encoding an IC protein; (ii) mis-regulation of the gene; and (iii) abenant post-translational modification of an IC protein, wherein a wild-type form of the gene encodes a protein with an IC activity.
  • the invention provides methods for identifying a compound that binds to or modulates the activity of an IC protein, by providing an indicator composition comprising an IC protein having IC activity, contacting the indicator composition with a test compound, and determining the effect of the test compound on IC activity in the indicator composition to identify a compound that modulates the activity of an IC protein.
  • Figures 1A-F depicts the cDNA sequence and predicted amino acid sequence of human IC23949.
  • the coding region without the 5 'or 3' untranslated region of the human IC23949 gene is shown in SEQ ID NO:3.
  • Figures 2 A-C depicts the cDNA sequence and predicted amino acid sequence of human IC32391.
  • the coding region without the 5' or 3' untranslated region of the human IC32391 gene is shown in SEQ ID NO:6.
  • Figure 3 depicts a hydrophobicity plot of the amino acid sequence of human IC23949 (SEQ ID NO:2).
  • Figure 4 depicts a hydrophobicity plot of the amino acid sequence of human IC32391 (SEQ ID NO:5).
  • Figure 5 depicts the results of a search which was performed against the MEMS AT database and which resulted in the identification of eighteen (18) "transmembrane domains" in the human IC23949 protein (SEQ ID NO:2).
  • Figure 6 depicts the results of a search which was performed against the MEMS AT database and which resulted in the identification of four (4) "transmembrane domains" in the human IC32391 protein (SEQ ID NO:5).
  • Figures 7 A-C depicts the results of a search which was performed against the HMM database and which resulted in the identification of four (4) "ion transport protein domains" in the human IC23949 protein (SEQ ID NO:2).
  • Figures 8A-D depicts the results of a search which was performed against the HMM database and which resulted in the identification of a "ligand-gated ion channel domain" in the human IC32391 protein (SEQ ID NO:5).
  • Figures 9A-F depicts the results of a search which was performed against the ProDom database.
  • Figures 10A-E depicts the results of a search which was performed against the
  • Figure 11 is a graphic depiction of the relative levels of human IC32391 mRNA expression in a human normal tissue panel, as determined using TaqmanTM analysis.
  • Figure 12 is a graphic depiction of the relative levels of human IC32391 mRNA expression in a rat normal tissue panel, as determined using TaqmanTM analysis.
  • Figure 13 is a graphic depiction of the relative levels of human IC32391 mRNA expression in a human and monkey normal tissue panel, as determined using TaqmanTM analysis.
  • the present invention is based, at least in part, on the discovery of novel molecules, refened to herein as IC (for ion channels 23949 and 32391) nucleic acid and protein molecules, which are novel members of the ion channel family.
  • IC for ion channels 23949 and 32391
  • novel molecules are capable of, for example, modulating ion transport in an electrically excitable cell (e.g., a neuronal or muscle cell, e.g., cardiac muscle cell), or in a non-electrically excitable cell, e.g., a spleen cell.
  • the term "ion channel” includes a protein or polypeptide which is involved in receiving, conducting, and transmitting signals in an cell (e.g., an electrically excitable cell, for example, a neuronal or muscle cell). Ion channels can determine membrane excitability (the ability of, for example, a cell to respond to a stimulus and to convert it into a sensory impulse). Ion channels can also influence the resting potential of membranes, wave forms and frequencies of action potentials, and thresholds of excitation. Ion channels are typically expressed in electrically excitable cells, e.g., neuronal cells, and may form heteromultimeric structures (e.g., composed of more than one type of subunit).
  • Ion channels may also be found in non-excitable cells (e.g., endothelial cells or spleen cells), where they may play a role in, for example, signal transduction.
  • ion channels include Ca 2+ ion channels, K + ion channels, and Na + ion channels.
  • the IC molecules of the present invention may modulate ion channel mediated activities, they may be useful for developing novel diagnostic and therapeutic agents for ion channel associated disorders.
  • an "ion channel associated disorder” includes a disorder, disease or condition which is characterized by a misregulation of an ion channel mediated activity. Ion channel associated disorders can detrimentally affect conveyance of sensory impulses from the periphery to the brain and/or conductance of motor impulses from the brain to the periphery; integration of reflexes; interpretation of sensory impulses; cellular proliferation, growth, differentiation, or migration, and emotional, intellectual (e.g., learning and memory), or motor processes.
  • ion channel associated disorders include CNS disorders such as cognitive and neurodegenerative disorders, examples of which include, but are not limited to, Alzheimer's disease, dementias related to Alzheimer's disease (such as Pick's disease), Parkinson's and other Lewy diffuse body diseases, senile dementia, Huntington's disease, Gilles de la Tourette's syndrome, multiple sclerosis, amyotrophic lateral sclerosis, progressive supranuclear palsy, epilepsy, and Jakob-Creutzfieldt disease; autonomic function disorders such as hypertension and sleep disorders, and neuropsychiatric disorders, such as depression, schizophrenia, schizoaffective disorder, korsakoff's psychosis, mania, anxiety disorders, or phobic disorders; learning or memory disorders, e.g., amnesia or age-related memory loss, attention deficit disorder, dysthymic disorder, major depressive disorder, mania, obsessive-compulsive disorder, psychoactive substance use disorders, anxiety, phobias, panic disorder,
  • Ion channel associated disorders also include pain disorders.
  • the IC molecules of the present invention may be present on sensory neurons and, thus, may be involved in detecting, for example, noxious chemical, mechanical, or thermal stimuli and transducing this information into membrane depolarization events.
  • the IC molecules by participating in pain signaling mechanisms, may modulate pain elicitation and act as targets for developing novel diagnostic targets and therapeutic agents to control pain.
  • pain disorders include posttherapeutic neuralgia, diabetic neuropathy, postmastectomy pain syndrome, stump pain, reflex sympathetic dystrophy, trigeminal neuralgia, neuropathic pain, orofacial neuropathic pain, osteoarthritis, rheumatoid arthritis, fibromyalgia syndrome, tension myalgia, Guillian-Ba ⁇ e syndrome, Meralgia paraesthetica, burning mouth syndrome, fibrbcitis.
  • myofascial pain syndrome idiopathic pain disorder, temporomandibular joint syndrome, atypical odontalgia, loin pain, haematuria syndrome, non-cardiac chest pain, low back pain, chronic nonspecific pain, psychogenic pain, musculoskeletal pain disorder, chronic pelvic pain, nonorganic chronic headache, tension- type headache, cluster headache, migraine, complex regional pain syndrome, vaginismus, nerve trunk pain, somatoform pain disorder, cyclical mastalgia, chronic fatigue syndrome, multiple somatization syndrome, chronic pain disorder, somatization disorder, Syndrome X, facial pain, idiopathic pain disorder, posttraumatic rheumatic pain modulation disorder (fibrositis syndrome), hyperalgesia, and Tangier disease.
  • Cardiovascular system disorders in which the IC molecules of the invention may be directly or indirectly involved include arteriosclerosis, ischemia reperfusion injury, restenosis, arterial inflammation, vascular wall remodeling, ventricular remodeling, rapid ventricular pacing, coronary microembolism, tachycardia, bradycardia, pressure overload, aortic bending, coronary artery ligation, vascular heart disease, atrial fibrilation, Jervell syndrome, Lange-Nielsen syndrome, long-QT syndrome, congestive heart failure, sinus node dysfunction, angina, heart failure, hypertension, atrial fibrillation, atrial flutter, dilated cardiomyopathy, idiopathic cardiomyopathy, myocardial infarction, coronary artery disease, coronary artery spasm, and anhythmia.
  • IC-mediated or related disorders also include disorders of the musculoskeletal system such as paralysis and muscle weakness, e.g., ataxia, myotonia, and myokymia.
  • Ion channel-associated disorders also include cellular proliferation, growth, differentiation, or migration disorders.
  • Cellular proliferation, growth, differentiation, or migration disorders include those disorders that affect cell proliferation, growth, differentiation, or migration processes.
  • a "cellular proliferation, growth, differentiation, or migration process" is a process by which a cell increases in number, size or content, by which a cell develops a specialized set of characteristics which differ from that of other cells, or by which a cell moves closer to or further from a particular location or stimulus.
  • the IC molecules of the present invention are involved in signal transduction mechanisms, which are known to be involved in cellular growth, differentiation, and migration processes.
  • the IC molecules may modulate cellular growth, differentiation, or migration, and may play a role in disorders characterized by abenantly regulated growth, differentiation, or migration.
  • Such disorders include cancer, e.g., carcinoma, sarcoma, or leukemia; tumor angiogenesis and metastasis; skeletal dysplasia; neuronal deficiencies resulting from impaired neural induction and patterning; neurodegenerative disorders, e.g., Alzheimer's disease, dementias related to Alzheimer's disease (such as Pick's disease), Parkinson's and other Lewy diffuse body diseases, multiple sclerosis, amyotrophic lateral sclerosis, progressive supranuclear palsy, epilepsy, Jakob-Creutzfieldt disease, or AIDS related dementia; hepatic disorders; cardiovascular disorders; and hematopoietic and/or myeloproliferative disorders.
  • IC-associated or related disorders also include disorders of tissues in which IC molecules are expressed, e.g., brain, including the hypothalamus and cortex, spinal cord, dorsal root ganglia, and heart.
  • an "ion channel mediated activity” includes an activity which involves an ion channel, e.g., an ion channel associated with receiving, conducting, and transmitting signals, in electrically excitable or non-electrically excitable cells.
  • Ion channel mediated activities include release of neurotransmitters or second messenger molecules, e.g., dopamine or norepinephrine, from cells, e.g., neuronal cells; modulation of resting potential of membranes, wave forms and frequencies of action potentials, and thresholds of excitation; participation in signal transduction pathways; and modulation of processes such as integration of sub-threshold synaptic responses and the conductance of back-propagating action potentials in, for example, neuronal cells (e.g., changes in those action potentials resulting in a morphological or differentiative response in the cell).
  • neurotransmitters or second messenger molecules e.g., dopamine or norepinephrine
  • family when referring to the protein and nucleic acid molecules of the invention is intended to mean two or more proteins or nucleic acid molecules having a common structural domain or motif and having sufficient amino acid or nucleotide sequence homology as defined herein.
  • family members can be naturally or non-naturally occurring and can be from either the same or different species.
  • a family can contain a first protein of human origin, as well as other, distinct proteins of human origin or alternatively, can contain homologues of non-human origin, e.g., monkey proteins.
  • Members of a family may also have common functional characteristics.
  • the family of IC proteins comprises at least one "transmembrane domain" and preferably two transmembrane domains.
  • transmembrane domain includes an amino acid sequence of about 20 amino acid residues in length which spans the plasma membrane. More preferably, a transmembrane domain includes about at least 15, 20, 25, 30, 35, 40, or 45 amino acid residues and spans the plasma membrane. Transmembrane domains are rich in hydrophobic residues, and typically have an alpha-helical structure. In a prefened embodiment, at least 50%, 60%, 70%, 80%, 90%, 95% or more of the amino acids of a transmembrane domain are hydrophobic, e.g., leucines, isoleucines, tyrosines, or tryptophans. Transmembrane domains are described in, for example, Zaklaklad.N.
  • IC23949 proteins having at least 50-60% homology preferably about 60-70%, more preferably about 70-80%, or about 80-90% homology with a transmembrane domain of human IC23949 are within the scope of the invention.
  • Amino acid residues 86-102, 563- 583, 637-658, and 831-855 of the native IC32391 protein are predicted to comprise transmembrane domains (see Figures 4 and 6).
  • IC32391 proteins having at least 50-60% homology preferably about 60-70%, more preferably about 70-80%, or about 80-90% homology with a transmembrane domain of human IC32391 are within the scope of the invention.
  • an IC molecule of the present invention is identified based on the presence of at least one "ion transport protein domain" in the protein or conesponding nucleic acid molecule.
  • the term "ion transport protein domain” includes a protein domain having an amino acid sequence of about 200-400 amino acid residues and having a bit score for the alignment of the sequence to the ion transport protein domain of at least 15, 25, 50, 75, 100, 150 or higher.
  • an ion transport protein domain includes at least about 200-300 amino acid residues, or more preferably about 250-300 amino acid residues, and has a bit score for the alignment of the sequence to the ion transport protein domain of at least 16.9.
  • the amino acid sequence of the protein is searched against a database of known protein domains (e.g., the HMM database) using the default parameters.
  • an IC molecule of the present invention is identified based on the presence of at least one "ligand-gated ion channel domain" in the protein or conesponding nucleic acid molecule.
  • ligand-gated ion channel domain includes a protein domain having an amino acid sequence of about 200-400 amino acid residues and having a bit score for the alignment of the sequence to ligand-gated ion channel domain of at least 50, 100, 150, 200, 250, 300 or higher.
  • a ligand-gated ion channel domain includes at least about 250-350 amino acid residues, or more preferably about 300-350 amino acid residues, and has a bit score for the alignment of the sequence to the ligand-gated ion channel domain of at least 214.9.
  • the amino acid sequence of the protein is searched against a database of known protein domains (e.g., the HMM database) using the default parameters.
  • a search was performed against the HMM database resulting in the identification of a ligand-gated ion channel domain in the amino acid sequence of human IC32391 (SEQ ID NO:5) at about residues 652-852 of SEQ ID NO:5.
  • the results of the search are set forth in Figure 8A-D.
  • Isolated proteins of the present invention preferably IC proteins, have an amino acid sequence sufficiently identical to the amino acid sequence of SEQ ID NO:2 or 5 or are encoded by a nucleotide sequence sufficiently identical to SEQ ID NO:l, 3, 4, or 6.
  • the term "sufficiently identical” refers to a first amino acid or nucleotide sequence which contains a sufficient or minimum number of identical or equivalent (e.g., an amino acid residue which has a similar side chain) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences share common structural domains or motifs and/or a common functional activity.
  • amino acid or nucleotide sequences which share common structural domains having at least 30%, 40%, or 50% homology, preferably 60% homology, more preferably 70%-80%, and even more preferably 90-95% homology across the amino acid sequences of the domains and contain at least one and preferably two structural domains or motifs, are defined herein as sufficiently identical.
  • amino acid or nucleotide sequences which share at least 30%, 40%, or 50%, preferably 60%, more preferably 70-80%, or 90-95% homology and share a common functional activity are defined herein as sufficiently identical.
  • an "IC activity”, “biological activity of IC “ or “functional activity of IC” refers to an activity exerted by an IC protein, polypeptide or nucleic acid molecule on an IC responsive cell or tissue, or on an IC protein substrate, as determined in vivo, or in vitro, according to standard techniques.
  • an IC activity is a direct activity, such as an association with an IC -target molecule.
  • a "target molecule” or “binding partner” is a molecule with which an IC protein binds or interacts in nature, such that IC -mediated function is achieved.
  • An IC target molecule can be a non-IC molecule or an IC protein or polypeptide of the present invention.
  • an IC target molecule is an IC ligand, e.g., an ion channel pore-forming subunit or an ion channel ligand.
  • an IC activity is an indirect activity, such as a cellular signaling activity mediated by interaction of the IC protein with an IC ligand. The biological activities of IC are described herein.
  • the IC proteins of the present invention can have one or more of the following activities: (1) modulation of membrane excitability; (2) modulation of intracellular ion concentration; (3) modulation of membrane polarization (e.g., membrane polarization and/or depolarization); (4) modulation of action potential; (5) modulation of cellular signal transduction; (6) modulation of neurotransmitter release (e.g., from neuronal cells); (7) modulation of synaptic transmission; (8) modulation of neuronal excitability and/or plasticity; (9) modulation of muscle contraction; (10) modulation of cell activation (e.g., T cell activation); and/or (11) modulation of cellular proliferation, growth, migration and/or differentiation.
  • modulation of membrane excitability modulation of intracellular ion concentration
  • modulation of membrane polarization e.g., membrane polarization and/or depolarization
  • modulation of action potential e.g., cellular signal transduction
  • (6) modulation of neurotransmitter release e.
  • IC proteins and polypeptides having an IC activity are isolated IC proteins and polypeptides having an IC activity.
  • Prefened proteins are IC proteins having one or more of the following domains: a transmembrane domain, an ion transport protein domain, a ligand- gated ion channel domain, and, preferably, an IC activity.
  • Yet other prefened proteins are IC proteins having at least one of the following domains: a transmembrane domain, an ion transport protein domain, a ligand-gated ion channel domain, and, preferably, an IC activity.
  • Additional prefened proteins have one or more of the following domains: a transmembrane domain, an ion transport protein domain, a ligand-gated ion channel domain, and are, preferably, encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1 , 3, 4, or 6.
  • nucleotide sequence of the isolated human IC cDNAs and the predicted amino acid sequences of the human IC polypeptides of the present invention are shown in Figures 1A-F and 2A-C and in SEQ ID NOs:l, 4, 2, and 5.
  • a plasmid containing the nucleotide sequence encoding human IC23949 was deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA 20110-2209, on and assigned Accession Number .
  • a plasmid containing the nucleotide sequence encoding human IC32391 was deposited with the American Type Culture Collection
  • the human IC23949 gene which is approximately 7007 nucleotides in length, encodes a protein having a molecular weight of approximately 191.2 kD and which is approximately 1738 amino acid residues in length.
  • the human IC32391 gene which is approximately 3534 nucleotides in length, encodes a protein having a molecular weight of approximately 111.1 kD and which is approximately 1010 amino acid residues in length.
  • nucleic acid molecules that encode IC proteins or biologically active portions thereof, as well as nucleic acid fragments sufficient for use as hybridization probes to identify IC-encoding nucleic acid molecules (e.g. , IC mRNA) and fragments for use as PCR primers for the amplification or mutation of IC nucleic acid molecules.
  • nucleic acid molecule is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs.
  • the nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.
  • isolated nucleic acid molecule includes nucleic acid molecules which are separated from other nucleic acid molecules which are present in the natural source of the nucleic acid.
  • isolated includes nucleic acid molecules which are separated from the chromosome with which the genomic DNA is naturally associated.
  • an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid (i.e. , sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.
  • the isolated IC nucleic acid molecule can contain less than about 5 kb, 4kb, 3kb, 2kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived.
  • an "isolated" nucleic acid molecule such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • a nucleic acid molecule of the present invention e.g., a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1 , 3 , 4, or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number or or a portion thereof, can be isolated using standard molecular biology techniques and the sequence information provided herein.
  • IC nucleic acid molecules can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).
  • nucleic acid molecule encompassing all or a portion of SEQ ID NO: 1, 3, 4, or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number or can be isolated by the polymerase chain reaction (PCR) using synthetic ohgonucleotide primers designed based upon the sequence of SEQ ID NO:l, 3, 4, or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number or .
  • PCR polymerase chain reaction
  • a nucleic acid of the invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate ohgonucleotide primers according to standard PCR amplification techniques.
  • the nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.
  • oligonucleotides conesponding to IC nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
  • an isolated nucleic acid molecule of the invention comprises the nucleotide sequence shown in SEQ ID NO: 1 or 4.
  • the cDNA of SEQ ID NO:l comprises sequences encoding the human IC23949 protein (i.e., "the coding region", from nucleotides 245-5461), as well as 5' untranslated sequences (nucleotides 1-244) and 3' untranslated sequences (nucleotides 5462-7007).
  • the nucleic acid molecule can comprise only the coding region of SEQ ID NO: 1 (e.g., nucleotides 245-5461, conesponding to SEQ ID NO:3).
  • the cDNA of SEQ ID NO:4 comprises sequences encoding the human IC32391 protein (i.e., "the coding region", from nucleotides 207-3236), as well as 5' untranslated sequences (nucleotides 1-206) and 3' untranslated sequences (nucleotides 3237-3534).
  • the nucleic acid molecule can comprise only the coding region of SEQ ID NO:4 (e.g., nucleotides 207-3236, conesponding to SEQ ID NO:6).
  • an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule which is a complement of the nucleotide sequence shown in SEQ ID NO:l, 3, 4, or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number or , or a portion of any of these nucleotide sequences.
  • a nucleic acid molecule which is complementary to the nucleotide sequence shown in SEQ ID NO:l, 3, 4, or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number or is one which is sufficiently complementary to the nucleotide sequence shown in SEQ ID NO:l, 3, 4, or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as
  • an isolated nucleic acid molecule of the present invention comprises a nucleotide sequence which is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%), 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the entire length of the nucleotide sequence shown in SEQ ID NO:l, 3, 4, or 6, or the entire length of the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as
  • nucleic acid molecule of the invention can comprise only a portion of the nucleic acid sequence of SEQ ID NO: 1 , 3, 4, or 6, or the nucleotide sequence of the
  • the nucleotide sequence determined from the cloning of the IC gene allows for the generation of probes and primers designed for use in identifying and/or cloning other IC family members, as well as IC homologues from other species.
  • the probe/primer typically comprises substantially purified ohgonucleotide.
  • the ohgonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12 or 15, preferably about 20 or 25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75 consecutive nucleotides of a sense sequence of SEQ ID NO: 1, 3, 4, or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number or , of an anti-sense sequence of SEQ ID NO: 1 , 3 , 4, or 6, or the nucleotide sequence of the
  • DNA insert of the plasmid deposited with ATCC as Accession Number or or of a naturally occurring allelic variant or mutant of SEQ ID NO:l, 3, 4, or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number or , or of a naturally occurring allelic variant or mutant of SEQ ID NO:l, 3, 4, or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession
  • a nucleic acid molecule of the present invention comprises a nucleotide sequence which is greater than 50-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, 900-1000, 1000-1100, 1100-1200, 1200-1300, 1300-1400, 1400-1500, 1500-1600, 1600-1700, 1700-1800, 1800-1900, 1900- 2000, 2000-2100, 2100-2200, 2200-2300, 2300-2400, 2400-2500, 2500-2600, 2600-2700, 2700-2800, 2800-2900, 2900-3000, 3000-3100, 3100-3200, 3200-3300, 3300-3400, 3400- 3500, 3500-3600, 3600-3700, 3700-3800, 3800-3900, 3900-4000, 4000-4100, 4100-4200, 4200-4300, 4300-4400, 4400-4500, 4500-4600, 4600-4700, 4700, 4700, 4700,
  • Probes based on the IC nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins.
  • the probe further comprises a label group attached thereto, e.g., the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.
  • Such probes can be used as a part of a diagnostic test kit for identifying cells or tissue which misexpress an IC protein, such as by measuring a level of an IC-encoding nucleic acid in a sample of cells from a subject e.g., detecting IC mRNA levels or determining whether a genomic IC gene has been mutated or deleted.
  • a nucleic acid fragment encoding a "biologically active portion of an IC protein” can be prepared by isolating a portion of the nucleotide sequence of SEQ ID NO:l, 3, 4, or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number or , which encodes a polypeptide having an IC biological activity (the biological activities of the IC proteins are described herein), expressing the encoded portion of the IC protein (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of the IC protein.
  • the invention further encompasses nucleic acid molecules that differ from the nucleotide sequence shown in SEQ ID NO:l, 3, 4, or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number or due to degeneracy of the genetic code and thus encode the same IC proteins as those encoded by the nucleotide sequence shown in SEQ ID NO:l, 3, 4, or 6, or the nucleotide sequence of the
  • an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in SEQ ID NO:2 or 5.
  • SEQ ID NO: 1 the amino acid sequence shown in SEQ ID NO: 1
  • SEQ ID NO: 2 the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number
  • DNA sequence polymorphisms that lead to changes in the amino acid sequences of the IC proteins may exist within a population (e.g., the human population). Such genetic polymorphism in the IC genes may exist among individuals within a population due to natural allelic variation.
  • the terms "gene” and “recombinant gene” refer to nucleic acid molecules which include an open reading frame encoding an IC protein, preferably a mammalian IC protein, and can further include non-coding regulatory sequences, and introns.
  • Allelic variants of human IC include both functional and non-functional IC proteins.
  • Functional allelic variants are naturally occurring amino acid sequence variants of the human IC protein that maintain the ability to bind an IC ligand or substrate and/or modulate cell proliferation and/or migration mechanisms.
  • Functional allelic variants will typically contain only conservative substitution of one or more amino acids of SEQ ID NO:2 or 5, or substitution, deletion or insertion of non-critical residues in non-critical regions of the protein.
  • Non-functional allelic variants are naturally occurring amino acid sequence variants of the human IC protein that do not have the ability to either bind an IC ligand and/or modulate any of the IC activities described herein.
  • Non-functional allelic variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO: 2 or 5, or a substitution, insertion or deletion in critical residues or critical regions.
  • the present invention further provides non-human orthologues of the human IC protein.
  • Orthologues of the human IC protein are proteins that are isolated from non-human organisms and possess the same IC ligand binding and/or modulation of membrane excitability activities of the human IC protein.
  • Orthologues of the human IC protein can readily be identified as comprising an amino acid sequence that is substantially identical to SEQ ID NO:2 or 5.
  • nucleic acid molecules encoding other IC family members and, thus, which have a nucleotide sequence which differs from the IC sequences of SEQ ID NO:l, 3, 4, or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number or are intended to be within the scope of the invention.
  • IC cDNA can be identified based on the nucleotide sequence of human IC.
  • nucleic acid molecules encoding IC proteins from different species and which, thus, have a nucleotide sequence which differs from the IC sequences of SEQ ID NO:l, 3, 4, or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with
  • a mouse IC cDNA can be identified based on the nucleotide sequence of a human IC.
  • Nucleic acid molecules conesponding to natural allelic variants and homologues of the IC cDNAs of the invention can be isolated based on their homology to the IC nucleic acids disclosed herein using the cDNAs disclosed herein, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions. Nucleic acid molecules conesponding to natural allelic variants and homologues of the IC cDNAs of the invention can further be isolated by mapping to the same chromosome or locus as the IC gene.
  • an isolated nucleic acid molecule of the invention is at least 15, 20, 25, 30 or more nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:l, 3, 4, or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number or .
  • the nucleic acid is at least 50-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, 900-1000, 1000-1100, 1100-1200, 1200-1300, 1300-1400, 1400-1500, 1500-1600, 1600-1700, 1700-1800, 1800-1900, 1900-2000, 2000-2100, 2100-2200, 2200- 2300, 2300-2400, 2400-2500, 2500-2600, 2600-2700, 2700-2800, 2800-2900, 2900-3000, 3000-3100, 3100-3200, 3200-3300, 3300-3400, 3400-3500, 3500-3600, 3600-3700, 3700- 3800, 3800-3900, 3900-4000, 4000-4100, 4100-4200, 4200-4300, 4300-4400, 4400-4500, 4500-4600, 4600-4700, 4700-4800, 4800-4900, 4900-5000, 5000-5100, 5100
  • hybridizes under stringent conditions is intended to describe conditions for hybridization and washing under which nucleotide sequences that are significantly identical or homologous to each other remain hybridized to each other.
  • the conditions are such that sequences at least about 70%, more preferably at least about 80%, even more preferably at least about 85% or 90% identical to each other remain hybridized to each other.
  • stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, Ausubel et al, eds., John Wiley & Sons, Inc. (1995), sections 2, 4 and 6.
  • stringent hybridization conditions includes hybridization in 4X sodium chloride/sodium citrate (SSC), at about 65-70°C (or hybridization in 4X SSC plus 50% formamide at about 42-50°C) followed by one or more washes in IX SSC, at about 65-70° C.
  • SSC sodium chloride/sodium citrate
  • a prefened, non-limiting example of highly stringent hybridization conditions includes hybridization in IX SSC, at about 65-70°C (or hybridization in IX SSC plus 50% formamide at about 42-50°C) followed by one or more washes in 0.3X SSC, at about 65-70° C.
  • a prefened, non-limiting example of reduced stringency hybridization conditions includes hybridization in 4X SSC, at about 50-60°C (or alternatively hybridization in 6X SSC plus 50% formamide at about 40-45°C) followed by one or more washes in 2X SSC, at about 50-60°C.
  • Ranges intermediate to the above-recited values, e.g., at 65-70°C or at 42- 50°C are also intended to be encompassed by the present invention.
  • SSPE lxSSPE is 0.15M NaCI, lOmM NaH 2 PO 4 , and 1.25mM EDTA, pH 7.4
  • SSC lxSSC is 0.15M NaCI and 15mM sodium citrate
  • the hybridization temperature for hybrids anticipated to be less than 50 base pairs in length should be 5-10°C less than the melting temperature (T m ) of the hybrid, where T ra is determined according to the following equations.
  • T m (°C) 2(# of A + T bases) + 4(# of G + C bases).
  • additional reagents may be added to hybridization and/or wash buffers to decrease non-specific hybridization of nucleic acid molecules to membranes, for example, nitrocellulose or nylon membranes, including but not limited to blocking agents (e.g., BSA or salmon or herring sperm carrier DNA), detergents (e.g., SDS), chelating agents (e.g., EDTA), Ficoll, PVP and the like.
  • blocking agents e.g., BSA or salmon or herring sperm carrier DNA
  • detergents e.g., SDS
  • chelating agents e.g., EDTA
  • Ficoll e.g., Ficoll, PVP and the like.
  • an additional prefened, non-limiting example of stringent hybridization conditions is hybridization in 0.25-0.5M NaH 2 PO , 7% SDS at about 65°C, followed by one or more washes at 0.02M NaH 2 PO 4 , 1% SDS at 65°C, see e.g., Church and Gilbert (1984) Proc. Natl. Acad. Sci. USA 81:1991-1995, (or alternatively 0.2X SSC, 1% SDS).
  • an isolated nucleic acid molecule of the invention hybridizes under stringent conditions to the sequence of SEQ ID NO: 1, 3, 4, or 6, and conesponds to a naturally-occurring nucleic acid molecule.
  • a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
  • allelic variants of the IC sequences that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequences of SEQ ID NO:l, 3, 4, or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number or , thereby leading to changes in the amino acid sequence of the encoded IC proteins, without altering the functional ability of the IC proteins.
  • nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues can be made in the sequence of SEQ ID NO:l, 3, 4, or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number or .
  • a "non- essential" amino acid residue is a residue that can be altered from the wild-type sequence of IC (e.g., the sequence of SEQ ID NO:2 or 5) without altering the biological activity, whereas an "essential" amino acid residue is required for biological activity.
  • amino acid residues that are conserved among the IC proteins of the present invention, e.g., those present in a transmembrane domain are predicted to be particularly unamenable to alteration.
  • nucleic acid molecules encoding IC proteins that contain changes in amino acid residues that are not essential for activity. Such IC proteins differ in amino acid sequence from SEQ ED NO:2 or 5, yet retain biological activity.
  • the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:2 or 5.
  • An isolated nucleic acid molecule encoding an IC protein identical to the protein of SEQ ID NO:2 or 5 can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ED NO: 1, 3, 4, or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession
  • Mutations can be introduced into SEQ ED NO: 1, 3, 4, or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with
  • conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues.
  • a "conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • a predicted nonessential amino acid residue in an IC protein is preferably replaced with another amino acid residue from the same side chain family.
  • mutations can be introduced randomly along all or part of an IC coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for IC biological activity to identify mutants that retain activity. Following mutagenesis of SEQ ED NO:l, 3, 4, or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with
  • a mutant IC protein can be assayed for the ability to (1) modulate membrane excitability, (2) regulate intracellular ion concentration, (3) modulate membrane polarization (e.g., membrane polarization and/or depolarization), (4) regulate action potential, (5) regulate cellular signal transduction, (6) regulate neurotransmitter release (e.g., from neuronal cells), (7) modulate synaptic transmission, (8) regulate neuronal excitability and/or plasticity, (9) regulate muscle contraction, (10) regulate cell activation and (11) regulate cellular proliferation, growth, migration and/or differentiation.
  • an antisense nucleic acid comprises a nucleotide sequence which is complementary to a "sense" nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bond to a sense nucleic acid.
  • the antisense nucleic acid can be complementary to an entire IC coding strand, or to only a portion thereof.
  • an antisense nucleic acid molecule is antisense to a "coding region" of the coding strand of a nucleotide sequence encoding IC.
  • the term “coding region” refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues (e.g., the coding region of human IC23949 conesponds to SEQ ED NO:3 and the coding region of human IC32391 conesponds to SEQ ED NO:6).
  • the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence encoding IC.
  • the term “noncoding region” refers to 5' and 3' sequences which flank the coding region that are not translated into amino acids (i.e., also refened to as 5' and 3 'untranslated regions).
  • antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick base pairing.
  • the antisense nucleic acid molecule can be complementary to the entire coding region of IC mRNA, but more preferably is an ohgonucleotide which is antisense to only a portion of the coding or noncoding region of IC mRNA.
  • the antisense ohgonucleotide can be complementary to the region sunounding the translation start site of IC mRNA.
  • An antisense ohgonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.
  • An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art.
  • an antisense nucleic acid e.g., an antisense ohgonucleotide
  • an antisense nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used.
  • modified nucleotides which can be used to generate the antisense nucleic acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl- 2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7- methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D- mannosylqueosine, 5 -methoxy
  • the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
  • the antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding an IC protein to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation.
  • the hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix.
  • An example of a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site.
  • antisense nucleic acid molecules can be modified to target selected cells and then administered systemically.
  • antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens.
  • the antisense nucleic acid molecules can also be delivered to cells using the vectors described herein.
  • vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol ll promoter are prefened.
  • the antisense nucleic acid molecule of the invention is an ⁇ -anomeric nucleic acid molecule.
  • An ⁇ -anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual ⁇ -units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625- 6641).
  • the antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) EESS Lett. 215:327-330).
  • an antisense nucleic acid of the invention is a ribozyme.
  • Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region.
  • ribozymes e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave IC mRNA transcripts to thereby inhibit translation of IC mRNA.
  • a ribozyme having specificity for an IC-encoding nucleic acid can be designed based upon the nucleotide sequence of an IC cDNA disclosed herein (i.e., SEQ ED NO:l, 3, 4, or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number or ).
  • a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in an IC-encoding mRNA. See, e.g., Cech et al. U.S. Patent No.
  • IC mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel, D. and Szostak, J.W. (1993) Science 261:1411-1418.
  • IC gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the IC (e.g., the IC promoter and/or enhancers; e.g., nucleotides 1-244 of SEQ ED NO:l or nucleotides 1-206 of SEQ TD NO:4) to form triple helical structures that prevent transcription of the IC gene in target cells.
  • nucleotide sequences complementary to the regulatory region of the IC e.g., the IC promoter and/or enhancers; e.g., nucleotides 1-244 of SEQ ED NO:l or nucleotides 1-206 of SEQ TD NO:4
  • the IC nucleic acid molecules of the present invention can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule.
  • the deoxyribose phosphate backbone of the nucleic acid molecules can be modified to generate peptide nucleic acids (see Hyrup B. et al. (1996) Bioorganic & Medicinal Chemistry 4 (1): 5-23).
  • peptide nucleic acids refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained.
  • the neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength.
  • the synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup B. et al. (1996) supra; Peny-OKeefe et al. Proc. Natl. Acad. Sci. 93: 14670-675.
  • PNAs of IC nucleic acid molecules can be used in therapeutic and diagnostic applications.
  • PNAs can be used as antisense or antigene agents for sequence- specific modulation of gene expression by, for example, inducing transcription or translation a ⁇ est or inhibiting replication.
  • PNAs of IC nucleic acid molecules can also be used in the analysis of single base pair mutations in a gene, (e.g., by PNA-directed PCR clamping); as 'artificial restriction enzymes' when used in combination with other enzymes, (e.g., S 1 nucleases (Hyrup B. (1996) supra)); or as probes or primers for DNA sequencing or hybridization (Hyrup B. et al.
  • PNAs of IC can be modified, (e.g., to enhance their stability or cellular uptake), by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art.
  • PNA-DNA chimeras of IC nucleic acid molecules can be generated which may combine the advantageous properties of PNA and DNA.
  • Such chimeras allow DNA recognition enzymes, (e.g., RNAse H and DNA polymerases), to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity.
  • PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup B. (1996) supra).
  • the synthesis of PNA-DNA chimeras can be performed as described in Hyrup B. (1996) supra and Finn P.J. et al. (1996) Nucleic Acids Res. 24 (17): 3357-63.
  • a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry and modified nucleoside analogs, e.g., 5'-(4-methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite, can be used as a between the PNA and the 5' end of DNA (Mag, M. et al. (1989) Nucleic Acid Res. 17: 5973-88).
  • modified nucleoside analogs e.g., 5'-(4-methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite
  • chimeric molecules are then coupled in a stepwise manner to produce a chimeric molecule with a 5'PNA segment and a 3'DNA segment (Finn P.J. et al. (1996) supra).
  • chimeric molecules can be synthesized with a 5 'DNA segment and a 3 'PNA segment (Peterser, K.H. et al. (1975) Bioorganic Med. Chem. Lett. 5: 1119-11124).
  • the ohgonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al.
  • oligonucleotides can be modified with hybridization-triggered cleavage agents (See, e.g., Krol et al. (1988) Bio-Techniques 6:958-976) or intercalating agents. (See, e.g., Zon (1988) Pharm. Res. 5:539-549).
  • the ohgonucleotide may be conjugated to another molecule, (e.g., a peptide, hybridization triggered cross- linking agent, transport agent, or hybridization-triggered cleavage agent).
  • another molecule e.g., a peptide, hybridization triggered cross- linking agent, transport agent, or hybridization-triggered cleavage agent.
  • the expression characteristics of an endogenous IC gene within a cell line or microorganism may be modified by inserting a heterologous DNA regulatory element into the genome of a stable cell line or cloned microorganism such that the inserted regulatory element is operatively linked with the endogenous IC gene.
  • an endogenous IC gene which is normally "transcriptionally silent”, i.e., an IC gene which is normally not expressed, or is expressed only at very low levels in a cell line or microorganism may be activated by inserting a regulatory element which is capable of promoting the expression of a normally expressed gene product in that cell line or microorganism.
  • a transcriptionally silent, endogenous IC gene may be activated by insertion of a promiscuous regulatory element that works across cell types.
  • a heterologous regulatory element may be inserted into a stable cell line or cloned microorganism, such that it is operatively linked with an endogenous IC gene, using techniques, such as targeted homologous recombination, which are well known to those of skill in the art, and described, e.g., in Chappel, U.S. Patent No. 5,272,071; PCT publication No. WO 91/06667, published May 16, 1991.
  • Isolated IC Proteins and Anti-IC Antibodies One aspect of the invention pertains to isolated IC proteins, and biologically active portions thereof, as well as polypeptide fragments suitable for use as immunogens to raise anti-IC antibodies.
  • native IC proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques.
  • IC proteins are produced by recombinant DNA techniques.
  • an IC protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques.
  • an “isolated” or “purified” protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the IC protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized.
  • the language “substantially free of cellular material” includes preparations of IC protein in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced.
  • the language "substantially free of cellular material” includes preparations of IC protein having less than about 30% (by dry weight) of non-IC protein (also refened to herein as a "contaminating protein"), more preferably less than about 20% of non-IC protein, still more preferably less than about 10% of non-IC protein, and most preferably less than about 5% non-IC protein.
  • IC protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of IC protein in which the protein is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein. In one embodiment, the language “substantially free of chemical precursors or other chemicals” includes preparations of IC protein having less than about 30% (by dry weight) of chemical . precursors or non-IC chemicals, more preferably less than about 20% chemical precursors or non-IC chemicals, still more preferably less than about 10% chemical precursors or non-IC chemicals, and most preferably less than about 5% chemical precursors or non-IC chemicals.
  • a "biologically active portion" of an IC protein includes a fragment of an IC protein which participates in an interaction between an IC molecule and a non-IC molecule.
  • Biologically active portions of an IC protein include peptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of the IC protein, e.g., the amino acid sequence shown in SEQ ED NO: 2 or 5, which include less amino acids than the full length IC proteins, and exhibit at least one activity of an IC protein.
  • biologically active portions comprise a domain or motif with at least one activity of the IC protein, e.g. , modulating membrane excitability.
  • a biologically active portion of an IC protein can be a polypeptide which is, for example, 25, 50, 75, 100, 125, 150, 175, 200 or more amino acids in length.
  • Biologically active portions of an IC protein can be used as targets for developing agents which modulate an IC mediated activity, e.g., modulation of membrane excitability.
  • a biologically active portion of an IC protein comprises at least one transmembrane domain. It is to be understood that a prefened biologically active portion of an IC protein of the present invention may contain one or more of the following domains: a transmembrane domain, an ion transport protein domain, and a ligand-gated ion channel domain.
  • other biologically active portions, in which other regions of the protein are deleted can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native IC protein.
  • the IC protein has an amino acid sequence shown in SEQ D NO:2 or 5.
  • the IC protein is substantially identical to SEQ ED NO:2 or 5, and retains the functional activity of the protein of SEQ ED NO:2 or 5, yet differs in amino acid sequence due to natural allelic variation or mutagenesis, as described in detail in subsection I above.
  • the IC protein is a protein which comprises an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%), 95%, 96%, 97%, 98%, 99% or more identical to SEQ TD NO:2 or 5.
  • sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-identical sequences can be disregarded for comparison purposes).
  • the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, or 90% of the length of the reference sequence (e.g., when aligning a second sequence to the IC amino acid sequence of SEQ ID NO:5 having 1010 amino acid residues, at least 100, preferably at least 150, more preferably at least 200, even more preferably at least 250, and even more preferably at least 300, or 400 or more amino acid residues are aligned).
  • the amino acid residues or nucleotides at conesponding amino acid positions or nucleotide positions are then compared.
  • amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid "homology”).
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
  • the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6.
  • the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4: 11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the nucleic acid and protein sequences of the present invention can further be used as a "query sequence" to perform a search against public databases to, for example, identify other family members or related sequences.
  • Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10.
  • Gapped BLAST can be utilized as described in Altschul et al. , (1997) Nucleic Acids Res. 25(17): 3389-3402.
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • XBLAST and NBLAST See http://www.ncbi.nlm.nih.gov.
  • an IC "chimeric protein” or “fusion protein” comprises an IC polypeptide operatively linked to a non-IC polypeptide.
  • An "IC polypeptide” refers to a polypeptide having an amino acid sequence conesponding to IC
  • a non-IC polypeptide refers to a polypeptide having an amino acid sequence conesponding to a protein which is not substantially homologous to the IC protein, e.g., a protein which is different from the IC protein and which is derived from the same or a different organism.
  • the IC polypeptide can conespond to all or a portion of an IC protein.
  • an IC fusion protein comprises at least one biologically active portion of an IC protein. In another prefened embodiment, an IC fusion protein comprises at least two biologically active portions of an IC protein.
  • the term "operatively linked" is intended to indicate that the IC polypeptide and the non-IC polypeptide are fused in-frame to each other. The non-IC polypeptide can be fused to the N-terminus or C-terminus of the IC polypeptide.
  • the fusion protein is a GST-IC fusion protein in which the IC sequences are fused to the C-terminus of the GST sequences.
  • Such fusion proteins can facilitate the purification of recombinant IC.
  • the fusion protein is an IC protein containing a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian host cells), expression and/or secretion of IC can be increased through use of a heterologous signal sequence.
  • the IC fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject in vivo.
  • the IC fusion proteins can.be used to affect the bioavailability of an IC substrate.
  • Use of IC fusion proteins maybe useful therapeutically for the treatment of disorders caused by, for example, (i) abenant modification or mutation of a gene encoding an IC protein; (ii) mis-regulation of the IC gene; and (iii) abenant post-translational modification of an IC protein.
  • the IC-fusion proteins of the invention can be used as immunogens to produce anti-IC antibodies in a subject, to purify IC ligands and in screening assays to identify molecules which inhibit the interaction of IC with an IC substrate.
  • an IC chimeric or fusion protein of the invention is produced by standard recombinant DNA techniques.
  • DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, for example by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).
  • anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence
  • many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide).
  • An IC-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the IC protein.
  • the present invention also pertains to variants of the IC proteins which function as either IC agonists (mimetics) or as IC antagonists.
  • Variants of the IC proteins can be generated by mutagenesis, e.g., discrete point mutation or truncation of an IC protein.
  • An agonist of the IC proteins can retain substantially the same, or a subset, of the biological activities of the naturally occurring form of an IC protein.
  • An antagonist of an IC protein can inhibit one or more of the activities of the naturally occurring form of the IC protein by, for example, competitively modulating an IC-mediated activity of an IC protein.
  • specific biological effects can be elicited by treatment with a variant of limited function.
  • treatment of a subject with a variant having a subset of the biological activities of the naturally occurring form of the protein has fewer side effects in a subject relative to treatment with the naturally occurring form of the IC protein.
  • variants of an IC protein which function as either IC agonists (mimetics) or as IC antagonists can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of an IC protein for IC protein agonist or antagonist activity.
  • a variegated library of IC variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library.
  • a variegated library of IC variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential IC sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of IC sequences therein.
  • a degenerate set of potential IC sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of IC sequences therein.
  • fusion proteins e.g., for phage display
  • degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential IC sequences.
  • Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang, S.A. (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477.
  • libraries of fragments of an IC protein coding sequence can be used to generate a variegated population of IC fragments for screening and subsequent selection of variants of an IC protein.
  • a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of an IC coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with SI nuclease, and ligating the resulting fragment library into an expression vector.
  • an expression library can be derived which encodes N-terminal, C-terminal and internal fragments of various sizes of the IC protein.
  • REM Recursive ensemble mutagenesis
  • cell based assays can be exploited to analyze a variegated IC library.
  • a library of expression vectors can be transfected into a cell line, e.g., a neuronal cell line, which ordinarily responds to an IC ligand in a particular IC ligand- dependent manner.
  • the transfected cells are then contacted with an IC ligand and the effect of expression of the mutant on, e.g., membrane excitability of IC can be detected.
  • Plasmid DNA can then be recovered from the cells which score for inhibition, or alternatively, potentiation of signaling by the IC ligand, and the individual clones further characterized.
  • an isolated IC protein, or a portion or fragment thereof, can be used as an immunogen to generate antibodies that bind IC using standard techniques for polyclonal and monoclonal antibody preparation.
  • a full-length IC protein can be used or, alternatively, the invention provides antigenic peptide fragments of IC for use as immunogens.
  • the antigenic peptide of IC comprises at least 8 amino acid residues of the amino acid sequence shown in SEQ ED NO:2 or 5 and encompasses an epitope of IC such that an antibody raised against the peptide forms a specific immune complex with IC.
  • the antigenic peptide comprises at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30 amino acid residues.
  • Prefened epitopes encompassed by the antigenic peptide are regions of IC that are located on the surface of the protein, e.g., hydrophilic regions, as well as regions with high antigenicity (see Figures 3 and 4).
  • An IC immunogen typically is used to prepare antibodies by immunizing a suitable subject, (e.g., rabbit, goat, mouse or other mammal) with the immunogen.
  • An appropriate immunogenic preparation can contain, for example, recombinantly expressed IC protein or a chemically synthesized IC polypeptide.
  • the preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or similar immunostimulatory agent. Immunization of a suitable subject with an immunogenic IC preparation induces a polyclonal anti-IC antibody response.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds (immunoreacts with) an antigen, such as IC.
  • immunologically active portions of immunoglobulin molecules include F(ab) and F(ab') 2 fragments which can be generated by treating the antibody with an enzyme such as pepsin.
  • the invention provides polyclonal and monoclonal antibodies that bind IC.
  • monoclonal antibody or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of IC.
  • a monoclonal antibody composition thus typically displays a single binding affinity for a particular IC protein with which it immunoreacts.
  • Polyclonal anti-IC antibodies can be prepared as described above by immunizing a suitable subject with an IC immunogen.
  • the anti-IC antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized IC.
  • ELISA enzyme linked immunosorbent assay
  • the antibody molecules directed against IC can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as protein A chromatography to obtain the IgG fraction.
  • antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein (1975) Nature 256:495-497) (see also, Brown et al. (1981) J. Immunol. 127:539-46; Brown et al. (1980) J. Biol. Chem .255:4980-83; Yeh et al. (1976) Proc. Natl. Acad. Sci. USA 76:2927-31; and Yeh et al. (1982) Int. J.
  • an immortal cell line typically a myeloma
  • lymphocytes typically splenocytes
  • IC immunogen as described above
  • the culture supematants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that binds IC.
  • Any of the many well known protocols used for fusing lymphocytes and immortalized cell lines can be applied for the purpose of generating an anti-IC monoclonal antibody (see, e.g., G. Galfre et al.
  • the immortal cell line e.g., a myeloma cell line
  • murine hybridomas can be made by fusing lymphocytes from a mouse immunized with an immunogenic preparation of the present invention with an immortalized mouse cell line.
  • Prefened immortal cell lines are mouse myeloma cell lines that are sensitive to culture medium containing hypoxanthine, aminopterin and thymidine ("HAT medium"). Any of a number of myeloma cell lines can be used as a fusion partner according to standard techniques, e.g., the P3-NSl/l-Ag4-l, P3-x63-Ag8.653 or Sp2/O- Agl4 myeloma lines. These myeloma lines are available from ATCC. Typically, HAT- sensitive mouse myeloma cells are fused to mouse splenocytes using polyethylene glycol (“PEG").
  • PEG polyethylene glycol
  • Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused and unproductively fused myeloma cells (unfused splenocytes die after several days because they are not transformed).
  • Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supematants for antibodies that bind IC, e.g., using a standard ELISA assay.
  • a monoclonal anti-IC antibody can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with IC to thereby isolate immunoglobulin library members that bind IC.
  • Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene SurfZAPTM Phage Display Kit, Catalog No. 240612). Additionally, examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, Ladner et al. U.S. Patent No.
  • recombinant anti-IC antibodies such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention.
  • chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in Robinson et al. International Application No. PCT/US 86/02269; Akira, et al. European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al. European Patent Application 173,494; Neuberger et al. PCT International Publication No. WO 86/01533; Cabilly et al.
  • An anti-IC antibody (e.g., monoclonal antibody) can be used to isolate IC by standard techniques, such as affinity chromatography or immunoprecipitation.
  • An anti-IC antibody can facilitate the purification of natural IC from cells and of recombinantly produced IC expressed in host cells.
  • an anti-IC antibody can be used to detect IC protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the IC protein.
  • Anti-IC antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen.
  • Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance.
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
  • an example of a luminescent material includes luminol;
  • bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include I, I,
  • vectors preferably expression vectors, containing a nucleic acid encoding an IC protein (or a portion thereof).
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • vectors e.g., non-episomal mammalian vectors
  • Other vectors are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • certain vectors are capable of directing the expression of genes to which they are operatively linked.
  • Such vectors are refened to herein as "expression vectors".
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and vector can be used interchangeably as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
  • the recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed.
  • operably linked is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • regulatory sequence is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).
  • Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cells and those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like.
  • the expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., IC proteins, mutant forms of IC proteins, fusion proteins, and the like).
  • the recombinant expression vectors of the invention can be designed for expression of IC proteins in prokaryotic or eukaryotic cells.
  • IC proteins can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).
  • the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
  • Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein.
  • Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification.
  • a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein.
  • enzymes, and their cognate recognition sequences include Factor Xa, thrombin and enterokinase.
  • Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D.B. and Johnson, K.S.
  • GST glutathione S-transferase
  • Purified fusion proteins can be utilized in IC activity assays, (e.g., direct assays or competitive assays described in detail below), or to generate antibodies specific for IC proteins, for example.
  • an IC fusion protein expressed in a retroviral expression vector of the present invention can be utilized to infect bone manow cells which are subsequently transplanted into inadiated recipients. The pathology of the subject recipient is then examined after sufficient time has passed (e.g., six (6) weeks).
  • Suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al., (1988) Gene 69:301-315) and pET l id (Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 60-89).
  • Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter.
  • Target gene expression from the pET 1 Id vector relies on transcription from a T7 gnlO-lac fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gnl). This viral polymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from a resident prophage harboring a T7 gnl gene under the transcriptional control of the lacUV 5 promoter.
  • One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 119-128).
  • Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al., (1992) Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.
  • the IC expression vector is a yeast expression vector.
  • yeast S. cerevisiae examples include pYepSecl (Baldari, et al, (1987) Embo J. 6:229-234), pMFa (Kurjan and Herskowiiz, (1982) Cell 30:933-943), pJRY88 (Schultz et al, (1987) Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego, CA), and picZ (InVitrogen Corp, San Diego, CA).
  • IC proteins can be expressed in insect cells using baculovirus expression vectors.
  • Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39).
  • a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector.
  • mammalian expression vectors include pCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187-195).
  • the expression vector's control functions are often provided by viral regulatory elements.
  • commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.
  • suitable expression systems for both prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.
  • the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid).
  • tissue-specific regulatory elements are known in the art.
  • suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto and Baltimore (1989) EMBO J.
  • promoters are also encompassed, for example the murine hox promoters (Kessel and Grass (1990) Science 249:374-379) and the ⁇ -fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3:537-546).
  • the invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively linked to a regulatory sequence in a manner which allows for expression (by transcription of the DNA molecule) of an RNA molecule which is antisense to IC mRNA.
  • Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen which direct constitutive, tissue specific or cell type specific expression of antisense RNA.
  • the antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced.
  • a high efficiency regulatory region the activity of which can be determined by the cell type into which the vector is introduced.
  • Another aspect of the invention pertains to host cells into which an IC nucleic acid molecule of the invention is introduced, e.g., an IC nucleic acid molecule within a recombinant expression vector or an IC nucleic acid molecule containing sequences which allow it to homologously recombine into a specific site of the host cell's genome.
  • host cell and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell.
  • a host cell can be any prokaryotic or eukaryotic cell.
  • an IC protein ca be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such a Chinese hamster ovary cells (CHO) or COS cells).
  • bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such a Chinese hamster ovary cells (CHO) or COS cells).
  • mammalian cells such as a Chinese hamster ovary cells (CHO) or COS cells.
  • Other suitable host cells are known to those skilled in the art.
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
  • transformation and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, D ⁇ A ⁇ -dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found ii Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), and other laboratory manuals.
  • a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest.
  • selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methotrexat.
  • Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding an IC protein or can be introduced on a separate vector.
  • Cells stabl) transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
  • a host cell of the invention such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) an IC protein.
  • the invention further provides methods for producing an IC protein using the host cells of the invention.
  • the method comprises culturing the host cell of the invention (into which a recombinant expression vector encoding an IC protein has been introduced) in a suitable medium such that an IC protein is produced.
  • the method further comprises isolating an IC protein from the medium or the host cell.
  • the host cells of the invention can also be used to produce non-human transgenic animals.
  • a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which IC-coding sequences have been introduced.
  • Such host cells can then be used to create non-human transgenic animals in which exogenous IC sequences have been introduced into their genome or homologous recombinant animals in which endogenous IC sequences have been altered.
  • Such animals are useful for studying the function and/or activity of an IC and for identifying and/or evaluating modulators of IC activity.
  • a "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.
  • Other examples of 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 IC 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.
  • a transgenic animal of the invention can be created by introducing an IC-encoding nucleic acid into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal.
  • the IC cDNA sequence of SEQ ED NO: 1 or 4 can be introduced as a transgene into the genome of a non-human animal.
  • a nonhuman homologue of a human IC gene such as a mouse or rat IC gene, can be used as a transgene.
  • an IC gene homologue such as another IC family member, can be isolated based on hybridization to the IC cDNA sequences of SEQ ED NO: 1, 3, 4, or 6, or the DNA insert of the plasmid deposited with ATCC as Accession Number or (described further in subsection I above) and used as a transgene.
  • Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene.
  • a tissue- specific regulatory sequence(s) can be operably linked to an IC transgene to direct expression of an IC protein to particular cells.
  • transgenic founder animal can be identified based upon the presence of an IC transgene in its genome and/or expression of IC mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals canying the transgene. Moreover, transgenic animals carrying a transgene encoding an IC protein can further be bred to other transgenic animals carrying other transgenes.
  • a vector is prepared which contains at least a portion of an IC gene into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the IC gene.
  • the IC gene can be a human gene (e.g., the cDNA of SEQ TD NO:3 or 6), but more preferably, is a non-human homologue of a human IC gene (e.g., a cDNA isolated by stringent hybridization with the nucleotide sequence of SEQ TD NO:l or 4).
  • a mouse IC gene can be used to construct a homologous recombination nucleic acid molecule, e.g., a vector, suitable for altering an endogenous IC gene in the mouse genome.
  • the homologous recombination nucleic acid molecule is designed such that, upon homologous recombination, the endogenous IC gene is functionally disrupted (i.e., no longer encodes a functional protein; also refened to as a "knock out" vector).
  • the homologous recombination nucleic acid molecule can be designed such that, upon homologous recombination, the endogenous IC gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous IC protein).
  • the altered portion of the IC gene is flanked at its 5' and 3 ' ends by additional nucleic acid sequence of the IC gene to allow for homologous recombination to occur between the exogenous IC gene carried by the homologous recombination nucleic acid molecule and an endogenous IC gene in a cell, e.g., an embryonic stem cell.
  • the additional flanking IC nucleic acid sequence is of sufficient length for successful homologous recombination with the endogenous gene.
  • homologous recombination nucleic acid molecule typically, several kilobases of flanking DNA (both at the 5' and 3' ends) are included in the homologous recombination nucleic acid molecule (see, e.g., Thomas, K.R. and Capecchi, M. R. (1987) Cell 51:503 for a description of homologous recombination vectors).
  • the homologous recombination nucleic acid molecule is introduced into a cell, e.g., an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced IC gene has homologously recombined with the endogenous IC gene are selected (see e.g., Li, E. et al. (1992) Cell 69:915).
  • the selected cells can then be injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras (see e.g., Bradley, A. in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E.J. Robertson, ed. (ERL, Oxford, 1987) pp. 113-152).
  • a chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term.
  • Progeny harboring the homologously recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously recombined DNA by germline transmission of the transgene.
  • transgenic non-human animals can be produced which contain selected systems which allow for regulated expression of the transgene.
  • creAoxP recombinase system of bacteriophage PI.
  • cre ⁇ oxP recombinase system see, e.g., Lakso et al. (1992) Proc. Natl. Acad. Sci USA 89:6232-6236.
  • FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al. (1991) Science 251 : 1351-1355.
  • creAoxP 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.
  • Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut, I. et al. (1997) Nature 385:810-813 and PCT International Publication Nos. WO 97/07668 and WO 97/07669.
  • a cell e.g., a somatic cell
  • the quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated.
  • the reconstructed oocyte is then cultured such that it develops to morula or blastocyte and then transfened to pseudopregnant female foster animal.
  • the offspring borne of this female foster animal will be a clone of the animal from which the cell, e.g., the somatic cell, is isolated.
  • compositions suitable for administration can be incorporated into pharmaceutical compositions suitable for administration.
  • Such compositions typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a fragment of an IC protein or an anti-IC antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • the active compound e.g., a fragment of an IC protein or an anti-IC antibody
  • dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the prefened methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • suppositories e.g., with conventional suppository bases such as cocoa butter and other glycerides
  • retention enemas for rectal delivery.
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50.
  • Compounds which exhibit large therapeutic indices are prefened. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • IC50 i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms
  • levels in plasma may be measured, for example, by high performance liquid chromatography.
  • a therapeutically effective amount of protein or polypeptide ranges from about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight.
  • an effective dosage ranges from about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight.
  • treatment of a subject with a therapeutically effective amount of a protein, polypeptide, or antibody can include a single treatment or, preferably, can include a series of treatments.
  • a subject is treated with antibody, protein, or polypeptide in the range of between about 0.1 to 20 mg/kg body weight, one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks.
  • the effective dosage of antibody, protein, or polypeptide used for treatment may increase or decrease over the course of a particular treatment. Changes in dosage may result and become apparent from the results of diagnostic assays as described herein.
  • the present invention encompasses agents which modulate expression or activity.
  • An agent may, for example, be a small molecule.
  • small molecules include, but are not limited to, peptides, peptidomimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic compounds (i.e,.
  • heteroorganic and organometallic compounds having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds. It is understood that appropriate doses of small molecule agents depends upon a number of factors within the ken of the ordinarily skilled physician, veterinarian, or researcher.
  • the dose(s) of the small molecule will vary, for example, depending upon the identity, size, and condition of the subject or sample being treated, further depending upon the route by which the composition is to be administered, if applicable, and the effect which the practitioner desires the small molecule to have upon the nucleic acid or polypeptide of the invention.
  • Exemplary doses include milligram or microgram amounts of the small molecule per kilogram of subject or sample weight (e.g., about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram.
  • appropriate doses of a small molecule depend upon the potency of the small molecule with respect to the expression or activity to be modulated. Such appropriate doses may be determined using the' assays described herein.
  • an animal e.g., a human
  • a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained.
  • the specific dose level for any particular animal subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.
  • an antibody may be conjugated to a therapeutic moiety such as a cytotoxin, a therapeutic agent or a radioactive metal ion.
  • a cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.
  • Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6- thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g
  • the drug moiety can be used for modifying a given biological response, the drug moiety is not to be construed as limited to classical chemical therapeutic agents.
  • the drug moiety may be a protein or polypeptide possessing a desired biological activity.
  • proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, alpha- interferon, beta-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; or, biological response modifiers such as, for example, lymphokines, interleukin-1 ("IL-1"), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.
  • IL-1 interleukin-1
  • IL-2 interleukin-2
  • an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,980.
  • the nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors.
  • Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Patent 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057).
  • the pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded.
  • the pharmaceutical preparation can include one or more cells which produce the gene delivery system.
  • compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • nucleic acid molecules, proteins, protein homologues, and antibodies described herein can be used in one or more of the following methods: a) screening assays; b) predictive medicine (e.g., diagnostic assays, prognostic assays, monitoring clinical trials, and pharmacogenetics); and c) methods of treatment (e.g., therapeutic and prophylactic).
  • an IC protein of the invention has one or more of the following activities: (1) modulates membrane excitability, (2) regulates intracellular ion concentration, (3) modulates membrane polarization (e.g., membrane polarization and/or depolarization), (4) regulates action potential, (5) regulates cellular signal transduction, (6) regulates neurotransmitter release (e.g., from neuronal cells), (7) modulates synaptic transmission, (8) regulates neuronal excitability and/or plasticity, (9) regulates muscle contraction, (10) regulate cell activation and (11) regulates cellular proliferation, growth, migration and/or differentiation.
  • the isolated nucleic acid molecules of the invention can be used, for example, to express IC protein (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect IC mRNA (e.g., in a biological sample) or a genetic alteration in an IC gene, and to modulate IC activity, as described further below.
  • IC proteins can be used to treat disorders characterized by insufficient or excessive production of an IC substrate or production of IC inhibitors.
  • the IC proteins can be used to screen for naturally occurring IC substrates, to screen for drugs or compounds which modulate IC activity, as well as to treat disorders characterized by insufficient or excessive production of IC protein or production of IC protein forms which have decreased, abenant or unwanted activity compared to IC wild type protein (e.g., proliferative disorders, CNS disorders such as cognitive and neurodegenerative disorders (e.g., Alzheimer's disease, dementias related to Alzheimer's disease (such as Pick's disease), Parkinson's and other Lewy diffuse body diseases, senile dementia, Huntington's disease, Gilles de la Tourette's syndrome, multiple sclerosis, amyotrophic lateral sclerosis, progressive supranuclear palsy, epilepsy, and Jakob- Creutzfieldt disease; autonomic function disorders such as hypertension and sleep disorders, and neuropsychiatric disorders, such as depression, schizophrenia, schizoaffective disorder, korsakoff's psychosis, mania, anxiety disorders, or
  • the invention provides a method (also refened to herein as a "screening assay") for identifying modulators, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drags) which bind to IC proteins, have a stimulatory or inhibitory effect on, for example, IC expression or IC activity, or have a stimulatory or inhibitory effect on, for example, the expression or activity of IC substrate.
  • the invention provides assays for screening candidate or test compounds which are substrates of an IC protein or polypeptide or biologically active portion thereof.
  • the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of an IC protein or polypeptide or biologically active portion thereof.
  • the test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the 'one-bead one-compound' library method; and synthetic library methods using affinity chromatography selection.
  • 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 (Lam, K.S. (1997) Anticancer Drug Des. 12:145).
  • an assay is a cell-based assay in which a cell which expresses an
  • IC protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to modulate IC activity is determined. Determining the ability of the test compound to modulate IC activity can be accomplished by monitoring, for example, the release of a neurotransmitter from a cell which expresses IC.
  • the cell for example, can be of mammalian origin, e.g., a neuronal cell or a thymus cell.
  • the ability of the test compound to modulate IC binding to a substrate or to bind to IC can also be determined.
  • Determining the ability of the test compound to modulate IC binding to a substrate can be accomplished, for example, by coupling the IC substrate with a radioisotope or enzymatic label such that binding of the IC substrate to IC can be determined by detecting the labeled IC substrate in a complex.
  • IC could be coupled with a radioisotope or enzymatic label to monitor the ability of a test compound to modulate IC binding to an IC substrate in a complex.
  • Determining the ability of the test compound to bind IC can be accomplished, for example, by coupling the compound with a radioisotope or enzymatic label such that binding of the compound to IC can be determined by detecting the labeled IC compound in a complex.
  • compounds e.g., IC substrates
  • compounds can be labeled with 125 I, 35 S, l C, or 3 H, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting.
  • compounds can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.
  • a microphysiometer can be used to detect the interaction of a compound with IC without the labeling of either the compound or the IC. McConnell, H. M. et al. (1992) Science 257:1906-1912.
  • a "microphysiometer” e.g., Cytosensor
  • LAPS light-addressable potentiometric sensor
  • an assay is a cell-based assay comprising contacting a cell expressing an IC target molecule (e.g., an IC substrate) with a test compound and determining the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the IC target molecule. Determining the ability of the test compound to modulate the activity of an IC target molecule can be accomplished, for example, by determining the ability of the IC protein to bind to or interact with the IC target molecule.
  • an IC target molecule e.g., an IC substrate
  • Determining the ability of the test compound to modulate the activity of an IC target molecule can be accomplished, for example, by determining the ability of the IC protein to bind to or interact with the IC target molecule.
  • Determining the ability of the IC protein, or a biologically active fragment thereof, to bind to or interact with an IC target molecule can be accomplished by one of the methods described above for determining direct binding. In a prefened embodiment, determining the ability of the IC protein to bind to or interact with an IC target molecule can be accomplished by determining the activity of the target molecule.
  • the activity of the target molecule can be determined by detecting induction of a cellular second messenger of the target (i.e., intracellular Ca , diacylglycerol, EP 3 , and the like), detecting catalytic/enzymatic activity of the target using an appropriate substrate, detecting the induction of a reporter gene (comprising a target-responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker, e.g., luciferase), or detecting a target- regulated cellular response.
  • a cellular second messenger of the target i.e., intracellular Ca , diacylglycerol, EP 3 , and the like
  • detecting catalytic/enzymatic activity of the target using an appropriate substrate detecting the induction of a reporter gene (comprising a target-responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker, e.g., luciferase), or detecting a
  • an assay of the present invention is a cell-free assay in which an IC protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to bind to the IC protein or biologically active portion thereof is determined.
  • Prefened biologically active portions of the IC proteins to be used in assays of the present invention include fragments which participate in interactions with non- IC molecules, e.g., fragments with high surface probability scores (see, for example, Figure 2A-C). Binding of the test compound to the IC protein can be determined either directly or indirectly as described above.
  • the assay includes contacting the IC protein or biologically active portion thereof with a known compound which binds IC to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with an IC protein, wherein determining the ability of the test compound to interact with an IC protein comprises determining the ability of the test compound to preferentially bind to IC or biologically active portion thereof as compared to the known compound.
  • the assay is a cell-free assay in which an IC protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the IC protein or biologically active portion thereof is determined.
  • Determining the ability of the test compound to modulate the activity of an IC protein can be accomplished, for example, by determining the ability of the IC protein to bind to an IC target molecule by one of the methods described above for determining direct binding. Determining the ability of the IC protein to bind to an IC target molecule can also be accomplished using a technology such as real-time Biomolecular Interaction Analysis (BIA). Sjolander, S. and Urbaniczky, C.
  • BIOA is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore). Changes in the optical phenomenon of surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.
  • SPR surface plasmon resonance
  • determining the ability of the test compound to modulate the activity of an IC protein can be accomplished by determining the ability of the IC protein to further modulate the activity of a downstream effector of an IC target molecule.
  • the activity of the effector molecule on an appropriate target can be determined or the binding of the effector to an appropriate target can be determined as previously described.
  • the cell-free assay involves contacting an IC protein or biologically active portion thereof with a known compound which binds the IC protein to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with the IC protein, wherein determining the ability of the test compound to interact with the IC protein comprises determining the ability of the ICprotein to preferentially bind to or modulate the activity of an IC target molecule.
  • Binding of a test compound to an IC protein, or interaction of an IC protein with a target molecule in the presence and absence of a candidate compound can be accomplished in any vessel suitable for containing the reactants.
  • vessels include microtitre plates, test tubes, and micro- centrifuge tubes.
  • a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix.
  • glutathione-S- transferase/IC fusion proteins or glutathione-S-transferase/target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St.
  • the test compound or the test compound and either the non-adsorbed target protein or IC protein are then combined with the test compound or the test compound and either the non-adsorbed target protein or IC protein, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH).
  • the beads or microtitre plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described above.
  • the complexes can be dissociated from the matrix, and the level of IC binding or activity determined using standard techniques. Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention.
  • an IC protein or an IC target molecule can be immobilized utilizing conjugation of biotin and streptavidin.
  • Biotinylated IC protein or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, IL), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).
  • antibodies reactive with IC protein or target molecules but which do not interfere with binding of the IC protein to its target molecule can be derivatized to the wells of the plate, and unbound target or IC protein trapped in the wells by antibody conjugation.
  • Methods for detecting such complexes include immunodetection of complexes using antibodies reactive with the IC protein or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the IC protein or target molecule.
  • modulators of IC expression are identified in a method wherein a cell is contacted with a candidate compound and the expression of IC mRNA or protein in the cell is determined. The level of expression of IC mRNA or protein in the presence of the candidate compound is compared to the level of expression of IC mRNA or protein in the absence of the candidate compound. The candidate compound can then be identified as a modulator of IC expression based on this comparison. For example, when expression of IC mRNA or protein is greater (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of IC mRNA or protein expression.
  • the candidate compound when expression of IC mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of IC mRNA or protein expression.
  • the level of IC mRNA or protein expression in the cells can be determined by methods described herein for detecting IC mRNA or protein.
  • the IC proteins can be used as "bait proteins" in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Patent No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al.
  • IC-binding proteins proteins which bind to or interact with IC
  • IC-binding proteins are also likely to be involved in the propagation of signals by the IC proteins or IC targets as, for example, downstream elements of an IC-mediated signaling pathway.
  • IC-binding proteins are likely to be IC inhibitors.
  • the two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains.
  • the assay utilizes two different DNA constructs.
  • the gene that codes for an IC protein is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4).
  • a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein (“prey" or "sample”) is fused to a gene that codes for the activation domain of the known transcription factor.
  • the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with the IC protein.
  • a reporter gene e.g., LacZ
  • the invention pertains to a combination of two or more of the assays described herein.
  • a modulating agent can be identified using a cell- based or a cell free assay, and the ability of the agent to modulate the activity of an IC protein can be confirmed in vivo, e.g., in an animal such as an animal model for cellular pain disorders.
  • This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein in an appropriate animal model.
  • an agent identified as described herein e.g., an IC modulating agent, an antisense IC nucleic acid molecule, an IC-specific antibody, or an IC-binding partner
  • an agent identified as described herein can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent.
  • an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent.
  • this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein.
  • Models for studying pain in vivo include rat models of neuropathic pain caused by methods such as intraperitoneal administration of Taxol (Authier et al. (2000) Brain Res. 887:239-249), chronic constriction injury (CCI), partial sciatic nerve transection (Linenlaub and Sommer (2000) Pain 89:97-106), transection of the tibial and sural nerves (Lee et al. (2000) Neurosci. Lett. 291:29-32), the spared nerve injury model (Decosterd and Woolf (2000) Pain 87:149-158), cuffing the sciatic nerve (Pitcher and Henry (2000) Eur. J.
  • Taxol Authier et al. (2000) Brain Res. 887:239-249
  • CCI chronic constriction injury
  • partial sciatic nerve transection Linenlaub and Sommer (2000) Pain 89:97-106
  • transection of the tibial and sural nerves Lee et al. (2000) Neuros
  • CFA (Abdi et al. (2000) Anesth. Analg. 91:955-959); rat models of post-incisional pain caused by incising the skin and fascia of a hind paw (Olivera and Prado (2000) Braz. J.
  • Examples of such methods include, but are not limited to brief intense exposure to a focused heat source, administration of a noxious chemical subcutaneously, the tail flick test, the hot plate test, the formalin test, Von Frey threshold, and testing for stress-induced analgesia (et al, by restraint, foot shock, and/or cold water swim) (Crawley (2000) Wfiat's Wrong With
  • Portions or fragments of the cDNA sequences identified herein (and the conesponding complete gene sequences) can be used in numerous ways as polynucleotide reagents. For example, these sequences can be used to: (i) map their respective genes on a chromosome; and, thus, locate gene regions associated with genetic disease; (ii) identify an individual from a minute biological sample (tissue typing); and (iii) aid in forensic identification of a biological sample. These applications are described in the subsections below.
  • Chromosome Mapping Once the sequence (or a portion of the sequence) of a gene has been isolated, this sequence can be used to map the location of the gene on a chromosome. This process is called chromosome mapping. Accordingly, portions or fragments of the IC nucleotide sequences, described herein, can be used to map the location of the IC genes on a chromosome. The mapping of the IC sequences to chromosomes is an important first step in conelating these sequences with genes associated with disease.
  • IC genes can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp in length) from the IC nucleotide sequences. Computer analysis of the IC sequences can be used to predict primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers can then be used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene conesponding to the IC sequences will yield an amplified fragment.
  • Somatic cell hybrids are prepared by fusing somatic cells from different mammals (e.g., human and mouse cells). As hybrids of human and mouse cells grow and divide, they gradually lose human chromosomes in random order, but retain the mouse chromosomes. By using media in which mouse cells cannot grow, because they lack a particular enzyme, but human cells can, the one human chromosome that contains the gene encoding the needed enzyme, will be retained. By using various media, panels of hybrid cell lines can be established. Each cell line in a panel contains either a single human chromosome or a small number of human chromosomes, and a full set of mouse chromosomes, allowing easy mapping of individual genes to specific human chromosomes. (D ⁇ ustachio P. et al. (1983) Science 220:919-924). Somatic cell hybrids containing only fragments of human chromosomes can also be produced by using human chromosomes with translocations and deletions.
  • PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular sequence to a particular chromosome. Three or more sequences can be assigned per day using a single thermal cycler. Using the IC nucleotide sequences to design ohgonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomes. Other mapping strategies which can similarly be used to map an IC sequence to its chromosome include in situ hybridization (described in Fan, Y. et al. (1990) Proc. Natl Acad. Sci. USA, 87:6223-27), pre-screening with labeled flow-sorted chromosomes, and pre-selection by hybridization to chromosome specific cDNA libraries.
  • Fluorescence in situ hybridization (FISH) of a DNA sequence to a metaphase chromosomal spread can further be used to provide a precise chromosomal location in one step.
  • Chromosome spreads can be made using cells whose division has been blocked in metaphase by a chemical such as colcemid that disrupts the mitotic spindle.
  • the chromosomes can be treated briefly with trypsin, and then stained with Giemsa. A pattern of light and dark bands develops on each chromosome, so that the chromosomes can be identified individually.
  • the FISH technique can be used with a DNA sequence as short as 500 or 600 bases.
  • clones larger than 1,000 bases have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection.
  • 1,000 bases, and more preferably 2,000 bases will suffice to get good results at a reasonable amount of time.
  • Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents conesponding to noncoding regions of the genes actually are prefened for mapping purposes. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.
  • differences in the DNA sequences between individuals affected and unaffected with a disease associated with the IC gene can be determined. If a mutation is observed in some or all of the affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent of the particular disease. Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that DNA sequence. Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymorphisms.
  • the IC sequences of the present invention can also be used to identify individuals from minute biological samples.
  • the United States military, for example, is considering the use of restriction fragment length polymorphism (RFLP) for identification of its personnel.
  • RFLP restriction fragment length polymorphism
  • an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identification.
  • This method does not suffer from the cunent limitations of "Dog Tags" which can be lost, switched, or stolen, making positive identification difficult.
  • the sequences of the present invention are useful as additional DNA markers for RFLP (described in U.S. Patent 5,272,057).
  • sequences of the present invention can be used to provide an alternative technique which determines the actual base-by-base DNA sequence of selected portions of an individual's genome.
  • the IC nucleotide sequences described herein can be used to prepare two PCR primers from the 5' and 3' ends of the sequences. These primers can then be used to amplify an individual's DNA and subsequently sequence it.
  • Panels of conesponding DNA sequences from individuals, prepared in this manner, can provide unique individual identifications, as each individual will have a unique set of such DNA sequences due to allelic differences.
  • the sequences of the present invention can be used to obtain such identification sequences from individuals and from tissue.
  • the IC nucleotide sequences of the invention uniquely represent portions of the human genome. Allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions. It is estimated that allelic variation between individual humans occurs with a frequency of about once per each 500 bases.
  • Each of the sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification purposes.
  • the noncoding sequences of SEQ ED NO:l or 4 can comfortably provide positive individual identification with a panel of perhaps 10 to 1,000 primers which each yield a noncoding amplified sequence of 100 bases. If predicted coding sequences, such as that in SEQ ED NO: 3 or 6 are used, a more appropriate number of primers for positive individual identification would be 500-2,000.
  • a panel of reagents from IC nucleotide sequences described herein is used to generate a unique identification database for an individual, those same reagents can later be used to identify tissue from that individual.
  • Using the unique identification database positive identification of the individual, living or dead, can be made from extremely small tissue samples.
  • DNA-based identification techniques can also be used in forensic biology. Forensic biology is a scientific field employing genetic typing of biological evidence found at a crime scene as a means for positively identifying, for example, a perpetrator of a crime.
  • PCR technology can be used to amplify DNA sequences taken from very small biological samples such as tissues, e.g., hair or skin, or body fluids, e.g., blood, saliva, or semen found at a crime scene. The amplified sequence can then be compared to a standard, thereby allowing identification of the origin of the biological sample.
  • sequences of the present invention can be used to provide polynucleotide reagents, e.g., PCR primers, targeted to specific loci in the human genome, which can enhance the reliability of DNA-based forensic identifications by, for example, providing another "identification marker" (i.e. another DNA sequence that is unique to a particular individual).
  • an "identification marker” i.e. another DNA sequence that is unique to a particular individual.
  • actual base sequence information can be used for identification as an accurate alternative to patterns formed by restriction enzyme generated fragments.
  • Sequences targeted to noncoding regions of SEQ ED NO:l or 4 are particularly appropriate for this use as greater numbers of polymorphisms occur in the noncoding regions, making it easier to differentiate individuals using this technique.
  • polynucleotide reagents include the IC nucleotide sequences or portions thereof, e.g., fragments derived from the noncoding regions of SEQ ED NO:l or 4 having a length of at least 20 bases, preferably at least 30 bases.
  • the IC nucleotide sequences described herein can further be used to provide polynucleotide reagents, e.g., labeled or labelable probes which can be used in, for example, an in situ hybridization technique, to identify a specific tissue, e.g., thymus or brain tissue. This can be very useful in cases where a forensic pathologist is presented with a tissue of unknown origin. Panels of such IC probes can be used to identify tissue by species and/or by organ type.
  • polynucleotide reagents e.g., labeled or labelable probes which can be used in, for example, an in situ hybridization technique, to identify a specific tissue, e.g., thymus or brain tissue. This can be very useful in cases where a forensic pathologist is presented with a tissue of unknown origin. Panels of such IC probes can be used to identify tissue by species and/or by organ type.
  • these reagents e.g., IC primers or probes can be used to screen tissue culture for contamination (i.e. screen for the presence of a mixture of different types of cells in a culture).
  • the present invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, and monitoring clinical trials are used for prognostic (predictive) purposes to thereby treat an individual prophylactically. Accordingly, one aspect of the present invention relates to diagnostic assays for determining IC protein and/or nucleic acid expression as well as IC activity, in the context of a biological sample (e.g., blood, serum, cells, tissue) to thereby determine whether an individual is afflicted with a disease or disorder, or is at risk of developing a disorder, associated with abenant or unwanted IC expression or activity.
  • a biological sample e.g., blood, serum, cells, tissue
  • the invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with IC protein, nucleic acid expression or activity. For example, mutations in an IC gene can be assayed in a biological sample. Such assays can be used for prognostic or predictive purpose to thereby phophylactically treat an individual prior to the onset of a disorder characterized by or associated with IC protein, nucleic acid expression or activity. Another aspect of the invention pertains to monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of IC in clinical trials.
  • agents e.g., drugs, compounds
  • An exemplary method for detecting the presence or absence of IC protein or nucleic acid in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting IC protein or nucleic acid (e.g., mRNA, or genomic DNA) that encodes IC protein such that the presence of IC protein or nucleic acid is detected in the biological sample.
  • a prefened agent for detecting IC mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to IC mRNA or genomic DNA.
  • the nucleic acid probe can be, for example, the IC nucleic acid set forth in SEQ ID NO:l, 3, 4, or 6, or the DNA insert of the plasmid deposited with ATCC as Accession Number or , or a portion thereof, such as an ohgonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to IC mRNA or genomic DNA.
  • Other suitable probes for use in the diagnostic assays of the invention are described herein.
  • a prefened agent for detecting IC protein is an antibody capable of binding to IC protein, preferably an antibody with a detectable label.
  • Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab')2) can be used.
  • the term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled.
  • Examples of indirect labeling include detection of a primary antibody using a fiuorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fiuorescently labeled streptavidin.
  • biological sample is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. That is, the detection method of the invention can be used to detect IC mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo.
  • in vitro techniques for detection of IC mRNA include Northern hybridizations and in situ hybridizations.
  • In vitro techniques for detection of IC protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence.
  • In vitro techniques for detection of IC genomic DNA include
  • in vivo techniques for detection of IC protein include introducing into a subject a labeled anti-IC antibody.
  • the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
  • the biological sample contains protein molecules from the test subject.
  • the biological sample can contain mRNA molecules from the test subject or genomic DNA molecules from the test subject.
  • a prefened biological sample is a serum sample isolated by conventional means from a subject.
  • the methods further involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent capable of detecting IC protein, mRNA, or genomic DNA, such that the presence of IC protein, mRNA or genomic DNA is detected in the biological sample, and comparing the presence of IC protein, mRNA or genomic DNA in the control sample with the presence of IC protein, mRNA or genomic DNA in the test sample.
  • kits for detecting the presence of IC in a biological sample can comprise a labeled compound or agent capable of detecting IC protein or mRNA in a biological sample; means for determining the amount of IC in the sample; and means for comparing the amount of IC in the sample with a standard.
  • the compound or agent can be packaged in a suitable container.
  • the kit can further comprise instructions for using the kit to detect IC protein or nucleic acid.
  • the diagnostic methods described herein can furthermore be utilized to identify subjects having or at risk of developing a disease or disorder associated with abenant or unwanted IC expression or activity.
  • the term "abenant” includes an IC expression or activity which deviates from the wild type IC expression or activity.
  • Abenant expression or activity includes increased or decreased expression or activity, as well as expression or activity which does not follow the wild type developmental pattern of expression or the subcellular pattern of expression.
  • abenant IC expression or activity is intended to include the cases in which a mutation in the IC gene causes the IC gene to be under-expressed or over-expressed and situations in which such mutations result in a non-functional IC protein or a protein which does not function in a wild-type fashion, e.g., a protein which does not interact with an IC substrate, e.g., a non-IC channel subunit or ligand, or one which interacts with a non-IC substrate, e.g. a non-IC channel subunit or ligand.
  • the term "unwanted” includes an unwanted phenomenon involved in a biological response such as cellular proliferation.
  • unwanted includes an IC expression or activity which is undesirable in a subject.
  • the assays described herein can be utilized to identify a subject having or at risk of developing a disorder associated with a misregulation in IC protein activity or nucleic acid expression, such as a CNS disorder (e.g., a cognitive or neurodegenerative disorder), a pain disorder, a muscular disorder, a cellular proliferation, growth, differentiation, or migration disorder, or a cardiovascular disorder.
  • a CNS disorder e.g., a cognitive or neurodegenerative disorder
  • a pain disorder e.g., a muscular disorder
  • a cellular proliferation, growth, differentiation, or migration disorder e.g., a cardiovascular disorder.
  • the prognostic assays can be utilized to identify a subject having or at risk for developing a disorder associated with a misregulation in IC protein activity or nucleic acid expression, such as a CNS disorder, a pain disorder, a cellular proliferation, growth, differentiation, or migration disorder, a muscular disorder, or a cardiovascular disorder.
  • a disorder associated with a misregulation in IC protein activity or nucleic acid expression such as a CNS disorder, a pain disorder, a cellular proliferation, growth, differentiation, or migration disorder, a muscular disorder, or a cardiovascular disorder.
  • the present invention provides a method for identifying a disease or disorder associated with abenant or unwanted IC expression or activity in which a test sample is obtained from a subject and IC protein or nucleic acid (e.g., mRNA or genomic DNA) is detected, wherein the presence of IC protein or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with abenant or unwanted IC expression or activity.
  • a test sample refers to a biological sample obtained from a subject of interest.
  • a test sample can be a biological fluid (e.g., cerebrospinal fluid or serum), cell sample, or tissue.
  • the prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) to treat a disease or disorder associated with abenant or unwanted IC expression or activity.
  • an agent e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate
  • agents e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate
  • agents e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate
  • such methods can be used to determine whether a subject can be effectively treated with an agent for a CNS disorder, a pain disorder, a muscular disorder, a cardiovascular disorder, or a
  • the present invention provides methods for determining whether a subject can be effectively treated with an agent for a disorder associated with abenant or unwanted IC expression or activity in which a test sample is obtained and IC protein or nucleic acid expression or activity is detected (e.g., wherein the abundance of IC protein or nucleic acid expression or activity is diagnostic for a subject that can be administered the agent to treat a disorder associated with abenant or unwanted IC expression or activity).
  • the methods of the invention can also be used to detect genetic alterations in an IC gene, thereby determining if a subject with the altered gene is at risk for a disorder characterized by misregulation in IC protein activity or nucleic acid expression, such as a CNS disorder, a pain disorder, a cellular proliferation, growth, differentiation, or migration disorder, a muscular disorder, or cardiovascular disorder.
  • the methods include detecting, in a sample of cells from the subject, the presence or absence of a genetic alteration characterized by at least one of an alteration affecting the integrity of a gene encoding an IC -protein, or the mis-expression of the IC gene.
  • such genetic alterations can be detected by ascertaining the existence of at least one of 1) a deletion of one or more nucleotides from an IC gene; 2) an addition of one or more nucleotides to an IC gene; 3) a substitution of one or more nucleotides of an IC gene, 4) a chromosomal reanangement of an IC gene; 5) an alteration in the level of a messenger RNA transcript of an IC gene, 6) abenant modification of an IC gene, such as of the methylation pattern of the genomic DNA, 7) the presence of a non-wild type splicing pattern of a messenger RNA transcript of an IC gene, 8) a non-wild type level of an IC -protein, 9) allelic loss of an IC gene, and 10) inappropriate post-translational modification of an IC-protein.
  • assays known in the art which can be used for detecting alterations in an IC gene.
  • detection of the alteration involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Patent Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) Proc.
  • PCR polymerase chain reaction
  • LCR ligation chain reaction
  • This method can include the steps of collecting a sample of cells from a subject, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to an IC gene under conditions such that hybridization and amplification of the IC-gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample.
  • nucleic acid e.g., genomic, mRNA or both
  • primers which specifically hybridize to an IC gene under conditions such that hybridization and amplification of the IC-gene (if present) occurs
  • detecting the presence or absence of an amplification product or detecting the size of the amplification product and comparing the length to a control sample.
  • PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations
  • mutations in an IC gene from a sample cell can be identified by alterations in restriction enzyme cleavage patterns.
  • sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA.
  • sequence specific ribozymes see, for example, U.S. Patent No. 5,498,531 can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.
  • genetic mutations in IC can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, to high density anays containing hundreds or thousands of oligonucleotides probes (Cronin, M.T. et al. (1996) Human Mutation 7: 244-255; Kozal, M.J. et al. (1996) Nature Medicine 2: 753-759).
  • a sample and control nucleic acids e.g., DNA or RNA
  • high density anays containing hundreds or thousands of oligonucleotides probes e.g., DNA or RNA
  • genetic mutations in IC can be identified in two dimensional anays containing light- generated DNA probes as described in Cronin, M.T. et al. supra.
  • a first hybridization anay of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear anays of sequential overlapping probes. This step allows the identification of point mutations. This step is followed by a second hybridization anay that allows the characterization of specific mutations by using smaller, specialized probe anays complementary to all variants or mutations detected.
  • Each mutation anay is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.
  • any of a variety of sequencing reactions known in the art can be used to directly sequence the IC gene and detect mutations by comparing the sequence of the sample IC with the conesponding wild-type (control) sequence.
  • sequencing reactions include those based on techniques developed by Maxam and Gilbert ((1977) Proc. Natl Acad. Sci. USA 74:560) or Sanger ((1977) Proc. Natl. Acad. Sci. USA 74:5463). It is also contemplated that any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays ((1995) Biotechniques 19:448), including sequencing by mass spectrometry (see, e.g., PCT International Publication No. WO 94/16101 ; Cohen et al (1996) Adv. Chromatogr. 36: 127-162; and Griffin et al (1993) Appl Biochem. Biotechnol. 38:147-159).
  • RNA/RNA or RNA/DNA heteroduplexes Other methods for detecting mutations in the IC gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science 230:1242).
  • the art technique of "mismatch cleavage" starts by providing heteroduplexes of formed by hybridizing (labeled) RNA or DNA containing the wild-type IC sequence with potentially mutant RNA or DNA obtained from a tissue sample.
  • the double-stranded duplexes are treated with an agent which cleaves single-stranded regions of the duplex such as which will exist due to basepair mismatches between the control and sample strands.
  • RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with SI nuclease to enzymatically digesting the mismatched regions.
  • either DNA DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. See, for example, Cotton et al (1988) Proc. Natl Acad Sci USA 85:4397; Saleeba et al. (1992) Methods Enzymol. 217:286- 295.
  • the control DNA or RNA can be labeled for detection.
  • the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called "DNA mismatch repair" enzymes) in defined systems for detecting and mapping point mutations in IC cDNAs obtained from samples of cells.
  • DNA mismatch repair enzymes
  • the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662).
  • a probe based on an IC sequence e.g., a wild-type IC sequence
  • a cDNA or other DNA product from a test cell(s).
  • the duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like. See, for example, U.S. Patent No. 5,459,039.
  • alterations in electrophoretic mobility will be used to identify mutations in IC genes.
  • SSCP single strand conformation polymorphism
  • Single- stranded DNA fragments of sample and control IC nucleic acids will be denatured and allowed to renature.
  • the secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change.
  • the DNA fragments may be labeled or detected with labeled probes.
  • the sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence.
  • the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al. (1991) Trends Genet 7:5).
  • the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGG ⁇ ) (Myers et al (1985) Nature 313:495).
  • DGG ⁇ denaturing gradient gel electrophoresis
  • DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high-melting GC-rich DNA by PCR.
  • a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem 265:12753).
  • ohgonucleotide primers may be prepared in which the known mutation is placed centrally and then hybridized to target DNA under conditions which permit hybridization only if a perfect match is found (Saiki et al. (1986) Nature 324:163); Saiki et al. (1989) Proc. Natl Acad. Sci USA 86:6230).
  • Such allele specific oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA.
  • Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization) (Gibbs et al. (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3' end of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (Prossner (1993) Tibtech 11:238).
  • amplification may also be performed using Taq ligase for amplification (Barany (1991) Proc. Natl. Acad. Sci USA 88: 189). In such cases, ligation will occur only if there is a perfect match at the 3' end of the 5' sequence making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.
  • the methods described herein may be performed, for example, by utilizing prepackaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving an IC gene.
  • any cell type or tissue in which IC is expressed may be utilized in the prognostic assays described herein.
  • Monitoring the influence of agents (e.g., drugs) on the expression or activity of an IC protein can be applied not only in basic drug screening, but also in clinical trials.
  • agents e.g., drugs
  • the effectiveness of an agent determined by a screening assay as described herein to increase IC gene expression, protein levels, or upregulate IC activity can be monitored in clinical trials of subjects exhibiting decreased IC gene expression, protein levels, or downregulated IC activity.
  • the effectiveness of an agent determined by a screening assay to decrease IC gene expression, protein levels, or downregulate IC activity can be monitored in clinical trials of subjects exhibiting increased IC gene expression, protein levels, or upregulated IC activity.
  • the expression or activity of an IC gene, and preferably, other genes that have been implicated in, for example, an IC-associated disorder can be used as a "read out" or markers of the phenotype of a particular cell.
  • genes, including IC that are modulated in cells by treatment with an agent (e.g., compound, drug or small molecule) which modulates IC activity (e.g., identified in a screening assay as described herein) can be identified.
  • an agent e.g., compound, drug or small molecule
  • IC activity e.g., identified in a screening assay as described herein
  • cells can be isolated and RNA prepared and analyzed for the levels of expression of IC and other genes implicated in the IC-associated disorder, respectively.
  • the levels of gene expression can be quantified by northern blot analysis or RT-PCR, as described herein, or alternatively by measuring the amount of protein produced, by one of the methods as described herein, or by measuring the levels of activity of IC or other genes.
  • the gene expression pattern 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 points during treatment of the individual with the agent.
  • 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) obtaining a pre-administration sample from a subject prior to administration of the agent; (ii) detecting the level of expression of an IC protein, mRNA, or genomic DNA in the preadministration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level of expression or activity of the IC protein, mRNA, or genomic DNA in the post-administration samples; (v) comparing the level of expression or activity of the IC protein, mRNA, or genomic DNA in the pre-administration sample with the IC protein, mRNA, or genomic DNA in the post administration sample or samples; and (vi) altering the administration of the agent to the subject accordingly.
  • an agent e.g., an agonist
  • increased administration of the agent may be desirable to increase the expression or activity of IC to higher levels than detected, i.e., to increase the effectiveness of the agent.
  • decreased administration of the agent may be desirable to decrease expression or activity of IC to lower levels than detected, i.e. to decrease the effectiveness of the agent.
  • IC expression or activity may be used as an indicator of the effectiveness of an agent, even in the absence of an observable phenotypic response.
  • the present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with abenant or unwanted IC expression or activity, e.g. a CNS disorder, a pain disorder, a cellular proliferation, growth, differentiation, or migration disorder, a muscular disorder, or a cardiovascular disorder.
  • a CNS disorder e.g. a CNS disorder, a pain disorder, a cellular proliferation, growth, differentiation, or migration disorder, a muscular disorder, or a cardiovascular disorder.
  • Treatment is defined as the application or administration of a therapeutic agent to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has a disease or disorder, a symptom of disease or disorder or a predisposition toward a disease or disorder, with the purpose of curing, healing, alleviating, relieving, altering, remedying, ameliorating, improving or affecting the disease or disorder, the symptoms of disease or disorder or the predisposition toward a disease or disorder.
  • a therapeutic agent includes, but is not limited to, small molecules, peptides, antibodies, ribozymes and antisense oligonucleotides.
  • prophylactic and therapeutic methods of treatment such treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics.
  • “Pharmacogenomics” refers to the application of genomics technologies such as gene sequencing, statistical genetics, and gene expression analysis to drugs in clinical development and on the market. More specifically, the term refers the study of how a patient's genes determine his or her response to a drug (e.g., a patient's "drug response phenotype", or “drug response genotype”).
  • another aspect of the invention provides methods for tailoring an individual's prophylactic or therapeutic treatment with either the IC molecules of the present invention or IC modulators according to that individual's drag response genotype.
  • Pharmacogenomics allows a clinician or physician to target prophylactic or therapeutic treatments to patients who will most benefit from the treatment and to avoid treatment of patients who will experience toxic drug-related side effects.
  • the invention provides a method for preventing in a subject, a disease or condition associated with an abenant or unwanted IC expression or activity, by administering to the subject an IC or an agent which modulates IC expression or at least one IC activity.
  • Subjects at risk for a disease which is caused or contributed to by abenant or unwanted IC expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein.
  • Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the IC abenancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression.
  • an IC, IC agonist or IC antagonist agent can be used for treating the subject. The appropriate agent can be determined based on screening assays described herein.
  • the modulatory method of the invention involves contacting a cell with an IC or agent that modulates one or more of the activities of IC protein activity associated with the cell.
  • An agent that modulates IC protein activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring target molecule of an IC protein (e.g., an IC substrate), an IC antibody, an IC agonist or antagonist, a peptidomimetic of an IC agonist or antagonist, or other small molecule.
  • the agent stimulates one or more IC activities.
  • stimulatory agents include active IC protein and a nucleic acid molecule encoding IC that has been introduced into the cell.
  • the agent inhibits one or more IC activities.
  • inhibitory agents include antisense IC nucleic acid molecules, anti-IC antibodies, and IC inhibitors.
  • the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., upregulates or downregulates) IC expression or activity.
  • an agent e.g., an agent identified by a screening assay described herein
  • the method involves administering an IC protein or nucleic acid molecule as therapy to compensate for reduced, abenant, or unwanted IC expression or activity.
  • Stimulation of IC activity is desirable in situations in which IC is abnormally downregulated and/or in which increased IC activity is likely to have a beneficial effect.
  • inhibition of IC activity is desirable in situations in which IC is abnormally upregulated and/or in which decreased IC activity is likely to have a beneficial effect.
  • IC molecules of the present invention as well as agents, or modulators which have a stimulatory or inhibitory effect on IC activity (e.g., IC gene expression) as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) IC-associated disorders (e.g., proliferative disorders) associated with abenant or unwanted IC activity.
  • IC-associated disorders e.g., proliferative disorders
  • pharmacogenomics i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug
  • Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug.
  • a physician or clinician may consider applying knowledge obtained in relevant pharmacogenomics studies in determining whether to administer an IC molecule or IC modulator as well as tailoring the dosage and/or therapeutic regimen of treatment with an IC molecule or IC modulator.
  • Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See, for example, Eichelbaum, M. et al. (1996) Clin. Exp. Pharmacol. Physiol. 23(10-11): 983-985 and Linder, M.W. et al. (1997) Clin. Chem. 43(2): 254-266.
  • 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 drag metabolism). These pharmacogenetic conditions can occur either as rare genetic defects or as naturally-occurring polymorphisms.
  • G6PD glucose-6-phosphate dehydrogenase deficiency
  • oxidant drugs anti-malarials, sulfonamides, analgesics, nitrofurans
  • a genome-wide association relies primarily on a high-resolution map of the human genome consisting of already known gene-related markers (e.g., a "bi-allelic” gene marker map which consists of 60,000-100,000 polymorphic or variable sites on the human genome, each of which has two variants.)
  • gene-related markers e.g., a "bi-allelic” gene marker map which consists of 60,000-100,000 polymorphic or variable sites on the human genome, each of which has two variants.
  • Such a high-resolution genetic map can be compared to a map of the genome of each of a statistically significant number of patients taking part in a Phase TTJTTT drug trial to identify markers associated with a particular observed drag response or side effect.
  • such a high resolution map can be generated from a combination of some ten-million known single nucleotide polymorphisms (SNPs) in the human genome.
  • SNPs single nucleotide polymorphisms
  • a "SNP" is a common alteration that occurs in a single nucleotide base in a stretch of DNA. For example, a SNP may occur once per every 1000 bases of DNA.
  • a SNP may be involved in a disease process, however, the vast majority may not be disease-associated.
  • individuals Given a genetic map based on the occunence of such SNPs, individuals can be grouped into genetic categories depending on a particular pattern of SNPs in their individual genome.
  • treatment regimens can be tailored to groups of genetically similar individuals, taking into account traits that may be common among such genetically similar individuals.
  • a method termed the "candidate gene approach” can be utilized to identify genes that predict drug response. According to this method, if a gene that encodes a drugs target is known (e.g., an IC protein of the present invention), all common variants of that gene can be fairly easily identified in the population and it can be determined if having one version of the gene versus another is associated with a particular drug response.
  • the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action.
  • drug metabolizing enzymes e.g., N-acetyltransf erase 2 (NAT 2) and cytochrome P450 enzymes CYP2D6 and CYP2C19
  • NAT 2 N-acetyltransf erase 2
  • CYP2D6 and CYP2C19 cytochrome P450 enzymes
  • the gene coding for CYP2D6 is highly polymorphic and several mutations have been identified in PM, which all lead to the absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quite frequently experience exaggerated drug response and side effects when they receive standard doses. If a metabolite is the active therapeutic moiety, PM show no therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6-formed metabolite morphine. The other extreme is the so called ultra-rapid metabolizers who do not respond to standard doses. Recently, the molecular basis of ultra-rapid metabolism has been identified to be due to CYP2D6 gene amplification.
  • a method termed the "gene expression profiling" can be utilized to identify genes that predict drug response.
  • a drug e.g., an IC molecule or IC modulator of the present invention
  • the gene expression of an animal dosed with a drug can give an indication whether gene pathways related to toxicity have been turned on.
  • Information generated from more than one of the above pharmacogenomics approaches can be used to determine appropriate dosage and treatment regimens for prophylactic or therapeutic treatment an individual. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with an IC molecule or IC modulator, such as a modulator identified by one of the exemplary screening assays described herein.
  • DHY sequence information refers to any nucleotide and/or amino acid sequence information particular to the DHY molecules of the present invention, including but not limited to full-length nucleotide and/or amino acid sequences, partial nucleotide and/or amino acid sequences, polymorphic sequences including single nucleotide polymorphisms (SNPs), epitope sequences, and the like.
  • SNPs single nucleotide polymorphisms
  • information "related to" said DHY sequence information includes detection of the presence or absence of a sequence (e.g., detection of expression of a sequence, fragment, polymorphism, etc.), determination of the level of a sequence (e.g., detection of a level of expression, for example, a quantative detection), detection of a reactivity to a sequence (e.g., detection of protein expression and/or levels, for example, using a sequence-specific antibody), and the like.
  • electronic apparatus readable media refers to any suitable medium for storing, holding or containing data or information that can be read and accessed directly by an electronic apparatus.
  • Such media can include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as compact disc; electronic storage media such as RAM, ROM, EPROM, EEPROM and the like; general hard disks and hybrids of these categories such as magnetic/optical storage media.
  • the medium is adapted or configured for having recorded thereon DHY sequence information of the present invention.
  • the term "electronic apparatus” is intended to include any suitable computing or processing apparatus or other device configured or adapted for storing data or information.
  • Examples of electronic apparatus suitable for use with the present invention include stand-alone computing apparatus; networks, including a local area network (LAN), a wide area network (WAN) Internet, Intranet, and Extranet; electronic appliances such as a personal digital assistants (PDAs), cellular phone, pager and the like; and local and distributed processing systems.
  • “recorded” refers to a process for storing or encoding information on the electronic apparatus readable medium. Those skilled in the art can readily adopt any of the presently known methods for recording information on known media to generate manufactures comprising the DHY sequence information.
  • sequence information can be represented in a word processing text file, formatted in commercially- available software such as WordPerfect and MicroSoft Word, or represented in the form of an ASCII file, stored in a database application, such as DB2, Sybase, Oracle, or the like, as well as in other forms.
  • a database application such as DB2, Sybase, Oracle, or the like
  • Any number of data processor structuring formats e.g., text file or database
  • DHY sequence information By providing DHY sequence information in readable form, one can routinely access the sequence information for a variety of purposes.
  • sequence information in readable form to compare a target sequence or target structural motif with the sequence information stored within the data storage means.
  • Search means are used to identify fragments or regions of the sequences of the invention which match a particular target sequence or target motif.
  • the present invention therefore provides a medium for holding instructions for performing a method for determining whether a subject has a DHY- associated disease or disorder or a pre-disposition to a DHY-associated disease or disorder, wherein the method comprises the steps of determining DHY sequence information associated with the subject and based on the DHY sequence information, determining whether the subject has a DHY - associated disease or disorder or a pre-disposition to a DHY-associated disease or disorder and/or recommending a particular treatment for the disease, disorder or pre-disease condition.
  • the present invention further provides in an electronic system and/or in a network, a method for determining whether a subject has a DHY-associated disease or disorder or a pre-disposition to a disease associated with a DHY wherein the method comprises the steps of determining DHY sequence information associated with the subject, and based on the DHY sequence information, determining whether the subject has a DHY -associated disease or disorder or a pre-disposition to a DHY-associated disease or disorder, and/or recommending a particular treatment for the disease, disorder or pre-disease condition.
  • the method may further comprise the step of receiving phenotypic information associated with the subject and/or acquiring from a network phenotypic information associated with the subject.
  • the present invention also provides in a network, a method for determining whether a subject has a DHY-associated disease or disorder or a pre-disposition to a DHY associated disease or disorder associated with DHY, said method comprising the steps of receiving DHY sequence information from the subject and/or information related thereto, receiving phenotypic information associated with the subject, acquiring information from the network conesponding to DHY and/or a DHY-associated disease or disorder, and based on one or more of the phenotypic information, the DHY information (e.g., sequence information and/or information related thereto), and the acquired information, determining whether the subject has a DHY-associated disease or disorder or a pre-disposition to a DHY-associated disease or disorder (e.g., as a CNS disorder, a pain disorder, a cellular proliferation, growth, differentiation, or migration disorder, a muscular disorder, or a cardiovascular disorder).
  • a DHY-associated disease or disorder e.g., as a CNS disorder,
  • the method may further comprise the step of recommending a particular treatment for the disease, disorder or pre-disease condition.
  • the present invention also provides a business method for determining whether a subject has a DHY-associated disease or disorder or a pre-disposition to a DHY-associated disease or disorder, said method comprising the steps of receiving information related to DHY (e.g., sequence information and/or information related thereto), receiving phenotypic information associated with the subject, acquiring information from the network related to DHY and/or related to a DHY-associated disease or disorder, and based on one or more of the phenotypic information, the DHY information, and the acquired information, determining whether the subject has a DHY-associated disease or disorder or a predisposition to a DHY-associated disease or disorder.
  • the method may further comprise the step of recommending a particular treatment for the disease, disorder or pre-disease condition.
  • the invention also includes an anay comprising a DHY sequence of the present invention.
  • the anay can be used to assay expression of one or more genes in the anay.
  • the anay can be used to assay gene expression in a tissue to ascertain tissue specificity of genes in the anay. In this manner, up to about 7600 genes can be simultaneously assayed for expression, one of which can be DHY. This allows a profile to be developed showing a battery of genes specifically expressed in one or more tissues.
  • the invention allows the quantitation of gene expression.
  • tissue specificity but also the level of expression of a battery of genes in the tissue is ascertainable.
  • genes can be grouped on the basis of their tissue expression per se and level of expression in that tissue. This is useful, for example, in ascertaining the relationship of gene expression between or among tissues.
  • one tissue can be perturbed and the effect on gene expression in a second tissue can be determined.
  • the effect of one cell type on another cell type in response to a biological stimulus can be determined.
  • Such a determination is useful, for example, to know the effect of cell-cell interaction at the level of gene expression.
  • the invention provides an assay to determine the molecular basis of the undesirable effect and thus provides the opportunity to co-administer a counteracting agent or otherwise treat the undesired effect.
  • undesirable biological effects can be determined at the molecular level.
  • the effects of an agent on expression of other than the target gene can be ascertained and counteracted.
  • the anay can be used to monitor the time course of expression of one or more genes in the anay. This can occur in various biological contexts, as disclosed herein, for example development of a DHY-associated disease or disorder, progression of DHY-associated disease or disorder, and processes, such a cellular transformation associated with the DHY-associated disease or disorder.
  • the anay is also useful for ascertaining the effect of the expression of a gene on the expression of other genes in the same cell or in different cells (e.g., ascertaining the effect of DHY expression on the expression of other genes). This provides, for example, for a selection of alternate molecular targets for therapeutic intervention if the ultimate or downstream target cannot be regulated.
  • the anay is also useful for ascertaining differential expression patterns of one or more genes in normal and abnormal cells.
  • This provides a battery of genes (e.g., including DHY) that could serve as a molecular target for diagnosis or therapeutic intervention.
  • the invention is based, at least in part, on the discovery of two human genes encoding novel proteins, refened to herein as IC23949 and IC32391.
  • the entire sequence of the human clone Fbh23949FL was determined and found to contain an open reading frame termed human "IC23949.”
  • the nucleotide sequence of the human IC23949 gene is set forth in Figure 1 A-F and in SEQ ED NOs: 1 and 3.
  • the amino acid sequence of the human IC23949 expression product is set forth in Figure 1 A-F and in SEQ ED NO:2.
  • the nucleotide sequence encoding the human IC23949 protein is shown in Figure
  • SEQ TD NO:l The protein encoded by this nucleic acid comprises about 1739 amino acids and has the amino acid sequence shown in Figure 1 A-F and set forth as SEQ TD NO:2.
  • the coding region (open reading frame) of SEQ ED NO:l is set forth as SEQ ED NO:3.
  • Clone Fbh23949FL comprising the coding region of human IC23949, was deposited with the American Type Culture Collection (ATCC®), 10801
  • the entire sequence of the human clone Fbh32391FL was determined and found to contain an open reading frame termed human "IC32391."
  • the nucleotide sequence of the human IC32391 gene is set forth in Figure 2A-C and in SEQ ID NOs:4 and 6.
  • the amino acid sequence of the human IC32391 expression product is set forth in Figure 2A-C and in SEQ TD NO:5.
  • the nucleotide sequence encoding the human IC32391 protein is shown in Figure 2A-C and is set forth as SEQ TD NO:4.
  • the protein encoded by this nucleic acid comprises about 1010 amino acids and has the amino acid sequence shown in Figure 2A-C and set forth as SEQ ED NO:5.
  • the coding region (open reading frame) of SEQ TD NO:4 is set forth as SEQ ED NO:6.
  • Clone Fbh32391FL comprising the coding region of human IC32391, was deposited with the American Type Culture Collection (ATCC®), 10801
  • the amino acid sequences of human IC23949 and human IC32391 were analyzed using the program PSORT (http://www. psort.nibb.ac.jp) to predict the localization of the proteins within the cell. This program assesses the presence of different targeting and localization amino acid sequences within the query sequence.
  • the results of the analysis show that human IC23949 (SEQ ED NO:2) may be localized to the endoplasmic reticulum, to the mitochondrion, or to the vacuoles. A signal peptide may be present.
  • human IC32391 (SEQ ED NO: 5) may be localized to the endoplasmic reticulum, to the mitochondrion, to vacuoles, to the nucleus, to the cytoplasm, or to the Golgi apparatus.
  • a signal peptide may be present.
  • the results of the search are set forth in Figure 9A-F.
  • the results of the search are set forth in Figure 10A-E.
  • This example describes the tissue distribution of human IC32391 and IC23949 mRNA in a variety of cells and tissues, as determined using the TaqManTM procedure.
  • the TaqmanTM procedure is a quantitative, reverse transcription PCR-based approach for detecting mRNA.
  • the RT-PCR reaction exploits the 5' nuclease activity of AmpliTaq GoldTM DNA Polymerase to cleave a TaqManTM probe during PCR.
  • cDNA was generated from the samples of interest, e.g., various human, rat, and monkey tissues, and used as the starting material for PCR amplification.
  • a gene-specific ohgonucleotide probe (complementary to the region being amplified) was included in the reaction (i.e., the TaqmanTM probe).
  • the TaqManTM probe includes the ohgonucleotide with a fluorescent reporter dye covalently linked to the 5' end of the probe (such as FAM (6-carboxyfluorescein), TET (6-carboxy-4,7,2',7'- tetrachlorofluorescein), JOE (6-carboxy-4,5-dichloro-2,7-dimethoxyfluorescein), or VIC) and a quencher dye (TAMRA (6-carboxy-N,N,N',N'-tetramethylrhodamine) at the 3' end of the probe.
  • a fluorescent reporter dye covalently linked to the 5' end of the probe
  • TET 6-carboxy-4,7,2',7'- tetrachlorofluorescein
  • cleavage of the probe separates the reporter dye and the quencher dye, resulting in increased fluorescence of the reporter. Accumulation of PCR products is detected directly by monitoring the increase in fluorescence of the reporter dye. When the probe is intact, the proximity of the reporter dye to the quencher dye results in suppression of the reporter fluorescence.
  • the probe specifically anneals between the forward and reverse primer sites. The 5 '-3' nucleolytic activity of the AmpliTaqTM Gold DNA Polymerase cleaves the probe between the reporter and the quencher only if the probe hybridizes to the target. The probe fragments are then displaced from the target, and polymerization of the strand continues.
  • a human tissue panel was tested revealing highest expression of human IC32391 mRNA in the brain and the spinal cord (see Figure 11).
  • a rat panel indicated highest expression of human IC32391 mRNA in the trigeminal root ganglion (TRG), striatum, thalamus, dorsal nuclei, spinal cord, brain stem, brain, cortex, and cerebellum, with weaker expression in the dorsal root ganglia (DRG) and superior cervical ganglia (SCG) (see Figure 12).
  • TRG trigeminal root ganglion
  • striatum striatum
  • thalamus dorsal nuclei
  • spinal cord spinal cord
  • brain stem brain stem
  • brain cortex
  • cerebellum cerebellum
  • a third panel containing various monkey and human tissues indicated highest expression of human IC32391 in the monkey cortex and spinal cord and in human brain and spinal cord (see Figure 13).
  • Both IC32391 and IC23949 show highest expression in neural tissue (e.g., brain, spinal cord, and DRG), indicating a role for these molecules in ion channel-associated disorders involving sensory neurons, including, but not limited to, pain disorders.
  • neural tissue e.g., brain, spinal cord, and DRG
  • IC32391 and IC23949 may play a role in CNS disorders and/or other diseases or disorders related to the brain, spinal cord, or sensory neurons.
  • IC32391 mRNA as may be determined by Polymerase Chain Reaction (PCR) on cDNA libraries using ohgonucleotide primers based on the human IC sequence.
  • PCR Polymerase Chain Reaction
  • tissues e.g. tissues obtained from brain
  • various tissues are first frozen on dry ice.
  • Ten-micrometer-thick sections of the tissues are postfixed with 4% formaldehyde in DEPC treated IX phosphate- buffered saline at room temperature for 10 minutes before being rinsed twice in DEPC IX phosphate-buffered saline and once in 0.1 M triethanolamine-HCl (pH 8.0).
  • sections are rinsed in DEPC 2X SSC (IX SSC is 0.15M NaCI plus 0.015M sodium citrate).
  • Tissue is then dehydrated through a series of ethanol washes, incubated in 100% chloroform for 5 minutes, and then rinsed in 100% ethanol for 1 minute and 95% ethanol for 1 minute and allowed to air dry.
  • Hybridizations are performed with 35s-radiolabeled (5 X 10 ⁇ cpm/ml) cRNA, probes. Probes are incubated in the presence of a solution containing 600 mM NaCI, 10 mM Tris (pH 7.5), 1 mM EDTA, 0.01% sheared salmon sperm DNA, 0.01% yeast tRNA, 0.05%) yeast total RNA type XI, IX Denhardt's solution, 50%) formamide, 10% dextran sulfate, 100 mM dithiothreitol, 0.1% sodium dodecyl sulfate (SDS), and 0.1% sodium thiosulfate for 18 hours at 55°C.
  • SDS sodium dodecyl sulfate
  • slides are washed with 2X SSC. Sections are then sequentially incubated at 37°C in TNE (a solution containing 10 mM Tris-HCl (pH 7.6), 500 mM NaCI, and 1 mM EDTA), for 10 minutes, in TNE with lO ⁇ g of RNase A per ml for 30 minutes, and finally in TNE for 10 minutes. Slides are then rinsed with 2X SSC at room temperature, washed with 2X SSC at 50°C for 1 hour, washed with 0.2X SSC at 55°C for 1 hour, and 0.2X SSC at 60°C for 1 hour.
  • TNE a solution containing 10 mM Tris-HCl (pH 7.6), 500 mM NaCI, and 1 mM EDTA
  • human IC23949 or human IC32391 is expressed as a recombinant glutathione-S-transferase (GST) fusion polypeptide in E. coli and the fusion polypeptide is isolated and characterized.
  • GST glutathione-S-transferase
  • human IC23949 or human IC32391 are fused to GST and this fusion polypeptide is expressed in E. coli, e.g., strain PEB199. Expression of the GST-IC fusion protein in PEB199 is induced with EPTG.
  • the recombinant fusion polypeptide is purified from crude bacterial lysates of the induced PEB199 strain by affinity chromatography on glutathione beads. Using polyacrylamide gel electrophoretic analysis of the polypeptide purified from the bacterial lysates, the molecular weight of the resultant fusion polypeptide is determined.
  • the pcDNA/Amp vector by Invitrogen Corporation (San Diego, CA) is used.
  • This vector contains an S V40 origin of replication, an ampicillin resistance gene, an E. coli replication origin, a CMV promoter followed by a polylinker region, and an SV40 intron and polyadenylation site.
  • S V40 origin of replication an ampicillin resistance gene
  • E. coli replication origin an E. coli replication origin
  • CMV promoter followed by a polylinker region
  • an SV40 intron and polyadenylation site a DNA fragment encoding the entire human IC23949 or human IC32391 protein and an HA tag (Wilson et al.
  • the human IC23949 or human IC32391 DNA sequence is amplified by PCR using two primers.
  • the 5' primer contains the restriction site of interest followed by approximately twenty nucleotides of the human IC23949 or human IC32391 coding sequence starting from the initiation codon; the 3' end sequence contains complementary sequences to the other restriction site of interest, a translation stop codon, the HA tag or FLAG tag and the last 20 nucleotides of the human IC23949 or human
  • the PCR amplified fragment and the pCDNA/Amp vector are digested with the appropriate restriction enzymes and the vector is dephosphorylated using the CLAP enzyme (New England Biolabs, Beverly, MA).
  • CLAP enzyme New England Biolabs, Beverly, MA
  • the two restriction sites chosen are different so that the IC gene is inserted in the conect orientation.
  • the ligation mixture is transformed into E. coli cells (strains HB101, DH5 ⁇ , SURE, available from
  • Plasmid DNA is isolated from transformants and examined by restriction analysis for the presence of the conect fragment.
  • COS cells are subsequently transfected with the human IC23949-pcDNA/Amp plasmid DNA or human IC32391-pcDNA/Amp plasmid DNA using the calcium phosphate or calcium chloride co-precipitation methods, DEAE-dextran-mediated transfection, lipofection, or electroporation.
  • Other suitable methods for transfecting host cells can be found in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.
  • the expression of the polypeptide is detected by radiolabelling ( 35 S-methionine or 35 S-cysteine available from NEN, Boston, MA, can be used) and immunoprecipitation (Hariow, E. and Lane, D. Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988) using an HA specific monoclonal antibody. Briefly, the cells are labelled for 8 hours with 35 S- methionine (or 35 S-cysteine). The culture media are then collected and the cells are lysed using detergents (RIPA buffer, 150 mM NaCI, 1 % NP-40, 0.1 % SDS, 0.5% DOC, 50 mM Tris, pH 7.5). Both the cell lysate and the culture media are precipitated with an HA specific monoclonal antibody. Precipitated polypeptides are then analyzed by SDS-PAGE.
  • DNA containing the human IC23949 or human IC32391 coding sequence is cloned directly into the polylinker of the pCDNA/Amp vector using the appropriate restriction sites.
  • the resulting plasmid is transfected into COS cells in the manner described above, and the expression of the human IC23949 or human IC32391 polypeptide is detected by radiolabelling and immunoprecipitation using a human IC23949 or human IC32391 specific monoclonal antibody.

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Abstract

L'invention concerne des molécules d'acides nucléiques isolées, dites molécules d'acides nucléiques isolées IC, codant pour de nouvelles molécules liées à un canal ionique IC. L'invention concerne également des molécules d'acides nucléiques antisens, des vecteurs d'expression de recombinaison contenant les molécules d'acides nucléiques IC, des cellules hôtes dans lesquelles on a introduit des vecteurs d'expression et des animaux transgéniques non humains dans lesquels on a introduit ou interrompu un gène IC. L'invention concerne aussi des protéines isolées IC, des protéines hybrides, des peptides antigéniques, et des anticorps anti-IC. On décrit enfin des méthodes diagnostiques utilisant les compositions décrites dans cette invention.
EP01930885A 2000-04-29 2001-04-27 23949 et 32391, nouveaux canaux ioniques humains, et utilisation associee Withdrawn EP1280902A2 (fr)

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US20020042100A1 (en) * 2000-07-03 2002-04-11 Chunhua Yan Isolated human ion channel proteins, nucleic acid molecules encoding human ion channel proteins, and uses thereof
UA79927C2 (en) * 2000-12-05 2007-08-10 Serono Genetics Inst Sa Polynucleotide, coding a polypeptide of potential-depending portal ionic human channel (canion), polypeptyde, antibody, method for identification of candidate modulator of canion-polypeptyde, method for treatment of bipolar disorder or schizophrenia and use of an antibody for production of drugs for treatment of schizophrenia or bipolar disorder
CA2446708A1 (fr) * 2001-05-10 2002-12-19 Mochida Pharmaceutical Co., Ltd. Nouvelle proteine du canal cation dependant du voltage
EP1532452A4 (fr) 2002-05-31 2006-11-29 Sloan Kettering Inst Cancer Canaux ioniques heterologues actives par un stimulus et leurs procedes d'utilisation
EP3716990A4 (fr) 2017-11-27 2021-12-08 Coda Biotherapeutics, Inc. Compositions et procédés de traitement de maladies neurologiques

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WO1999066041A1 (fr) * 1998-06-16 1999-12-23 Human Genome Sciences, Inc. 94 proteines humaines secretees
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WO1999028342A2 (fr) * 1997-12-03 1999-06-10 Merck & Co., Inc. Compositions a canaux calcium actives a basse tension et procedes correspondants
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