EP1200586A2 - Human nervous system-associated proteins - Google Patents

Human nervous system-associated proteins

Info

Publication number
EP1200586A2
EP1200586A2 EP00948833A EP00948833A EP1200586A2 EP 1200586 A2 EP1200586 A2 EP 1200586A2 EP 00948833 A EP00948833 A EP 00948833A EP 00948833 A EP00948833 A EP 00948833A EP 1200586 A2 EP1200586 A2 EP 1200586A2
Authority
EP
European Patent Office
Prior art keywords
nsprt
polynucleotide
polypeptide
sequence
sequences
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00948833A
Other languages
German (de)
French (fr)
Inventor
Y. Tom Tang
Henry Yue
Dyung Aina M. Lu
Junming Yang
Roopa Reddy
Yalda Azimzai
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Incyte Corp
Original Assignee
Incyte Genomics Inc
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Filing date
Publication date
Application filed by Incyte Genomics Inc filed Critical Incyte Genomics Inc
Publication of EP1200586A2 publication Critical patent/EP1200586A2/en
Withdrawn legal-status Critical Current

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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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A61P21/04—Drugs for disorders of the muscular or neuromuscular system for myasthenia gravis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/08—Antiepileptics; Anticonvulsants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/38—Drugs for disorders of the endocrine system of the suprarenal hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • PNS neurotrophic system
  • the PNS can be further divided into the somauc nervous system, which regulates voluntary motor ac ⁇ vity such as for skeletal muscle, and the autonomic nervous system, which regulates involuntary motor acuvity for internal organs such as the heart, lungs, and viscera
  • somauc nervous system which regulates voluntary motor ac ⁇ vity such as for skeletal muscle
  • autonomic nervous system which regulates involuntary motor acuvity for internal organs such as the heart, lungs, and viscera
  • the sympatheuc nervous system and the parasympatheuc nervous system.
  • a nerve cell contains tour regions, the cell body, axon, dend ⁇ tes, and axon terminal.
  • the cell body contains the nucleus and other organelles.
  • the dendrites are processes which extend outward from the cell body and receive signals from sense organs or from the axons ot other neurons. These signals are converted to electrical impulses and transmitted to the cell body
  • the axon whose size can range from one millimeter to more than one meter, is a single process that conducts the nerve impulse away from the cell body.
  • a change in electrical potential at the nerve terminal resulting from the electrical impulse triggers the release of the neurotransmitter from the synaptic vesicle by exocytosis
  • the neurotransmitter rapidly diffuses across the synaptic cleft separating the presynaptic nerve cell from the postsynaptic cell
  • the neurotransmitter then binds receptors and opens transmitter-gated ion channels located in the plasma membrane of the postsynaptic cell, provoking a change in the cell's electrical potential
  • This change in membrane potential of the postsynaptic cell may serve either to excite or inhibit further transmission of the nerve impulse
  • GABA is the major inhibitory neurotransmitter m the CNS
  • GABA receptors are the principal target of sedatives such as benzodiazepines and barbiturates which act by enhancing GABA-mediated effects (Katzung, B G (1995) Basic and Clinical Pharmacology.
  • ECM extracellular matrix
  • Many ECM molecules including fibronectin, vitronectin members of the laminin tenascin, collagen, and thrombospondin families, and a variety ot proteoglycans, can act either as promoters or inhibitors ot neu ⁇ te outgrowth and extension (Tessier- Lavigne et al , supra)
  • Receptors for ECM molecules include integnns immunoglobulin superfamily members, and proteoglycans.
  • ECM molecules and their receptors have also been implicated in the adhesion, maintenance, and differentiation of neurons (Reichardt, L.F et al. (1991) Ann. Rev Neurosci. 14.531-571).
  • SMA spinal muscular atrophy
  • PCD5 PCD5 ' s expression is restricted to the cerebellum and the eye.
  • the gene encoding PCD5 was localized to mouse chromosome 8 (Nordquist, D.T. et al (1988) J. Neurosci. 8(12).4780-4789).
  • the invention features purified polypeptides, human nervous system-associated proteins, referred to collectively as “NSPRT” and individually as “NSPRT- 1. " "NSPRT-2. " “NSPRT-3, “' and “NSPRT-4 ""
  • the invention provides an isolated polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected trom the group consisting of SEQ ID NO 1 -4. b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO 1 -4. c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO 1 -4. and d) an immunogemc fragment ot an amino acid sequence selected from the group consisting WO 01/07470 PCT/USOO/l 9837
  • the invention provides an isolated polypepude comp ⁇ smg the ammo acid sequence of SEQ ID NO 1 -4
  • the invention further provides an isolated polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO 1 -4, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an ammo acid sequence selected from the group consisting of SEQ ID NO 1-4, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO 1-4, and d) an immunogemc fragment ot an amino acid sequence selected from the group consisting of SEQ ID NO 1-4
  • the polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NO 1-4
  • the polynucleotide is selected from the group consisting of SEQ ID NO 5-8
  • the invention provides a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding a polypeptide comprising an ammo acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO 1-4, b) a naturally occurring ammo acid sequence having at least 90% sequence identity to an am o acid sequence selected from the group consisting of SEQ ID NO 1-4, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO 1-4, and d) an immunogemc fragment of an amino acid sequence selected from the group consisting of SEQ ID NO 1-4
  • the invention provides a cell transformed with the recombinant polynucleotide
  • the invention provides a transgenic orgamsm comprising the recombinant polynucleotide
  • the invention also provides a method for producing a polypeptide comprising an amino acid sequence selected from the group consisting ot a) an amino acid sequence selected from the group consisting of SEQ ID NO 1-4, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO 1-4, c) a biologically active fragment of an am o acid sequence selected from the group consisting of SEQ ID NO 1-4, and d) an immunogemc fragment of an amino acid sequence selected from the group consisting of SEQ ID NO 1-4
  • the method comprises a) cultu ⁇ ng a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide comprising a promoter sequence operably linked to a polvnucleotide encoding the polypepude and b) recovering the polypeptide so expressed
  • the invention provides an isolated antibodv which specifically binds to a polypeptide comprising an amino acid sequence selected from the group consisting ot a) an amino acid sequence selected from the group consisting of SEQ ID NO 1 -4 b) a naturally occurring amino acid sequence having at least 90% sequence identity to an am o acid sequence selected from the group consisting ot SEQ ID NO 1-4, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO 1-4, and d) an immunogemc fragment of an amino acid sequence selected from the group consisting of SEQ ID NO 1-4
  • the invention further provides an isolated polynucleotide comprising a polynucleotide sequence selected from the group consisting of a) a polynucleotide sequence selected from the group consisting of SEQ ID NO 5-8, b) a naturally occurring polynucleotide sequence having at least 70% sequence identity to a polynucleotide sequence selected trom the group consisting of SEQ
  • the invention provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide comprising a polynucleotide sequence selected from the group consisting of a) a polynucleotide sequence selected from the group consisting ot SEQ ID NO 5-8, b) a naturally occurring polynucleotide sequence having at least 70% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NO 5-8.
  • the method comprises a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex, and optionally, if present, the amount thereof
  • the probe comprises at least 60 contiguous nucleotides
  • the invention further provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide comprising a polynucleotide sequence selected from the group consist
  • the method comprises a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereol, and.
  • the invention further provides a pharmaceutical composition comp ⁇ sing an effective amount of a polypeptide comprising an amino acid sequence selected from the group consisting of a) an am o acid sequence selected from the group consisting of SEQ ID NO 1-4, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO 1-4, c) a biologically active fragment ot an amino acid sequence selected from the group consisting of SEQ ID NO' 1-4, and d) an immunogemc fragment of an amino acid sequence selected from the group consisting of SEQ ID NO.1-4. and a pharmaceutically acceptable exc ⁇ ient.
  • the pharmaceutical composition comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1 -4
  • the invention additionally provides a method of treating a disease or condition associated with decreased expression of functional NSPRT, comprising administering to a patient in need of such treatment the pharmaceutical composition
  • the invention also provides a method for screemng a compound for effectiveness as an agomst of a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO.1-4, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO.1-4, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-4, and d) an immunogemc fragment of an ammo acid sequence selected from the group consisting of SEQ ID NO.1-4.
  • the method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting agomst activity in the sample.
  • the invention provides a pharmaceutical composition comp ⁇ sing an agomst compound identified by the method and a pharmaceutically acceptable excipient
  • the invention provides a method of treating a disease or condition associated with decreased expression of functional NSPRT.
  • the invention provides a method for screemng a compound for effectiveness as an antagonist of a polypepude comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO 1-4, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO 1-4.
  • the invenuon provides a pharmaceuucal composition comprising an antagomst compound identified by the method and a pharmaceutically acceptable excipient
  • the invenuon provides a method of treating a disease or condition associated with overexpression of functional NSPRT, comprising administering to a patient in need of such treatment the pharmaceuucal composition.
  • the invenuon further provides a method of screemng for a compound that modulates the activity of a polypepude comp ⁇ sing an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO.1 -4, b) a naturally occur ⁇ ng amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-4, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO.1-4, and d) an immunogemc fragment of an amino acid sequence selected from the group consisting of SEQ ID NO.1-4.
  • the method comp ⁇ ses a) combimng the polypeptide with at least one test compound under conditions permissive for the activity of the polypeptide, b) assessing the activity of the polypeptide in the presence of the test compound, and c) comparing the activity of the polypepude in the presence of the test compound with the activity of the polypeptide in the absence of the test compound, wherein a change m the activity of the polypeptide in the presence ot the test compound is indicative of a compound that modulates the activity of the polypeptide.
  • Table 1 shows polypeptide and nucleotide sequence identification numbers (SEQ ID NOs), clone identification numbers (clone IDs). cDNA libraries, and cDNA fragments used to assemble full- length sequences encoding NSPRT Table 2 shows features of each polypeptide sequence, including potential moufs, homologous sequences, and methods, algorithms, and searchable databases used tor analysis of NSPRT
  • Table 3 shows selected fragments of each nucleic acid sequence; the tissue-specific expression patterns ot each nucleic acid sequence as determined by northern analysis: diseases, disorders, or conditions associated with these tissues: and the vector into which each cDNA was cloned.
  • Table 4 describes the tissues used to construct the cDNA libraries from which cDNA clones encoding NSPRT were isolated
  • Table 5 shows the tools, programs, and algorithms used to analyze the polynucleotides and polypeptides of the invention, along with applicable descriptions, references, and threshold parameters.
  • Allelic variant * is an alternative form of the gene encoding NSPRT Allelic variants may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or m polypeptides whose structure or function may or may not be altered
  • a gene may have none, one, or many allelic variants of its naturally occurring form
  • Common mutational changes which give rise to allelic variants are generally ascribed to natural deletions, additions, or substitutions of nucleotides Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence
  • Amino acids with uncharged side chains having similar hydrophilicity values may include leucine, isoleucine, and valine. glycme and alanine, and phenylalamne and tyrosine
  • Amplification relates to the production of additional copies ot a nucleic acid sequence Amplification is generally carried out using polymerase chain reaction (PCR) technologies well known
  • antagomst refers to a molecule which inhibits or attenuates the biological activity ol NSPRT.
  • Antagonists may include proteins such as antibodies, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of NSPRT either by directly interacting with NSPRT or by acting on components of the biological pathway in which NSPRT participates.
  • ⁇ antibody refers to intact immunoglobulin molecules as well as to fragments thereof, such as Fab, F(ab ' ) 2 , and Fv fragments, which are capable of binding an epitopic determinant
  • Antibodies that bind NSPRT polypeptides can be prepared using intact polypeptides or using fragments containing small peptides of interest as the immunizing antigen
  • the polypeptide or ohgopeptide used to immumze an ammal e.g., a mouse, a rat, or a rabbit
  • ca ⁇ ier protein e.g., a ca ⁇ ier protein
  • Commonly used ca ⁇ iers that are chemically coupled to peptides include bovine serum albumin, thyroglobuhn. and keyhole limpet hemocyanin (KLH) The coupled peptide is then used to immumze the ammal.
  • antigenic determinant refers to that region of a molecule (i.e., an epitope) that makes contact with a particular antibody.
  • a protein or a fragment of a protein is used to immumze a host ammal, numerous regions of the protein may induce the production of antibodies which bind specifically to antigenic determinants (particular regions or three-dimensional structures on the protein).
  • An antigenic determinant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) for binding to an antibody.
  • antisense refers to any composition capable of base-pairing with the "sense" (coding) strand of a specific nucleic acid sequence.
  • Antisense compositions may include DNA, RNA. peptide nucleic acid (PNA), o gonucleotides having modified backbone linkages such as phosphor othioates, methylphosphonates, or benzylphosphonates.
  • PNA peptide nucleic acid
  • ohgonucleotides having modified sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars
  • ohgonucleotides having modified bases such as 5-methyl cytosine, 2'-deoxyurac ⁇ l, or 7-deaza-2'-deoxyguanos ⁇ ne.
  • Antisense molecules may be produced by any method including chemical synthesis or transcription Once introduced into a cell, the complementary antisense molecule base-pairs with a naturally occumng nucleic acid sequence produced by the cell to form duplexes which block either transcription or translation
  • the designation "negative “ or “minus” can reter to the anusense strand, and the designation " positive “ or “plus * can reter to the sense strand of a reference DNA molecule
  • biologically active refers to a protein having structural, regulatory, or biochemical functions of a naturally occumng molecule
  • “lmmunologically active " or “immunogemc” refers to the capability of the natural, recombinant, or synthetic NSPRT.
  • Complementary describes the relationship between two single-stranded nucleic acid sequences that anneal by base-pairing. For example, 5'-AGT-3' pairs with its complement, 3'-TCA-5'
  • composition comprising a given polynucleotide sequence " and a “composition comprising a given amino acid sequence "” refer broadly to any composition contaimng the given polynucleotide or amino acid sequence.
  • the composition may comprise a dry formulation or an aqueous solution.
  • Compositions comprising polynucleotide sequences encoding NSPRT or fragments of NSPRT may be employed as hybridization probes.
  • the probes may be stored in freeze-d ⁇ ed form and may be associated with a stabilizing agent such as a carbohydrate.
  • the probe may be deployed in an aqueous solution contaimng salts (e.g., NaCl), detergents (e g , sodium dodecyl sulfate, SDS), and other components (e g , Denhardt's solution, dry milk, salmon sperm DNA. etc.).
  • aqueous solution contaimng salts e.g., NaCl
  • detergents e.g , sodium dodecyl sulfate, SDS
  • other components e g , Denhardt's solution, dry milk, salmon sperm DNA. etc.
  • Consensus sequence refers to a nucleic acid sequence which has been subjected to repeated DNA sequence analysis to resolve uncalled bases, extended using the XL-PCR kit (PE Biosystems, Foster City CA) in the 5' and/or the 3' direction, and resequenced, or which has been assembled from one or more overlapping cDNA, EST, or genomic DNA fragments using a computer program for fragment assembly, such as the GEL VIEW fragment assembly system (GCG, Madison WI) or Phrap (Umversity of Washington, Seattle WA).
  • Constant am o acid substitutions are those substitutions that are predicted to least interfere with the properties of the original protein, I e., the structure and especially the function of the protein is conserved and not significantly changed by such substitutions
  • the table below shows amino acids which may be substituted for an original amino acid in a protein and which are regarded as conservative ammo acid substitutions Original Residue Conservative Substitution Ala Gly, Ser
  • Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, tor example, as a beta sheet or alpha helical conformation, (b) the charge or hydrophobicity of the molecule at the site of the substitution, and/or (c) the bulk of the side chain.
  • a “deletion" refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides.
  • derivative '* refers to a chemically modified polynucleotide or polypeptide.
  • Chemical modifications of a polynucleotide sequence can include, for example, replacement of hydrogen by an alkyl. acyl, hydroxyl, or amino group.
  • a derivative polynucleotide encodes a polypeptide which retains at least one biological or immunological function of the natural molecule.
  • a derivative polypeptide is one modified by glycosylation, pegylation. or any similar process that retains at least one biological or immunological function of the polypeptide from which it was derived.
  • a “detectable label” refers to a reporter molecule or enzyme that is capable ot generating a measurable signal and is covalently or noncovalently joined to a polynucleotide or polypepude.
  • a “fragment” is a umque portion of NSPRT or the polynucleotide encoding NSPRT which is identical in sequence to but shorter in length than the parent sequence.
  • a fragment may comp ⁇ se up to the entire length of the defined sequence, minus one nucleotide/amino acid residue For example, a fragment may comprise from 5 to 1000 contiguous nucleotides or amino acid residues
  • a fragment used as a probe, p ⁇ mer. antigen, therapeutic molecule, or for other purposes, may be at least 5, 10, 15.
  • a polypeptide fragment may comp ⁇ se a certain length of contiguous amino acids selected from the first 250 or 500 amino acids (or first 25% or 50% of a polypeptide) as shown in a certain defined sequence Clearly these lengths are exemplary, and any length that is supported by the specification, including the Sequence Listing, tables, and figures, may be encompassed by the present embodiments
  • a fragment of SEQ ID NO 5-8 comp ⁇ ses a region ol umque polynucleotide sequence that specifically identifies SEQ ID NO 5-8. lor example, as distinct from any other sequence in the genome trom which the tragment was obtained
  • a fragment ot SEQ ID NO 5-8 is useful, tor example, in hybridization and amphtication technologies and in analogous methods that distinguish SEQ ID NO 5-8 from related polynucleotide sequences
  • the precise length ot a tragment of SEQ ID NO:5-8 and the region of SEQ ID NO.5-8 to which the fragment co ⁇ esponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment.
  • a "full- length" polynucleotide sequence encodes a "full-length” polypeptide sequence
  • “Homology” refers to sequence similarity or, interchangeably, sequence identity, between two or more polynucleotide sequences or two or more polypeptide sequences.
  • NCBI National Center for Biotechnology Information
  • BLAST Basic Local Alignment Search Tool
  • the BLAST software suite includes various sequence analysis programs including "blastn, " that is used to align a known polynucleotide sequence with other polynucleotide sequences from a variety of databases Also available is a tool called "BLAST 2 Sequences * ' that is used for direct pairwise comparison ot two nucleotide sequences. "BLAST 2 Sequences " can be accessed and used interactively at http://www.ncbi.nlm.mh.gov/gorf/bl2.html. The "BLAST 2 Sequences ' ' tool can be used for both blastn and blastp (discussed below).
  • BLAST programs are commonly used with gap and other parameters set to default settings. For example, to compare two nucleotide sequences, one may use blastn with the "BLAST 2 Sequences "" tool Version 2.0.12 (Ap ⁇ l-21-2000) set at default parameters. Such default parameters may be, for example. Matrix: BLOSUM62 Reward for match: 1 Penalty for mismatch- -2 Open Gap: 5 and Extension Gap: 2 penalties
  • Percent identity may be measured over the length of an entire defined sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides.
  • Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures, or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
  • NCBI BLAST software suite may be used. For example, for a pairwise comparison of two polypepude sequences, one may use the "BLAST 2 Sequences "' tool Version 2.0.12 (Apr-21-2000) with blastp set at default parameters. Such default parameters may be, for example
  • Gap x drop-off 50
  • Percent identity may be measured over the length of an enure defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, tor instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues.
  • Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
  • HACs Human artificial chromosomes
  • HACs are linear microchromosomes which may contain DNA sequences of about 6 kb to 10 Mb in size, and which contain all of the elements required for chromosome replication, segregation and maintenance.
  • humanized antibody refers to an antibody molecule in which the amino acid sequence in the non-antigen binding regions has been altered so that the antibody more closely resembles a human antibody, and still retains its original binding ability
  • Hybridization refers to the process by which a polynucleotide strand anneals with a complementary strand through base pairing under defined hybridization conditions Specific hybridization is an indication that two nucleic acid sequences share a high degree of complementarity Specific hybridization complexes form under permissive annealing conditions and remain hybridized after the "washing " step(s) The washing step(s) is particularly important in determimng the stringency of the hybridization process, with more stringent conditions allowing less non-specific binding, l e .
  • Permissive annealing conditions occur, for example, at 68°C in the presence of about 6 x SSC. about 1 % (w/v) SDS. and about 100 ⁇ g/ l sheared, denatured salmon sperm DNA.
  • High stringency conditions for hybridization between polynucleotides ot the present invention include wash conditions of 68 °C in the presence of about 0.2 x SSC and about 0.1 % SDS, for 1 hour. Alternatively, temperatures of about 65°C. 60°C, 55°C, or 42°C may be used. SSC concentration may be varied from about 0 1 to 2 x SSC, with SDS being present at about 0.1 % Typically, blocking reagents are used to block non-specific hybridization. Such blocking reagents include, for instance, sheared and denatured salmon sperm DNA at about 100-200 ⁇ g/ml.
  • Organic solvent such as formamide at a concentration of about 35-50% v/v
  • Organic solvent such as formamide at a concentration of about 35-50% v/v
  • Hybridization particularly under high stringency conditions, may be suggestive of evolutionary similarity between the nucleotides Such similarity is strongly indicative of a similar role for the nucleotides and their encoded polypeptides
  • hybridization complex refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary bases
  • a hybridization complex may be formed in solution (e g , C n t or R 0 t analysis) or formed between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e g , paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids have been fixed)
  • insertion " and “addition” refer to changes in an amino acid or nucleotide sequence resulting in the addition ot one or more amino acid residues or nucleotides. respectively
  • Methods for DNA sequencing are well known in the art and may be used to practice any of the embodiments of the invention.
  • the methods may employ such enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase (PE Biosystems, Foster City CA), thermostable T7 polymerase (Amersham Pharmacia Biotech, Piscataway NJ), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE amplification system (Life Technologies, Gaithersburg MD).
  • sequence preparation is automated with machines such as the MICROLAB 2200 liquid transfer system (Hamilton, Reno NV), PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal cycler (PE Biosystems). Sequencing is then ca ⁇ ied out using either the ABI 373 or 377 DNA sequencing system (PE Biosystems), the MEGABACE 1000 DNA sequencing system (Molecular Dynamics, Sunnyvale CA), or other systems known in the art. The resulting sequences are analyzed using a variety of algorithms which are well known in the art. (See, e.g., Ausubel, F.M. (1997) Short Protocols in Molecular Biology, John Wiley & Sons, New York NY, unit 7.7; Meyers, R.A.
  • nucleic acid sequences encoding NSPRT may be extended utilizing a partial nucleotide sequence and employing various PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements.
  • restriction-site PCR uses universal and nested primers to amplify unknown sequence from genomic DNA within a cloning vector.
  • inverse PCR uses primers that extend in divergent directions to amplify unknown sequence from a circularized template.
  • primers may be designed using commercially available software, such as OLIGO 4.06 Primer Analysis software (National Biosciences, Plymouth MN) or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the template at temperatures of about 68 °C to 72°C.
  • Capillary electrophoresis systems which are commercially available may be used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products.
  • capillary sequencing may employ flowable polymers for electrophoretic separation, four different nucleotide- specific, laser-stimulated fluorescent dyes, and a charge coupled device camera for detection of the emitted wavelengths.
  • Output/light intensity may be converted to electrical signal using appropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, PE Biosystems), and the entire process from loading of samples to computer analysis and electronic data display may be computer controlled.
  • Capillary electrophoresis is especially preferable for sequencing small DNA fragments which may be present in limited amounts in a particular sample.
  • DNA shuffling is a process by which a library of gene va ⁇ ants is produced using PCR-mediated recombination of gene fragments.
  • the library is then subjected to selection or screemng procedures that identify those gene va ⁇ ants with the desired properties
  • These prefe ⁇ ed va ⁇ ants may then be pooled and further subjected to recursive rounds of DNA shuffling and selection/screening.
  • genetic diversity is created through "artificial" breeding and rapid molecular evolution. For example, fragments of a single gene contaimng random point mutations may be recombined, screened, and then reshuffled until the desired properties are optimized. Alternatively, fragments of a given gene may be recombined with fragments of homologous genes in the same gene family, either from the same or different species, thereby maximizing the genetic diversity of multiple naturally occumng genes in a directed and controllable manner.
  • the peptide may be substantially purified by preparative high performance liquid chromatography (See. e g., Chiez, R M. and F.Z. Regmer (1990) Methods Enzymol 182.392-421.)
  • the composition of the synthetic peptides may be confirmed by amino acid analysis or by sequencing. (See, e g., Creighton, supra, pp 28-53 )
  • the nucleotide sequences encoding NSPRT or derivatives thereof may be inserted into an appropriate expression vector, I e .
  • Such signals may also be used to achieve more efficient translation of sequences encoding NSPRT.
  • Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence.
  • sequences encoding NSPRT and its initiation codon and upstream regulatory sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed.
  • exogenous translational control signals including an in-frame ATG initiation codon should be provided by the vector.
  • Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers appropriate for the particular host cell system used. (See, e.g., Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162.)
  • a variety of expression vector/host systems may be utilized to contain and express sequences encoding NSPRT. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors (e.g., baculovirus); plant cell systems transformed with viral expression vectors (e.g., cauliflower mosaic virus, CaMV. or tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems.
  • microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors
  • yeast transformed with yeast expression vectors insect cell systems infected with viral expression vectors (e.g., baculovirus)
  • plant cell systems transformed with viral expression vectors e.g., cauliflower mosaic virus, CaMV. or tobacco
  • nucleotide sequences may be used for delivery of nucleotide sequences to the targeted organ, tissue, or cell population.
  • Di Nicola, M. et al. (1998) Cancer Gen. Ther. 5(6):350-356: Yu. M. et al., (1993) Proc. Natl. Acad. Sci. USA 90(13):6340-6344: Buller. R.M. et al. (1985) Nature 317(6040):813-815; McGregor. D.P. et al. WO 01/07470 PCT/USOO/l 9837
  • clomng and expression vectors may be selected depending upon the use intended for polynucleotide sequences encoding NSPRT.
  • routine clomng, subclomng, and propagation of polynucleotide sequences encoding NSPRT can be achieved using a multifunctional E. coh vector such as PBLUESCRIPT (Stratagene, La Jolla CA) or PSPORT1 plasmid (Life Technologies).
  • PBLUESCRIPT Stratagene, La Jolla CA
  • PSPORT1 plasmid Life Technologies.
  • these vectors may be useful for in vitro transcription, dideoxy sequencing, single strand rescue with helper phage, and creation of nested deletions in the cloned sequence.
  • vectors which direct high level expression of NSPRT may be used.
  • vectors contaimng the strong, inducible T5 or T7 bacteriophage promoter may be used.
  • Yeast expression systems may be used for production of NSPRT.
  • a number of vectors contaimng constitutive or inducible promoters may be used in the yeast Saccharomyces cerevisiae or Pichia pasto ⁇ s.
  • such vectors direct either the secretion or lntracellular retention of expressed proteins and enable integration of foreign sequences into the host genome for stable propagation.
  • Plant systems may also be used for expression of NSPRT. Transcription of sequences encoding NSPRT may be driven viral promoters, e g , the 35S and 19S promoters of CaMV used alone or in combination with the omega leader sequence from TMV (Takamatsu, N (1987) EMBO J 6 307-311) Alternatively, plant promoters such as the small subumt of RUBISCO or heat shock promoters may be used (See, e g., Coruzzi, supra. Broghe. supra; and Winter, supra.) These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection (See, e.g.. The McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill. New York NY, pp 191-196 )
  • sequences encoding NSPRT may be hgated into an adenovirus transcription/translation complex consisting ot the late promoter and tripartite leader sequence Insertion in a non-essential El or E3 region ot the viral genome may be used to obtain infective virus which expresses NSPRT in host cells (See. e g . Logan.
  • HACs Human artificial chromosomes
  • plasmid HACs of about 6 kb to 10 Mb are constructed and delivered via conventional dehvery methods (hposomes, polycatiomc ammo polymers, or vesicles) for therapeutic purposes.
  • hposomes polycatiomc ammo polymers, or vesicles
  • hposomes polycatiomc ammo polymers, or vesicles
  • sequences encoding NSPRT can be transformed into cell lines using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for about 1 to 2 days in enriched media before being switched to selective media.
  • the purpose of the selectable marker is to confer resistance to a selective agent, and its presence allows growth and recovery of cells which successfully express the introduced sequences.
  • Resistant clones of stably transformed cells may be propagated using tissue culture techmques approp ⁇ ate to the cell type.
  • Any number of selection systems may be used to recover transformed cell lines. These include, but are not limited to, the he ⁇ es simplex virus thymidine kmase and ademne phospho ⁇ bosyltransferase genes, for use in tk ⁇ and apr cells, respectively. (See, e.g., Wigler, M. et al. (1977) Cell 11.223-232. Lowy, I. et al. (1980) Cell 22:817-823.) Also, antimetabo te, antibiotic, or herbicide resistance can be used as the basis for selection. For example, dhfr confers resistance to methotrexate.
  • ELISAs enzyme-linked lmmunosorbent assays
  • RIAs radioimmunoassays
  • FACS fluorescence activated cell sorting
  • NSPRT nucleic acid and ammo acid assays.
  • Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding NSPRT include ohgolabehng, mck translation, end-labeling, or PCR amphtication using a labeled nucleotide.
  • the sequences encoding NSPRT. or any fragments thereof may be cloned into a vector for the production of an mRNA probe.
  • the protein produced by a transformed cell may be secreted or retained intracellularly depending on the sequence and/or the vector used As will be understood by those of skill in the art.
  • expression vectors contaimng polynucleotides which encode NSPRT may be designed to contain signal sequences which direct secretion of NSPRT through a prokaryotic or eukaryotic cell membrane.
  • a host cell strain may be chosen for its ability to modulate expression of the inserted sequences or to process the expressed protein in the desired fashion.
  • modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation.
  • Post-translational processing which cleaves a "prepro” or “"pro " ' form of the protein may also be used to specify protein targeting, folding, and/or activity.
  • Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are available from the American Type Culture Collection (ATCC, Manassas VA) and may be chosen to ensure the correct modification and processing of the foreign protein.
  • ATCC American Type Culture Collection
  • natural, modified, or recombinant nucleic acid sequences encoding NSPRT may be ligated to a heterologous sequence resulting in translation of a fusion protein in any of the aforementioned host systems.
  • a chimeric NSPRT protein containing a heterologous moiety that can be recognized by a commercially available antibody may facilitate the screening of peptide libraries for inhibitors of NSPRT activity.
  • Heterologous protein and peptide moieties may also facilitate purification of fusion proteins using commercially available affinity matrices.
  • Such moieties include, but are not limited to, glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmoduUn binding peptide (CBP), 6-His, FLAG, c-myc, and hemagglutinin (HA).
  • GST, MBP, Trx, CBP, and 6-His enable purification of their cognate fusion proteins on immobiUzed glutathione, maltose, phenylarsine oxide, calmoduUn, and metal-chelate resins, respectively.
  • FLAG, c-myc, and hemagglutinin (HA) enable immunoaffinity purification of fusion proteins using commercially available monoclonal and polyclonal antibodies that specifically recognize these epitope tags.
  • a fusion protein may also be engineered to contain a proteolytic cleavage site located between the NSPRT encoding sequence and the heterologous protein sequence, so that NSPRT may be cleaved away from the heterologous moiety following purification. Methods for fusion protein expression and purification are discussed in Ausubel (1995, supra, ch. 10). A variety of commercially available kits may also be used to facilitate expression and purification of fusion proteins.
  • synthesis of radiolabeled NSPRT may be achieved in vitro using the TNT rabbit reticulocyte lysate or wheat germ extract system (Promega). These systems couple transcription and translation of protein-coding sequences operably associated with the T7, T3, or SP6 promoters. Translation takes place in the presence of a radiolabeled amino acid precursor, for example. 35 S-methionine.
  • NSPRT of the present invention or fragments thereof may be used to screen for compounds that specifically bind to NSPRT.
  • At least one and up to a plurality of test compounds may be screened for specific binding to NSPRT Examples of test compounds include antibodies, ohgonucleotides, proteins (e.g., receptors), or small molecules
  • the compound thus identified is closely related to the natural hgand of NSPRT, e.g., a hgand or fragment thereof, a natural substrate, a structural or functional mimetic, or a natural binding partner.
  • NSPRT natural hgand of NSPRT
  • the compound can be closely related to the natural receptor to which NSPRT binds, or to at least a fragment of the receptor, e.g., the hgand binding site. In either case, the compound can be rationally designed using known techmques.
  • screemng for these compounds involves producing approp ⁇ ate cells which express NSPRT, either as a secreted protein or on the cell membrane
  • Prefe ⁇ ed cells include cells from mammals, yeast, Drosophila, or E_ co .
  • Cells expressing NSPRT or cell membrane fractions which contain NSPRT are then contacted with a test compound and binding, stimulation, or inhibition of activity of either NSPRT or the compound is analyzed.
  • An assay may simply test binding of a test compound to the polypeptide, wherein binding is detected by a fluorophore, radioisotope, enzyme conjugate, or other detectable label.
  • the assay may comp ⁇ se the steps of combimng at least one test compound with NSPRT, either in solution or affixed to a solid support, and detecting the binding of NSPRT to the compound.
  • the assay may detect or measure binding of a test compound in the presence of a labeled competitor.
  • the assay may be ca ⁇ ied out using cell-free preparations, chemical libra ⁇ es, or natural product mixtures, and the test compound(s) may be free in solution or affixed to a solid support
  • NSPRT of the present invention or fragments thereof may be used to screen for compounds that modulate the activity of NSPRT Such compounds may include agonists, antagonists, or partial or inverse agomsts.
  • an assay is performed under conditions permissive for NSPRT activity, wherein NSPRT is combined with at least one test compound, and the activity ot NSPRT in the presence of a test compound is compared with the activity of NSPRT in the absence of the test compound.
  • a change in the activity of NSPRT in the presence of the test compound is indicative of a compound that modulates the activity of NSPRT
  • a test compound is combined with an in vitro or cell-free system comprising NSPRT under conditions suitable for NSPRT activity, and the assay is performed In either of these assays, a test compound which modulates the activity of NSPRT may do so indirectly and need not come in direct contact with the test compound At least one and up to a plurality of test compounds may be screened
  • polynucleotides encoding NSPRT or their mammalian homologs may be "knocked out" in an ammal model system using homologous recombination in embryonic stem (ES) cells Such techmques are well known in the art and are useful for the generation ot ammal
  • homologous recombination takes place using the Cre-loxP system to knockout a gene of interest in a tissue- or developmental stage-specific manner (Marth, J.D. (1996) Clin. Invest. 97:1999-2002: Wagner, K.U. et al. (1997) Nucleic Acids Res. 25:4323-4330).
  • Transformed ES cells are identified and microinjected into mouse cell blastocysts such as those from the C57BL/6 mouse strain. The blastocysts are surgically transferred to pseudopregnant dams, and the resulting chime ⁇ c progeny are genotyped and bred to produce heterozygous or homozygous strains. Transgenic animals thus generated may be tested with potential therapeutic or toxic agents.
  • Polynucleotides encoding NSPRT may also be manipulated in vitro in ES cells de ⁇ ved from human blastocysts.
  • Human ES cells have the potential to differentiate into at least eight separate cell lineages including endoderm, mesoderm, and ectodermal cell types. These cell hneages differentiate into, for example, neural cells, hematopoietic lineages, and cardiomyocytes (Thomson, J.A. et al. (1998) Science 282.1145-1147).
  • Polynucleotides encoding NSPRT can also be used to create "knockin” humamzed animals (pigs) or transgenic animals (mice or rats) to model human disease.
  • knockin technology a region of a polynucleotide encoding NSPRT is injected into ammal ES cells, and the injected sequence integrates into the ammal cell genome.
  • Transformed cells are injected into blastulae, and the blastulae are implanted as described above.
  • Transgemc progeny or inbred lines are studied and treated with potential pharmaceutical agents to obtain information on treatment of a human disease.
  • a mammal inbred to overexpress NSPRT e g., by secreting NSPRT in its milk, may also serve as a convement source of that protein (Janne, J. et al. (1998) Biotechnol. Annu. Rev. 4:55- 74) THERAPEUTICS
  • NSPRT or a fragment or derivative thereof may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of NSPRT.
  • disorders include, but are not limited to, a neurological disorder such as epilepsy, ischemic cerebrovascular disease, stroke, cerebral neoplasms, Alzheimer's disease, Pick's disease, Huntington s disease, dementia, Parkinson * s disease and other extrapyramidal disorders, amyottophic lateral sclerosis and other motor neuron disorders, progressive neural muscular atrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosis and other demyeUnating diseases, bacterial and viral meningitis, brain abscess, subdural empyema, epidural abscess, suppurative intracranial thrombophlebitis, myelitis and radicuUtis, viral central nervous system disease, prion diseases including kuru, Creutzfeldt-Jakob disease,
  • erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture ' s syndrome, gout, Graves * disease, Hashimoto ' s thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis.
  • a vector capable of expressing NSPRT or a fragment or derivative thereof may be admimstered to a subject to treat or prevent a disorder associated with decreased expression or activity of NSPRT including, but not Umited to, those described above
  • a pharmaceutical composition comprising a substantially purified NSPRT in conjunction with a suitable pharmaceutical carrier may be admimstered to a subject to treat or prevent a disorder associated with decreased expression or activity of NSPRT including, but not Umited to, those provided above
  • an agomst which modulates the activity of NSPRT may be admimstered to a subject to treat or prevent a disorder associated with decreased expression or activity of NSPRT including, but not limited to, those listed above
  • an antagonist of NSPRT may be admimstered to a subject to treat or prevent a disorder associated with increased expression or activity of NSPRT Examples of such disorders include, but are not limited to, those neurological, inflammatory, and cell proliferative disorders, including cancer, described above
  • an antibody which specifically binds NSPRT may be used directly as an antagonist or indirectly as a targeting or dehvery mechanism for bringing a pharmaceutical agent to cells or tissues which express NSPRT
  • a vector expressing the complement of the polynucleotide encoding NSPRT may be admimstered to a subject to treat or prevent a disorder associated with increased expression or activity of NSPRT including, but not limited to, those described above
  • any of the proteins, antagomsts, antibodies, ago sts, complementary sequences, or vectors of the invention may be admimstered in combination with other appropriate therapeutic agents Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skill in the art, according to conventional pharmaceutical principles The combination of therapeutic agents may act synergistically to effect the treatment or prevention of the various disorders described above Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent thus reducing the potential for adverse side effects
  • An antagomst of NSPRT may be produced using methods which are generally known in the art
  • purified NSPRT mav be used to produce antibodies or to screen libraries ot pharmaceutical agents to identify those which specifically bind NSPRT.
  • Antibodies to NSPRT may also be generated using methods that are well known in the art.
  • Such antibodies may include, but are not Umited to, polyclonal, monoclonal, chime ⁇ c, and single chain antibodies, Fab fragments, and fragments produced by a Fab expression Ubrary.
  • Neutralizing antibodies i.e., those which inhibit dimer formation
  • various hosts including goats, rabbits, rats, mice, humans, and others may be immunized by injection with NSPRT or with any fragment or ohgopeptide thereof which has immunogemc properties.
  • various adjuvants may be used to increase immunological response.
  • adjuvants include, but are not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluromc polyols, polyamons, peptides, oil emulsions, KLH, and dimtrophenol.
  • BCG Bacilli Calmette-Gue ⁇ n
  • Corvnebacte ⁇ um parvum are especially preferable.
  • the ohgopeptides, peptides, or fragments used to induce antibodies to NSPRT have an amino acid sequence consisting of at least about 5 amino acids, and generally will consist of at least about 10 amino acids. It is also preferable that these ohgopeptides, peptides, or fragments are identical to a portion of the am o acid sequence of the natural protein. Short stretches of NSPRT amino acids may be fused with those of another protein, such as KLH, and antibodies to the chime ⁇ c molecule may be produced.
  • Monoclonal antibodies to NSPRT may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not Umited to, the hyb ⁇ doma technique, the human B-cell hyb ⁇ doma technique, and the EBV-hyb ⁇ doma technique.
  • the hyb ⁇ doma technique the human B-cell hyb ⁇ doma technique
  • the EBV-hyb ⁇ doma technique See, e.g., Kohler, G. et al. (1975) Nature 256.495-497; Kozbor, D. et al. (1985) J. Immunol. Methods 81 31-42, Cote, R.J et al. (1983) Proc. Natl. Acad. Sci. USA 80.2026-2030; and Cole, S.P. et al. (1984) Mol. Cell Biol.
  • Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobuUn libraries or panels of highly specific binding reagents as disclosed in the Uterature. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. USA 86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299.)
  • Antibody fragments which contain specific binding sites for NSPRT may also be generated.
  • fragments include, but are not Umited to, F(ab " ) 2 fragments produced by pepsin digestion of the antibody molecule and Fab fragments generated by reducing the disulfide bridges of the F(ab * )2 fragments.
  • Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity. (See, e.g., Huse, W.D. et al. (1989) Science 246:1275-1281.)
  • Various immunoassays may be used for screening to identify antibodies having the desired specificity.
  • High-affinity antibody preparations with K ranging from about 10 9 to 10 12 L/mole are prefe ⁇ ed for use in immunoassays in which the NSPRT-antibody complex must withstand rigorous manipulations.
  • Low-affinity antibody preparations with K ranging from about 10 6 to 10 7 L/mole are prefe ⁇ ed for use in immunopurification and similar procedures which ultimately require dissociation of NSPRT, preferably in active form, from the antibody (Catty, D. (1988) Antibodies. Volume I: A Practical Approach, IRL Press, Washington DC: Liddell, J.E. and A. Cryer (1991) A Practical Guide to Monoclonal Antibodies. John Wiley & Sons, New York NY).
  • the polynucleotides encoding NSPRT may be used for therapeutic pu ⁇ oses.
  • modifications of gene expression can be achieved by designing complementary sequences or antisense molecules (DNA, RNA. PNA, or modified oligonucleotides) to the coding or regulatory regions of the gene encoding NSPRT.
  • complementary sequences or antisense molecules DNA, RNA. PNA, or modified oligonucleotides
  • antisense ohgonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding NSPRT. (See, e.g., Agrawal, S., ed.
  • Antisense sequences can be delivered intracellularly in the form of an expression plasmid which, upon transcription, produces a sequence complementary to at least a portion of the cellular sequence encoding the target protein.
  • Antisense sequences can also be introduced intracellularly through the use of viral vectors, such as retrovirus and adeno-associated virus vectors.
  • viral vectors such as retrovirus and adeno-associated virus vectors.
  • Other gene dehvery mechanisms include liposome-derived systems, artificial viral envelopes, and other systems known in the art.
  • polynucleotides encoding NSPRT may be used for somatic or germhne gene therapy.
  • Gene therapy may be performed to (i) co ⁇ eet a genetic deficiency (e.g., in the cases of severe combined immunodeficiency (SCID)-Xl disease characterized by X-linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672), severe combined immunodeficiency syndrome associated with an inherited adenosine deaminase (ADA) deficiency (Blaese, R.M. et al. (1995) Science 270:475-480; Bordignon, C. et al.
  • SCID severe combined immunodeficiency
  • ADA adenosine deaminase
  • NSPRT hepatitis B or C virus
  • fungal parasites such as Candida albicans and Paracoccidioides brasiliensis
  • protozoan parasites such as Plasmodium falciparum and Trypanosoma cruzi
  • diseases or disorders caused by deficiencies in NSPRT are treated by constructing mammaUan expression vectors encoding NSPRT and introducing these vectors by mechanical means into NSPRT-deficient cells.
  • Mechanical transfer technologies for use with cells in vivo or ex vitro include (i) direct DNA microinjection into individual cells, (ii) balUstic gold particle delivery, (iii) liposome-mediated transfection, (iv) receptor-mediated gene transfer, and (v) the use of DNA transposons (Morgan, R.A. and W.F. Anderson (1993) Annu. Rev. Biochem. 62:191- 217; Ivies, Z. (1997) Cell 91 :501-510; Boulay, J-L. and H.
  • Expression vectors that may be effective for the expression of NSPRT include, but are not limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX vectors (Invitrogen, Carlsbad CA), PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La Jolla CA), and PTET-OFF, PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA).
  • NSPRT may be expressed using (i) a constitutively active promoter, (e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or ⁇ -actin genes), (ii) an inducible promoter (e.g., the tetracycline-regulated promoter (Gossen, M. and H. Bujard (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551 : Gossen, M. et al. (1995) Science 268:1766-1769; Rossi, F.M.V. and H.M. Blau (1998) Cu ⁇ . Opin. Biotechnol.
  • a constitutively active promoter e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or ⁇ -actin
  • liposome transformation kits e.g., the PERFECT LIPID TRANSFECTION KIT, available from Invitrogen
  • PERFECT LIPID TRANSFECTION KIT available from Invitrogen
  • transformation is performed using the calcium phosphate method (Graham. F.L. and A.J. Eb (1973) Virology 52:456-467), or by electroporation (Neumann. E. et al. (1982) EMBO J. 1 :841-845).
  • the introduction of DNA to primary cells requires modification of these standardized mammalian transfection protocols.
  • diseases or disorders caused by genetic defects with respect to NSPRT expression are treated by constructing a refrovirus vector consisting of (i) the polynucleotide encoding NSPRT under the control of an independent promoter or the refrovirus long terminal repeat (LTR) promoter, (u) appropriate RNA packaging signals, and (in) a Rev-responsive element (RRE) along with additional refrovirus ⁇ s-acting RNA sequences and coding sequences required for efficient vector propagation.
  • Refrovirus vectors e.g., PFB and PFBNEO
  • PFB and PFBNEO are commercially available (Stratagene) and are based on pubhshed data (Riviere, I. et al. (1995) Proc. Natl.
  • the vector is propagated in an appropriate vector producing cell line (VPCL) that expresses an envelope gene with a tropism for receptors on the target cells or a promiscuous envelope protein such as VSVg (Armentano, D. et al (1987) J. Virol. 61 :1647-1650; Bender, M.A et al. (1987) J Virol. 61.1639-1646. Adam, M.A. and A.D. Miller (1988) J. Virol. 62.3802-3806. Dull, T. et al. (1998) J Virol 72.8463-8471 , Zufferey, R et al.
  • VSVg vector producing cell line
  • Propagation of refrovirus vectors, transduction of a population of cells (e.g., CD4 + T-cells), and the return of transduced cells to a patient are procedures well known to persons skilled in the art of gene therapy and have been well documented (Ranga, U. et al. (1997) J. Virol. 71 :7020-7029: Bauer, G. et al. (1997) Blood 89:2259-2267: Bonyhadi, M L (1997) J. Virol. 71 :4707-4716, Ranga. U et al (1998) Proc. Natl. Acad. Sci. USA 95:1201-1206: Su, L (1997) Blood 89:2283-2290)
  • an adenovirus-based gene therapy delivery system is used to deliver polynucleotides encoding NSPRT to cells which have one or more genetic abnormalities with respect to the expression of NSPRT.
  • adenovirus-based vectors are well known to those with ordinary skill in the art. Replication defective adenovirus vectors have proven to be versatile for importing genes encoding lmmunoregulatory proteins into intact islets in the pancreas (Csete, M.E. et al. (1995) Transplantation 27 263-268) Potentially useful adenoviral vectors are described in U.S.
  • a he ⁇ es-based. gene therapy delivery system is used to deliver polynucleotides encoding NSPRT to target cells which have one or more genetic abnormalities with respect to the expression of NSPRT.
  • HSV he ⁇ es simplex virus
  • Patent Number 5,804,413 teaches the use of recombinant HSV d92 which consists of a genome contaimng at least one exogenous gene to be transfe ⁇ ed to a cell under the control of the appropriate promoter for pu ⁇ oses including human gene therapy. Also taught by this patent are the construction and use of recombinant HSV strains deleted for ICP4, ICP27 and ICP22. For HSV vectors, see also Goms, W.F. et al. (1999) J Virol 73.519-532 and Xu. H. et al. (1994) Dev Biol. 163.152-161, hereby inco ⁇ orated by reference.
  • a particular embodiment of the present invention involves screening a combinatorial library of oligonucleotides (such as deoxyribonucleotides, ribonucleotides. peptide nucleic acids, and modified ohgonucleotides) for antisense activity against a specific polynucleotide sequence (Bnuce, T.W. et al. (1997) U.S. Patent No. 5,686,242, Bruice, T.W. et al. (2000) U.S. Patent No. 6,022,691).
  • oligonucleotides such as deoxyribonucleotides, ribonucleotides. peptide nucleic acids, and modified ohgonucleotides
  • nucleotide sequences which encode NSPRT may be used in any molecular biology techmques that have yet to be developed, provided the new techmques rely on properties of nucleotide sequences that are cu ⁇ ently known, including, but not limited to, such properties as the triplet genetic code and specific base pair interactions
  • plasmid DNA was amplified trom host cell lysates using direct link PCR in a WO 01/07470 PCT/USOO/l 9837
  • the first column of Table 5 shows the tools, programs, and algorithms used, the second column provides brief descriptions thereof, the third column presents appropriate references, all of which are inco ⁇ orated by reference herein in their entirety, and the fourth column presents, where applicable, the scores, probability values, and other parameters used to evaluate the strength of a match between two sequences (the higher the score, the greater the homology between two sequences)
  • Sequences were analyzed using MACDNASIS PRO software (Hitachi Software Engineering, South San Francisco CA) and LASERGENE software (DNASTAR). Polynucleotide and polypeptide sequence alignments were generated using the default parameters specified by the clustal algorithm as inco ⁇ orated into the MEGALIGN multisequence alignment program (DNASTAR), which also calculates the percent identity between aligned sequences
  • sequences were then queried against a selection of public databases such as the GenBank primate, rodent, mammahan, vertebrate, and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM, and PFAM to acquire annotation using programs based on BLAST, FASTA, and BLIMPS
  • GenBank primate rodent
  • mammahan mammahan
  • vertebrate vertebrate
  • eukaryote databases BLOCKS, PRINTS, DOMO, PRODOM, and PFAM
  • sequences were assembled into full length polynucleotide sequences using programs based on Phred, Phrap, and Consed, and were screened for open reading frames using programs based on GeneMark.
  • BLAST, and FASTA The full length polynucleotide sequences were translated to derive the co ⁇ esponding full length amino acid sequences, and these full length sequences were subsequently analyzed by querying against databases such as the GenBank databases (described above), SwissProt, BLOCKS, PRINTS, DOMO, PRODOM. Prosite, and Hidden Markov Model (HMM)-based protein family databases such as PFAM HMM is a probabilistic approach which analyzes consensus primary structures of gene families (See. e g , Eddy, S R (1996) Cu ⁇ Opin Struct Biol 6 361-365 )
  • Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled nucleotide sequence to a membrane on which RNAs from a particular cell type or tissue have been bound (See, e g , Sambrook. supra, ch 7 Ausubel 1995, supra, ch 4 and 16 )
  • Analogous computer techmques applying BLAST were used to search for identical or related molecules in cDNA databases such as GenBank or LIFESEQ (Incyte Genormcs) This analvsis is much faster than multiple membrane-based hybridizations
  • the sensitivity of the computer search can be modified to determine whether any particular match is categorized as exact or similar
  • the basis of the search is the product score, which is defined as
  • the product score represents a balance between fractional overlap and quality in a BLAST alignment For example, a product score of 100 is produced only for 100% identity over the entire length of the shorter of the two sequences being compared A product score of 70 is produced either by 100% identity and 70% overlap at one end, or by 88% identity and 100% overlap at the other A product score of 50 is produced either by 100% identity and 50% overlap at one end. or 79% identity and 100% overlap
  • the results of northern analyses are reported as a percentage distribution of libraries in which the transcript encoding NSPRT occu ⁇ ed Analysis involved the categorization of cDNA libraries by organ/tissue and disease
  • organ/tissue categories included cardiovascular, dermatologic, developmental endocrine, gastrointestinal, hematopoietic/immune, musculoskeletal. nervous reproductive, and urologic
  • disease/condition categories included cancer, inflammation, trauma, cell proliferation, neurological, and pooled
  • Percentage values of tissue-specific and disease- or condition-specific expression are reported in Table 3 V.
  • the concentration of DNA in each well was determined by dispensing 100 ⁇ l PICOGREEN quantitation reagent (0 25% (v/v) PICOGREEN, Molecular Probes, Eugene OR) dissolved in IX TE and 0 5 ⁇ l of undiluted PCR product into each well of an opaque fluo ⁇ meter plate (Corning Costar, Acton MA), allowing the DNA to bind to the reagent
  • the plate was scanned in a Fluoroskan II (Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample and to quantify the concentration of DNA
  • a 5 ⁇ l to 10 ⁇ l ahquot of the reaction mixture was analyzed by electrophoresis on a 1 % agarose mini-gel to determine which reactions were successful in extending the sequence
  • the extended nucleotides were desalted and concentrated, ttansfe ⁇ ed to 384-well plates, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison WI
  • Step 7 storage at 4°C DNA was quanufied by PICOGREEN reagent (Molecular Probes) as described above Samples with low DNA recoveries were reamplified using the same conditions as described above Samples were diluted with 20% dimethysulfoxide (1 2, v/v), and sequenced using DYENAMIC energy transfer sequencing primers and the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (PE Biosystems) In like manner, the polynucleotide sequences of SEQ ID NO 5-8 are used to obtain 5' regulatory sequences using the procedure above along with ohgonucleotides designed tor such extension, and an appropriate genomic library VI. Labeling and Use of Individual Hybridization Probes
  • the labeled oligonucleotides are substantially purified using a SEPHADEX G-25 superfine size exclusion dexttan bead column (Amersham Pharmacia Biotech). An aliquot contaimng 10 7 counts per minute of the labeled probe is used in a typical membrane-based hybridization analysis of human genomic DNA digested with one of the following endonucleases: Ase I, Bgl II, Eco RI. Pst I. Xba I, or Pvu II (DuPont NEN)
  • a typical a ⁇ ay may be produced using available methods and machines well known to those of ordinary skill in the art and may contain any appropriate number of elements (See, e g , Schena, M. et al. (1995) Science 270467-470. Shalon, D et al ( 1996) Genome Res 6 639-645. Marshall, A. and J. Hodgson (1998) Nat Biotechnol 16.27-31 )
  • laser desorbtion and mass specttometty may be used for detection of hybridization.
  • the degree of complementarity and the relative abundance of each polynucleotide which hybridizes to an element on the microa ⁇ ay may be assessed.
  • microa ⁇ ay preparation and usage is described in detail below.
  • the reverse transc ⁇ ption reaction is performed in a 25 ml volume contaimng 200 ng poly(A) + RNA with GEMB RIGHT kits (Incyte).
  • Specific control poly(A) + RNAs are synthesized by in vitro transcription from non-coding yeast genomic DNA. After incubation at 37 °C for 2 fir, each reaction sample (one with Cy3 and another with Cy5 labeling) is treated with 2.5 ml of 0.5M sodium hydroxide and incubated for 20 minutes at 85 °C to the stop the reaction and degrade the RNA.
  • Sequences of the present invenuon are used to generate a ⁇ ay elements
  • Each a ⁇ ay element is amplified from bacte ⁇ al cells contaimng vectors with cloned cDNA inserts
  • PCR amplification uses primers complementary to the vector sequences flanking the cDNA insert
  • a ⁇ ay elements are amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a final quantity greater than 5 ⁇ g. Amplified array elements are then purified using SEPHACRYL-400 (Amersham Pharmacia Biotech)
  • the chamber contaimng the a ⁇ ays is incubated for about 6.5 hours at 60°C
  • the a ⁇ ays are washed for 10 mm at 45 °C in a first wash buffer (IX SSC, 0.17c SDS), three times tor 10 minutes each at 45 °C in a second wash buffer (0 IX SSC), and dried. Detection
  • Reporter-labeled hybridization complexes are detected with a microscope equipped with an Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of generating specttal hnes at 488 nm for excitation ot Cy3 and at 632 nm for excitation ot Cy5
  • the excitation laser light is focused on the a ⁇ ay using a 20X microscope objective (Nikon. Inc., Melville NY)
  • the slide contaimng the a ⁇ ay is placed on a computer-controlled X-Y stage on the microscope and raster- scanned past the objective
  • the 1.8 cm x 1.8 cm array used in the present example is scanned with a resolution of 20 micrometers
  • a mixed gas multiline laser excites the two tluorophores sequentially Emitted light is split, based on wavelength, into two photomul ⁇ plier tube detectors (PMT R1477, Hamamatsu Photomcs Systems. Bndgewater NJ) co ⁇ esponding to the two tluorophores
  • Approp ⁇ ate filters positioned between the a ⁇ ay and the photomultipher tubes are used to filter the signals
  • the emission maxima ot the tluorophores used are 565 nm for Cy3 and 650 nm tor Cy5
  • Each a ⁇ ay is typically scanned twice, one scan per fluorophore using the appropriate filters at the laser source, although the apparatus is capable of recording the spectra trom both tluorophores simultaneously
  • the sensitivity ot the scans is typically calibrated using the signal intensity generated by a cDNA control species added to the sample mixture at a known concentration
  • a specific location on the a ⁇ ay contains a complementary DNA
  • the output of the photomultiplier tube is digitized using a 12-bit RTI-835H analog-to-digital (A/D) conversion board (Analog Devices. Inc.. Norwood MA) installed in an IBM-compatible PC computer
  • A/D analog-to-digital
  • the digitized data are displayed as an image where the signal intensity is mapped using a linear 20-color transformation to a pseudocolor scale ranging from blue (low signal) to red (high signal)
  • the data is also analyzed quantitatively Where two different fluorophores are excited and measured simultaneously, the data are first co ⁇ ected for optical crosstalk (due to overlapping emission spectra) between the fluorophores using each fluorophore * s emission spectrum
  • a grid is supe ⁇ mposed over the fluorescence signal image such that the signal from each spot is centered in each element of the g ⁇ d
  • the fluorescence signal within each element is then integrated to obtain a nume ⁇ cal value co ⁇ esponding to the average intensity of the signal
  • the software used for signal analysis is the GEMTOOLS gene expression analysis program (Incyte)
  • Sequences complementary to the NSPRT-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occumng NSPRT
  • ohgonucleotides comprising from about 15 to 30 base pairs
  • Appropriate ohgonucleotides are designed using OLIGO 4 06 software (National Biosciences) and the coding sequence of NSPRT
  • a complementary ohgonucleotide is designed from the most umque 5 " sequence and used to prevent promoter binding to the coding sequence
  • a complementary oligonucleotide is designed to prevent ⁇ bosomal binding to the NSPRT-encoding transcript
  • NSPRT expression and purification of NSPRT is achieved using bacterial or virus-based expression systems
  • cDNA is subcloned into an appropriate vector contaimng an antibiotic resistance gene and an inducible promoter that directs high levels of cDNA transcription
  • promoters include, but are not limited to. the trp-lac (tac) hybrid promoter and the T5 or T7 bacteriophage promoter in conjunction with the lac operator regulatory element Recombinant vectors are transformed into suitable bacterial hosts, e g .
  • NSPRT isopropyl beta-D- thiogalactopyranoside
  • IPTG isopropyl beta-D- thiogalactopyranoside
  • NSPRT is synthesized as a fusion protein with, e g , glutathione S- transferase (GST) or a peptide epitope tag.
  • GST glutathione S- transferase
  • a peptide epitope tag such as FLAG or 6-His, permitting rapid, single-step, affimty-based purification of recombinant fusion protein from crude cell lysates GST, a 26-k ⁇ lodalton enzyme from Schistosoma lapomcum.
  • Green Fluorescent Protein (GFP, Clontech), CD64, or a CD64-GFP fusion protein Row cytometry (FCM).
  • FCM Green Fluorescent Protein
  • an automated, laser optics- based technique is used to identity ttansfected cells expressing GFP or CD64-GFP and to evaluate the apoptotic state of the cells and other cellular properties
  • FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with cell death
  • These events include changes in nuclear DNA content as measured by staining of DNA with propidium iodide, changes in cell size and granularity as measured by forward light scatter and 90 degree side light scatter, down- regulation of DNA synthesis as measured by decrease in bromodeoxyu ⁇ dine uptake, alterations in expression of cell surface and intracellular proteins as measured by reactivity with specific antibodies, and alterations in plasma membrane composition as measured by the binding of fluorescem-conjugated Annex V protein to the cell surface
  • NSPRT Flow Cytometry, Oxford, New York NY
  • the influence of NSPRT on gene expression can be assessed using highly purified populations of cells ttansfected with sequences encoding NSPRT and either CD64 or CD64-GFP CD64 and CD64-GFP are expressed on the surface of ttansfected cells and bind to conserved regions of human immunoglobulin G (IgG)
  • Transfected cells are efficiently separated from nontransfected cells using magnetic beads coated with either human IgG or antibody against CD64 (DYNAL, Lake Success NY)
  • mRNA can be purified from the cells using methods well known by those of skill in the art
  • Expression of mRNA encoding NSPRT and other genes of interest can be analyzed by northern analysis or microa ⁇ ay techmques XII. Production of NSPRT Specific Antibodies
  • NSPRT ammo acid sequence is analyzed using LASERGENE software (DNASTAR) to determine regions of high lmmunogenicity, and a co ⁇ esponding ohgopeptide is synthesized and used to raise antibodies by means known to those of skill in the art Methods for selection of appropriate epitopes such as those near the C-terminus or in hydrophihc regions are well described in the art (See, e g , Ausubel, 1995, supra ch 11 )
  • ohgopeptides of about 15 residues in length are synthesized using an ABI 431 A peptide synthesizer (PE Biosystems) using FMOC chemistry and coupled to KLH (Sigma- Aldrich St Louis MO) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increase immunogemcity.
  • PES N-maleimidobenzoyl-N-hydroxysuccinimide ester
  • Rabbits are immumzed with the ohgopeptide- KLH complex in complete Freunds adjuvant. Resulting antisera are tested for antipeptide and anti-NSPRT activity by, for example, binding the peptide or NSPRT to a substtate. blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting with radio-iodinated goat anti-rabbit IgG.
  • XIII Purification of Naturally Occurring NSPRT Using Specific Antibodies
  • Naturally occurring or recombinant NSPRT is substantially purified by immunoaffimty chromatography using antibodies specific for NSPRT.
  • An immunoaffimty column is constructed by covalently couphng anti-NSPRT antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech) After the coupling, the resin is blocked and washed according to the manufacturer " s instructions.
  • NSPRT Media contaimng NSPRT are passed over the lmmunoatfinity column, and the column is washed under conditions that allow the preferential absorbance of NSPRT (e.g., high lomc strength buffers in the presence of detergent)
  • the column is eluted under conditions that disrupt antibody/NSPRT binding (e.g., a buffer of pH 2 to pH 3, or a high concentration of a chaotrope, such as urea or thiocyanate ion), and NSPRT is collected
  • NSPRT Molecules interacting with NSPRT are analyzed using the yeast two-hyb ⁇ d system as desc ⁇ bed in Fields, S. and O. Song (1989, Nature 340:245-246), or using commercially available kits based on the two-hyb ⁇ d system, such as the MATCHMAKER system (Clontech). NSPRT may also be used in the PATHCALLING process (CuraGen Co ⁇ .. New Haven CT) which employs the yeast two-hybrid system in a high-throughput manner to determine all interactions between the proteins encoded by two large libraries of genes (Nandabalan. K. et al (2000) U S. Patent No. 6,057.101)
  • ABI/PARACEI FDF A Tast Data Finder usul in comparing and PE Biosystems, Foster City, CA, Mismatch ⁇ 50% annotating ammo acid or nucleic acid sequences Paraccl Inc , Pasadena CA
  • ABI AuloAssembler A program that assembles nucleic acid sequences PE Biosystems, Foster City CA

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Abstract

The invention provides human nervous system-associated proteins (NSPRT) and polynucleotides which identify and encode NSPRT. The invention also provides expression vectors, host cells, antibodies, agonists, and antagonists. The invention also provides methods for diagnosing, treating, or preventing disorders associated with expression of NSPRT.

Description

HUMAN NERVOUS SYSTEM-ASSOCIATED PROTEINS
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of human nervous system- associated proteins and to the use of these sequences in the diagnosis, treatment, and prevenuon of neurological, inflammatory, and cell proliferauve disorders, including cancer.
BACKGROUND OF THE INVENTION
The human nervous system, which regulates all bodily funcUons. is composed of the central nervous system (CNS), consisung of the brain and spinal cord, and the peripheral nervous system
(PNS), consisting ot afferent neural pathways tor conducung nerve impulses from sensory organs to the CNS, and efferent neural pathways tor conducung motor impulses from the CNS to effector organs. The PNS can be further divided into the somauc nervous system, which regulates voluntary motor acϋvity such as for skeletal muscle, and the autonomic nervous system, which regulates involuntary motor acuvity for internal organs such as the heart, lungs, and viscera There are two subdivisions of the autonomic nervous system, the sympatheuc nervous system and the parasympatheuc nervous system.
A nerve cell (neuron) contains tour regions, the cell body, axon, dendπtes, and axon terminal. The cell body contains the nucleus and other organelles. The dendrites are processes which extend outward from the cell body and receive signals from sense organs or from the axons ot other neurons. These signals are converted to electrical impulses and transmitted to the cell body The axon, whose size can range from one millimeter to more than one meter, is a single process that conducts the nerve impulse away from the cell body. Cytoskeletal fibers, including microtubules and neurofilaments, run the length of the axon and funcuon in transporting proteins, membrane vesicles, and other macromolecules from the cell body along the axon to the axon terminal. Some axons are surrounded by a mye n sheath made up of membranes from either an oligodendrocyte cell (CNS) or a Schwann cell (PNS) Mye nated axons conduct electrical impulses faster than unmyehnated ones of the same diameter The axon terminal is at the tip of the axon away from the cell body (See Lodish. H et al (1986) Molecular Cell Biology Scientific American Books New York NY. pp 715-719 ) Contact from one neuron to another occurs at a specialized site called the synapse At this site. the axon terminal from one neuron (the presynaptic cell) sends a signal to another neuron (the postsynaptic cell) Synapses may be connected either electrically or chemically An electrical synapse consists of gap junctions connecting the two neurons, allowing electrical impulses to pass directly from the presynaptic to the postsynaptic cell In a chemical synapse, the axon terminal of the presynaptic cell WO 01/07470 PCT/USOO/l 9837
contains membrane vesicles containing a particular neurotransmitter molecule A change in electrical potential at the nerve terminal resulting from the electrical impulse triggers the release of the neurotransmitter from the synaptic vesicle by exocytosis The neurotransmitter rapidly diffuses across the synaptic cleft separating the presynaptic nerve cell from the postsynaptic cell The neurotransmitter then binds receptors and opens transmitter-gated ion channels located in the plasma membrane of the postsynaptic cell, provoking a change in the cell's electrical potential This change in membrane potential of the postsynaptic cell may serve either to excite or inhibit further transmission of the nerve impulse
Neurotransmitters comprise a diverse group of more than 30 small molecules which include acetylchohne, monoamines such as serotonin, dopamine, epinephrine, norepinephrme, and histamme, and amino acids such as gamma-aminobutyπc acid (GABA), glutamate, and aspartate, and neuropeptides such as endorphins and enkephahns (McCance, K L and Huether, S E (1994) PATHOPHYSIOLOGY. The Biologic Basis for Disease in Adults and Children, 2nd edition, Mosby, St Louis, MO, pp 403-404) Many of these molecules have more than one function and the effects may be excitatory, e g to depolarize the postsynaptic cell plasma membrane and stimulate nerve impulse transmission, or inhibitory, e g to hyperpolaπze the plasma membrane and inhibit nerve impulse transmission
Neurotransmitters and their receptors are targets of pharmacological agents aimed at controlling neurological function For example GABA is the major inhibitory neurotransmitter m the CNS, and GABA receptors are the principal target of sedatives such as benzodiazepines and barbiturates which act by enhancing GABA-mediated effects (Katzung, B G (1995) Basic and Clinical Pharmacology. 6th edition, Appleton & Lange, Norwalk, CT, pp 338-339) Aberrant activity of neurotransmitters and their receptors is involved in various neurological conditions, including Alzheimer s disease, myasthenia gravis, stroke, epilepsy, and Parkinson s disease (See Planells-Cases, R et al (1993) Proc Natl Acad Sci USA 90 5057-5061 )
Each of over a trillion neurons in adult humans connects with over a thousand target cells (Tessier-Lavigne, M et al (1996) Science 274 1123-1133) These neuronal connections form during embryonic development Each differentiating neuron sends out an axon tipped at the leading edge by a growth cone Aided by molecular guidance cues, the growth cone migrates through the embryonic environment to its synaptic target
Axon growth is guided in part by contact-mediated mechanisms involving cell surface and extracellular matrix (ECM) molecules Many ECM molecules, including fibronectin, vitronectin members of the laminin tenascin, collagen, and thrombospondin families, and a variety ot proteoglycans, can act either as promoters or inhibitors ot neuπte outgrowth and extension (Tessier- Lavigne et al , supra) Receptors for ECM molecules include integnns immunoglobulin superfamily members, and proteoglycans. ECM molecules and their receptors have also been implicated in the adhesion, maintenance, and differentiation of neurons (Reichardt, L.F et al. (1991) Ann. Rev Neurosci. 14.531-571).
The adrenal medulla is the central portion of the adrenal gland and is functionally related to the sympathetic nervous system. The neurotransmitters epinephrine and norepinephrme are secreted by the adrenal medullae into the blood stream which causes a systemic response. A cDNA encoding a 50 kD protein has been isolated from the adrenal medulla. The possible function of this protein has not been determined (NCBI Entrez Protein query, g483843 on 20 July 1999).
Another nervous system-associated protein is 4F5 The gene encoding the 4F5 protein has been identified as a candidate modifying gene for spinal muscular atrophy (SMA). a recessive disorder SMA is characterized by the loss of lower motor neurons in the spinal cord. The age of onset and severity of the disease allows it to be classified into three types. All three types of SMA have been mapped to chromosome 5ql3. The genetic basis for the phenotypic variability of SMA is unclear (Scharf, J.M. et al. (1998) Nat. Genet. 20:83-86). To understand the biology of specific neuron types, cell-type specific molecules are being identified. One group selected cDNA clones by comparing libraries of normal mouse cerebellar cDNA and cerebellar cDNA from Purkinje cell degeneration (pcd) mice. One clone identified to correspond with mRNA present in Purkinje neurons encodes a protein of 99 amino acids. PCD5 PCD5 's expression is restricted to the cerebellum and the eye. The gene encoding PCD5 was localized to mouse chromosome 8 (Nordquist, D.T. et al (1988) J. Neurosci. 8(12).4780-4789).
The discovery of new human nervous system-associated proteins and the polynucleotides encoding them satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention, and treatment ot neurological, inflammatory, and cell pro ferative disorders, including cancer
SUMMARY OF THE INVENTION The invention features purified polypeptides, human nervous system-associated proteins, referred to collectively as "NSPRT" and individually as "NSPRT- 1." "NSPRT-2." "NSPRT-3,"' and "NSPRT-4 "" In one aspect, the invention provides an isolated polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected trom the group consisting of SEQ ID NO 1 -4. b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO 1 -4. c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO 1 -4. and d) an immunogemc fragment ot an amino acid sequence selected from the group consisting WO 01/07470 PCT/USOO/l 9837
of SEQ ID NO 1-4 In one alternative, the invention provides an isolated polypepude compπsmg the ammo acid sequence of SEQ ID NO 1 -4
The invention further provides an isolated polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO 1 -4, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an ammo acid sequence selected from the group consisting of SEQ ID NO 1-4, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO 1-4, and d) an immunogemc fragment ot an amino acid sequence selected from the group consisting of SEQ ID NO 1-4 In one alternative, the polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NO 1-4 In another alternative, the polynucleotide is selected from the group consisting of SEQ ID NO 5-8
Additionally, the invention provides a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding a polypeptide comprising an ammo acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO 1-4, b) a naturally occurring ammo acid sequence having at least 90% sequence identity to an am o acid sequence selected from the group consisting of SEQ ID NO 1-4, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO 1-4, and d) an immunogemc fragment of an amino acid sequence selected from the group consisting of SEQ ID NO 1-4 In one alternative, the invention provides a cell transformed with the recombinant polynucleotide In another alternative, the invention provides a transgenic orgamsm comprising the recombinant polynucleotide
The invention also provides a method for producing a polypeptide comprising an amino acid sequence selected from the group consisting ot a) an amino acid sequence selected from the group consisting of SEQ ID NO 1-4, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO 1-4, c) a biologically active fragment of an am o acid sequence selected from the group consisting of SEQ ID NO 1-4, and d) an immunogemc fragment of an amino acid sequence selected from the group consisting of SEQ ID NO 1-4 The method comprises a) cultuπng a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide comprising a promoter sequence operably linked to a polvnucleotide encoding the polypepude and b) recovering the polypeptide so expressed
Additionally, the invention provides an isolated antibodv which specifically binds to a polypeptide comprising an amino acid sequence selected from the group consisting ot a) an amino acid sequence selected from the group consisting of SEQ ID NO 1 -4 b) a naturally occurring amino acid sequence having at least 90% sequence identity to an am o acid sequence selected from the group consisting ot SEQ ID NO 1-4, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO 1-4, and d) an immunogemc fragment of an amino acid sequence selected from the group consisting of SEQ ID NO 1-4 The invention further provides an isolated polynucleotide comprising a polynucleotide sequence selected from the group consisting of a) a polynucleotide sequence selected from the group consisting of SEQ ID NO 5-8, b) a naturally occurring polynucleotide sequence having at least 70% sequence identity to a polynucleotide sequence selected trom the group consisting of SEQ ID NO 5-8, c) a polynucleotide sequence complementary to a), d) a polynucleotide sequence complementary to b), and e) an RNA eqmvalent ot a)-d) In one alternative, the polynucleotide comprises at least 60 contiguous nucleotides
Additionally, the invention provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide comprising a polynucleotide sequence selected from the group consisting of a) a polynucleotide sequence selected from the group consisting ot SEQ ID NO 5-8, b) a naturally occurring polynucleotide sequence having at least 70% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NO 5-8. c) a polynucleotide sequence complementary to a), d) a polynucleotide sequence complementary to b), and e) an RNA equivalent of a)-d) The method comprises a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex, and optionally, if present, the amount thereof In one alternative, the probe comprises at least 60 contiguous nucleotides The invention further provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide comprising a polynucleotide sequence selected from the group consisting of a) a polynucleotide sequence selected from the group consisting of SEQ ID NO 5-8, b) a naturally occurring polynucleotide sequence having at least 70% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NO 5-8. c) a polynucleotide sequence complementary to a), d) a polynucleotide sequence complementary to b), and e) an RNA equivalent of a)-d) The method comprises a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereol, and. optionally, if present, the amount thereof The invention further provides a pharmaceutical composition compπsing an effective amount of a polypeptide comprising an amino acid sequence selected from the group consisting of a) an am o acid sequence selected from the group consisting of SEQ ID NO 1-4, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO 1-4, c) a biologically active fragment ot an amino acid sequence selected from the group consisting of SEQ ID NO' 1-4, and d) an immunogemc fragment of an amino acid sequence selected from the group consisting of SEQ ID NO.1-4. and a pharmaceutically acceptable excφient. In one embodiment, the pharmaceutical composition comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1 -4 The invention additionally provides a method of treating a disease or condition associated with decreased expression of functional NSPRT, comprising administering to a patient in need of such treatment the pharmaceutical composition
The invention also provides a method for screemng a compound for effectiveness as an agomst of a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO.1-4, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO.1-4, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-4, and d) an immunogemc fragment of an ammo acid sequence selected from the group consisting of SEQ ID NO.1-4. The method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting agomst activity in the sample. In one alternative, the invention provides a pharmaceutical composition compπsing an agomst compound identified by the method and a pharmaceutically acceptable excipient In another alternative, the invention provides a method of treating a disease or condition associated with decreased expression of functional NSPRT. compπsing administering to a patient in need of such treatment the pharmaceuucal composition Additionally, the invention provides a method for screemng a compound for effectiveness as an antagonist of a polypepude comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO 1-4, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO 1-4. c) a biologically active tragment of an amino acid sequence selected from the group consisting of SEQ ID NO 1-4, and d) an immunogemc fragment of an amino acid sequence selected from the group consisting of SEQ ID NO 1 -4 The method compπses a) exposing a sample compπsing the polypeptide to a compound, and b) detecting antagonist activity in the sample. In one alternative, the invenuon provides a pharmaceuucal composition comprising an antagomst compound identified by the method and a pharmaceutically acceptable excipient In another alternative, the invenuon provides a method of treating a disease or condition associated with overexpression of functional NSPRT, comprising administering to a patient in need of such treatment the pharmaceuucal composition.
The invention further provides a method of screemng for a compound that specifically binds to a polypeptide comprising an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO.1 -4, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO.1-4, c) a biologically acUve fragment of an amino acid sequence selected from the group consisting of SEQ ID NO.1 -4, and d) an immunogemc fragment of an amino acid sequence selected from the group consisting of SEQ ID NO.1-4. The method compπses a) combimng the polypeptide with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide to the test compound, thereby identifying a compound that specifically binds to the polypeptide.
The invenuon further provides a method of screemng for a compound that modulates the activity of a polypepude compπsing an amino acid sequence selected from the group consisting of a) an amino acid sequence selected from the group consisting of SEQ ID NO.1 -4, b) a naturally occurπng amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-4, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO.1-4, and d) an immunogemc fragment of an amino acid sequence selected from the group consisting of SEQ ID NO.1-4. The method compπses a) combimng the polypeptide with at least one test compound under conditions permissive for the activity of the polypeptide, b) assessing the activity of the polypeptide in the presence of the test compound, and c) comparing the activity of the polypepude in the presence of the test compound with the activity of the polypeptide in the absence of the test compound, wherein a change m the activity of the polypeptide in the presence ot the test compound is indicative of a compound that modulates the activity of the polypeptide.
The invenuon further provides a method for screemng a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a sequence selected from the group consisting of SEQ ID NO 5-8, the method comprising a) exposing a sample compπsing the target polynucleotide to a compound, and b) detecung altered expression of the target polynucleotide
BRIEF DESCRIPTION OF THE TABLES
Table 1 shows polypeptide and nucleotide sequence identification numbers (SEQ ID NOs), clone identification numbers (clone IDs). cDNA libraries, and cDNA fragments used to assemble full- length sequences encoding NSPRT Table 2 shows features of each polypeptide sequence, including potential moufs, homologous sequences, and methods, algorithms, and searchable databases used tor analysis of NSPRT
Table 3 shows selected fragments of each nucleic acid sequence; the tissue-specific expression patterns ot each nucleic acid sequence as determined by northern analysis: diseases, disorders, or conditions associated with these tissues: and the vector into which each cDNA was cloned.
Table 4 describes the tissues used to construct the cDNA libraries from which cDNA clones encoding NSPRT were isolated
Table 5 shows the tools, programs, and algorithms used to analyze the polynucleotides and polypeptides of the invention, along with applicable descriptions, references, and threshold parameters.
DESCRIPTION OF THE INVENTION
Before the present proteins, nucleotide sequences, and methods are described, it is understood that this invenuon is not limited to the particular machines, materials and methods described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invenuon which will be limited only by the appended claims
It must be noted that as used herein and in the appended claims, the singular forms "a." "an,'* and "the" include plural reference unless the context clearly dictates otherwise. Thus, tor example, a reference to "a host cell" includes a plurality of such host cells, and a reference to "an antibody" is a reference to one or more antibodies and equivalents thereof known to those skilled in the art. and so forth
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one ot ordinary skill in the art to which this invention belongs .Although any machines, materials, and methods similar or equivalent to those described herein can be used to practice or test the present invention, the preferred machines, materials and methods are now described All publications mentioned herein are cited for the purpose of describing and disclosing the cell lines, protocols, reagents and vectors which are reported in the publications and which might be used in connection with the invention Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention DEFINITIONS
"NSPRT" refers to the amino acid sequences of substantially purified NSPRT obtained from any species, particularly a mammalian species, including bovine, ovine, porcine, muπne. equine, and human, and from any source, whether natural, synthetic, semi-synthetic, or recombinant
The term agomst ' refers to a molecule which intensifies or mimics the biological acuvity ot NSPRT Agomsts may include proteins, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of NSPRT either by directly interacting with NSPRT or by acting on components of the biological pathway in which NSPRT participates
An "allelic variant * is an alternative form of the gene encoding NSPRT Allelic variants may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or m polypeptides whose structure or function may or may not be altered A gene may have none, one, or many allelic variants of its naturally occurring form Common mutational changes which give rise to allelic variants are generally ascribed to natural deletions, additions, or substitutions of nucleotides Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence
"'Altered-' nucleic acid sequences encoding NSPRT include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polypepude the same as NSPRT or a polypepude with at least one functional characteristic of NSPRT Included within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleoude probe of the polynucleotide encoding NSPRT, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding NSPRT The encoded protein may also be "'altered," and may contain deletions, insertions, or substitutions of ammo acid residues which produce a silent change and result in a functionally equivalent NSPRT Deliberate am o acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and or the amphipathic nature of the residues, as long as the biological or immunological activity of NSPRT is retained For example, negatively charged amino acids may include aspartic acid and glutamic acid, and posiuvely charged amino acids may include lysine and arginine Amino acids with uncharged polar side chains having similar hydrophilicity values may include asparagine and glutamine. and seπne and threonine Amino acids with uncharged side chains having similar hydrophilicity values may include leucine, isoleucine, and valine. glycme and alanine, and phenylalamne and tyrosine
The terms "amino acid" and "'amino acid sequence " refer to an ohgopeptide, peptide. polypeptide. or protein sequence, or a fragment of any of these, and to naturally occurring or synthetic molecules Where " amino acid sequence " is recited to refer to a sequence ot a naturally occurring protein molecule, am o acid sequence " and like terms are not meant to limit the amino acid sequence to the complete native ammo acid sequence associated with the recited protein molecule
"Amplification " relates to the production of additional copies ot a nucleic acid sequence Amplification is generally carried out using polymerase chain reaction (PCR) technologies well known
The term antagomst " refers to a molecule which inhibits or attenuates the biological activity ol NSPRT. Antagonists may include proteins such as antibodies, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of NSPRT either by directly interacting with NSPRT or by acting on components of the biological pathway in which NSPRT participates. The term "■antibody" refers to intact immunoglobulin molecules as well as to fragments thereof, such as Fab, F(ab')2, and Fv fragments, which are capable of binding an epitopic determinant Antibodies that bind NSPRT polypeptides can be prepared using intact polypeptides or using fragments containing small peptides of interest as the immunizing antigen The polypeptide or ohgopeptide used to immumze an ammal (e.g., a mouse, a rat, or a rabbit) can be derived from the translation of RNA, or synthesized chemically, and can be conjugated to a caπier protein if desired. Commonly used caπiers that are chemically coupled to peptides include bovine serum albumin, thyroglobuhn. and keyhole limpet hemocyanin (KLH) The coupled peptide is then used to immumze the ammal.
The term "antigenic determinant" refers to that region of a molecule (i.e., an epitope) that makes contact with a particular antibody. When a protein or a fragment of a protein is used to immumze a host ammal, numerous regions of the protein may induce the production of antibodies which bind specifically to antigenic determinants (particular regions or three-dimensional structures on the protein). An antigenic determinant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) for binding to an antibody.
The term "antisense" refers to any composition capable of base-pairing with the "sense" (coding) strand of a specific nucleic acid sequence. Antisense compositions may include DNA, RNA. peptide nucleic acid (PNA), o gonucleotides having modified backbone linkages such as phosphor othioates, methylphosphonates, or benzylphosphonates. ohgonucleotides having modified sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars, or ohgonucleotides having modified bases such as 5-methyl cytosine, 2'-deoxyuracιl, or 7-deaza-2'-deoxyguanosιne. Antisense molecules may be produced by any method including chemical synthesis or transcription Once introduced into a cell, the complementary antisense molecule base-pairs with a naturally occumng nucleic acid sequence produced by the cell to form duplexes which block either transcription or translation The designation "negative" or "minus" can reter to the anusense strand, and the designation " positive " or "plus * can reter to the sense strand of a reference DNA molecule The term "biologically active" refers to a protein having structural, regulatory, or biochemical functions of a naturally occumng molecule Likewise, "lmmunologically active" or "immunogemc " refers to the capability of the natural, recombinant, or synthetic NSPRT. or of any ohgopeptide thereof , to induce a specific immune response in appropriate ammals or cells and to bind with specific antibodies "Complementary" describes the relationship between two single-stranded nucleic acid sequences that anneal by base-pairing. For example, 5'-AGT-3' pairs with its complement, 3'-TCA-5'
A "composition comprising a given polynucleotide sequence" and a "composition comprising a given amino acid sequence"" refer broadly to any composition contaimng the given polynucleotide or amino acid sequence. The composition may comprise a dry formulation or an aqueous solution. Compositions comprising polynucleotide sequences encoding NSPRT or fragments of NSPRT may be employed as hybridization probes. The probes may be stored in freeze-dπed form and may be associated with a stabilizing agent such as a carbohydrate. In hybridizations, the probe may be deployed in an aqueous solution contaimng salts (e.g., NaCl), detergents (e g , sodium dodecyl sulfate, SDS), and other components (e g , Denhardt's solution, dry milk, salmon sperm DNA. etc.).
"Consensus sequence"" refers to a nucleic acid sequence which has been subjected to repeated DNA sequence analysis to resolve uncalled bases, extended using the XL-PCR kit (PE Biosystems, Foster City CA) in the 5' and/or the 3' direction, and resequenced, or which has been assembled from one or more overlapping cDNA, EST, or genomic DNA fragments using a computer program for fragment assembly, such as the GEL VIEW fragment assembly system (GCG, Madison WI) or Phrap (Umversity of Washington, Seattle WA). Some sequences have been both extended and assembled to produce the consensus sequence
"Conservative am o acid substitutions" are those substitutions that are predicted to least interfere with the properties of the original protein, I e., the structure and especially the function of the protein is conserved and not significantly changed by such substitutions The table below shows amino acids which may be substituted for an original amino acid in a protein and which are regarded as conservative ammo acid substitutions Original Residue Conservative Substitution Ala Gly, Ser
Asp Asn, Glu
Cys Ala, Ser
Gly Ala
His Asn. Arg, Gin. Glu
He Leu. Val Leu lie, Val
Lys Arg, Gin, Glu
Met Leu. He
Phe His, Met. Leu. Trp. Tyr
Ser Cys. Thr WO 01/07470 PCT/USOO/l 9837
Thr Ser, Val Trp Phe, Tyr Tyr His. Phe, Trp Val He, Leu, Thr
Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, tor example, as a beta sheet or alpha helical conformation, (b) the charge or hydrophobicity of the molecule at the site of the substitution, and/or (c) the bulk of the side chain. A "deletion" refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides.
The term "derivative'* refers to a chemically modified polynucleotide or polypeptide. Chemical modifications of a polynucleotide sequence can include, for example, replacement of hydrogen by an alkyl. acyl, hydroxyl, or amino group. A derivative polynucleotide encodes a polypeptide which retains at least one biological or immunological function of the natural molecule. A derivative polypeptide is one modified by glycosylation, pegylation. or any similar process that retains at least one biological or immunological function of the polypeptide from which it was derived.
A "detectable label" refers to a reporter molecule or enzyme that is capable ot generating a measurable signal and is covalently or noncovalently joined to a polynucleotide or polypepude. A "fragment" is a umque portion of NSPRT or the polynucleotide encoding NSPRT which is identical in sequence to but shorter in length than the parent sequence. A fragment may compπse up to the entire length of the defined sequence, minus one nucleotide/amino acid residue For example, a fragment may comprise from 5 to 1000 contiguous nucleotides or amino acid residues A fragment used as a probe, pπmer. antigen, therapeutic molecule, or for other purposes, may be at least 5, 10, 15. 16, 20, 25, 30, 40. 50. 60, 75, 100, 150. 250 or at least 500 contiguous nucleotides or amino acid residues in length. Fragments may be preferentially selected trom certain regions of a molecule. For example, a polypeptide fragment may compπse a certain length of contiguous amino acids selected from the first 250 or 500 amino acids (or first 25% or 50% of a polypeptide) as shown in a certain defined sequence Clearly these lengths are exemplary, and any length that is supported by the specification, including the Sequence Listing, tables, and figures, may be encompassed by the present embodiments
A fragment of SEQ ID NO 5-8 compπses a region ol umque polynucleotide sequence that specifically identifies SEQ ID NO 5-8. lor example, as distinct from any other sequence in the genome trom which the tragment was obtained A fragment ot SEQ ID NO 5-8 is useful, tor example, in hybridization and amphtication technologies and in analogous methods that distinguish SEQ ID NO 5-8 from related polynucleotide sequences The precise length ot a tragment of SEQ ID NO:5-8 and the region of SEQ ID NO.5-8 to which the fragment coπesponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment.
A fragment of SEQ ID NO.1 -4 is encoded by a fragment of SEQ ID NO.5-8 A fragment of SEQ ID NO.1-4 comprises a region of umque amino acid sequence that specifically identifies SEQ ID NO.1 -4 For example, a fragment of SEQ ID NO 1 -4 is useful as an immunogemc peptide for the development of antibodies that specifically recogmze SEQ ID NO 1-4 The precise length ot a fragment of SEQ ID NO.1-4 and the region of SEQ ID NO 1-4 to which the fragment coπesponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment A "full-length" polynucleotide sequence is one contaimng at least a translation initiation codon
(e.g., methiomne) followed by an open reading frame and a translation termination codon A "full- length" polynucleotide sequence encodes a "full-length" polypeptide sequence
"Homology" refers to sequence similarity or, interchangeably, sequence identity, between two or more polynucleotide sequences or two or more polypeptide sequences. The terms "percent identity*' and "% identity," as applied to polynucleotide sequences, refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences. Percent identity between polynucleotide sequences may be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN version 3 12e sequence alignment program. This program is part of the LASERGENE software package, a suite of molecular biological analysis programs (DNASTAR, Madison WI) CLUSTAL V is described in Higgins, D G and P.M. Sharp (1989) CABIOS 5.151-153 and in Higgins, D G. et al (1992) CABIOS 8 189-191 For pairwise alignments of polynucleotide sequences, the default parameters are set as follows.
Ktuple=2, gap penalty=5, wmdow=4, and "diagonals saved"=4 The "weighted"" residue weight table is selected as the default. Percent identity is reported by CLUSTAL V as the "percent similarity" between aligned polynucleotide sequences
Alternatively, a suite of commonly used and freely available sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) (Altschul. S F et al. (1990) J Mol Biol 215 403-410). which is available from several sources, including the NCBI. Bethesda. MD. and on the Internet at http.//www ncbi nlm run gov/BLAST/ The BLAST software suite includes various sequence analysis programs including "blastn," that is used to align a known polynucleotide sequence with other polynucleotide sequences from a variety of databases Also available is a tool called "BLAST 2 Sequences*' that is used for direct pairwise comparison ot two nucleotide sequences. "BLAST 2 Sequences" can be accessed and used interactively at http://www.ncbi.nlm.mh.gov/gorf/bl2.html. The "BLAST 2 Sequences'' tool can be used for both blastn and blastp (discussed below). BLAST programs are commonly used with gap and other parameters set to default settings. For example, to compare two nucleotide sequences, one may use blastn with the "BLAST 2 Sequences"" tool Version 2.0.12 (Apπl-21-2000) set at default parameters. Such default parameters may be, for example. Matrix: BLOSUM62 Reward for match: 1 Penalty for mismatch- -2 Open Gap: 5 and Extension Gap: 2 penalties
Gap x drop-off: 50 Expect: 10 Word Size: 11 Filter: on Percent identity may be measured over the length of an entire defined sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures, or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
Nucleic acid sequences that do not show a high degree ot identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein.
The phrases "percent identity** and "% identity," as applied to polypeptide sequences, refer to the percentage of residue matches between at least two polypeptide sequences aligned using a standardized algorithm. Methods of polypeptide sequence alignment are well-known Some alignment methods take into account conservative amino acid substitutions Such conservative substitutions, explained in more detail above, generally preserve the charge and hydrophobicity at the site of substitution, thus preserving the structure (and therefore function) of the polypeptide
Percent identity between polypeptide sequences may be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN version 3.12e sequence alignment program (described and referenced above) For pairwise alignments of polypepude sequences using CLUSTAL V, the default parameters are set as follows Ktuple=l, gap penalty=3, wιndow=5, and "diagonals saved"=5. The PAM250 matrix is selected as the default residue weight table. As with polynucleotide alignments, the percent identity is reported by CLUSTAL V as the "percent similarity"" between aligned polypeptide sequence pairs. Alternatively the NCBI BLAST software suite may be used. For example, for a pairwise comparison of two polypepude sequences, one may use the "BLAST 2 Sequences"' tool Version 2.0.12 (Apr-21-2000) with blastp set at default parameters. Such default parameters may be, for example
Matrix: BLOSUM62
Open Gap: 11 and Extension Gap: 1 penalties Gap x drop-off: 50
Expect: 10
Word Size: 3
Filter- on
Percent identity may be measured over the length of an enure defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, tor instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
"Human artificial chromosomes" (HACs) are linear microchromosomes which may contain DNA sequences of about 6 kb to 10 Mb in size, and which contain all of the elements required for chromosome replication, segregation and maintenance.
The term "humanized antibody" refers to an antibody molecule in which the amino acid sequence in the non-antigen binding regions has been altered so that the antibody more closely resembles a human antibody, and still retains its original binding ability
"Hybridization" refers to the process by which a polynucleotide strand anneals with a complementary strand through base pairing under defined hybridization conditions Specific hybridization is an indication that two nucleic acid sequences share a high degree of complementarity Specific hybridization complexes form under permissive annealing conditions and remain hybridized after the "washing " step(s) The washing step(s) is particularly important in determimng the stringency of the hybridization process, with more stringent conditions allowing less non-specific binding, l e . binding between pairs ot nucleic acid strands that are not perfectly matched Permissive conditions for annealing of nucleic acid sequences are routinely determinable by one of ordinary skill in the art and WO 01/07470 PCT/USOO/l 9837
may be consistent among hybridization experiments, whereas wash conditions may be varied among experiments to achieve the desired stringency, and therefore hybridization specificity. Permissive annealing conditions occur, for example, at 68°C in the presence of about 6 x SSC. about 1 % (w/v) SDS. and about 100 μg/ l sheared, denatured salmon sperm DNA. Generally, stringency ot hybridization is expressed, in part, with reference to the temperature under which the wash step is carried out Such wash temperatures are typically selected to be about 5°C to 20°C lower than the thermal melting point (TJ for the specific sequence at a defined lomc strength and pH The Tm is the temperature (under defined lomc strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe An equation for calculating Tm and conditions for nucleic acid hybridization are well known and can be found in Sambrook, J et al., 1989. Molecular Cloning A Laboratory Manual. 2nd ed.. vol 1-3, Cold Spring Harbor Press, Plainview NY. specifically see volume 2, chapter 9
High stringency conditions for hybridization between polynucleotides ot the present invention include wash conditions of 68 °C in the presence of about 0.2 x SSC and about 0.1 % SDS, for 1 hour. Alternatively, temperatures of about 65°C. 60°C, 55°C, or 42°C may be used. SSC concentration may be varied from about 0 1 to 2 x SSC, with SDS being present at about 0.1 % Typically, blocking reagents are used to block non-specific hybridization. Such blocking reagents include, for instance, sheared and denatured salmon sperm DNA at about 100-200 μg/ml. Organic solvent, such as formamide at a concentration of about 35-50% v/v, may also be used under particular circumstances, such as for RNA DNA hybridizations Useful variations on these wash conditions will be readily apparent to those of ordinary skill in the art. Hybridization, particularly under high stringency conditions, may be suggestive of evolutionary similarity between the nucleotides Such similarity is strongly indicative of a similar role for the nucleotides and their encoded polypeptides
The term "hybridization complex " refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary bases A hybridization complex may be formed in solution (e g , Cnt or R0t analysis) or formed between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e g , paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids have been fixed) The words "insertion" and "addition " refer to changes in an amino acid or nucleotide sequence resulting in the addition ot one or more amino acid residues or nucleotides. respectively
"Immune response" can reter to conditions associated with inflammation, trauma, immune disorders, or infectious or genetic disease, etc These conditions can be characterized by expression ot various tactors. e g . cytokines. chemokines. and other signaling molecules, which may affect cellular WO 01/07470 PCT/USOO/l 9837
and systemic defense systems.
An "immunogemc fragment"' is a polypeptide or ohgopeptide fragment of NSPRT which is capable of eliciting an immune response when introduced into a living orgamsm, for example, a mammal The term "immunogemc tragment" also includes any polypeptide or ohgopeptide fragment ot NSPRT which is useful in any of the antibody production methods disclosed herein or known in the art The term "microarray" refers to an aπangement of a plurality ot polynucleotides, polypeptides. or other chemical compounds on a substrate
The terms "element" and "array element" refer to a polynucleotide, polypeptide, or other chemical compound having a umque and defined position on a microaπay The term "modulate"" refers to a change in the activity ot NSPRT For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of NSPRT
The phrases "nucleic acid"' and "nucleic acid sequence*" refer to a nucleotide, ohgonucleotide. polynucleotide, or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA- ke or RNA- ke material
"Operably linked" refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with a second nucleic acid sequence For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence Operably linked DNA sequences may be in close proximity or contiguous and. where necessary to join two protein coding regions, in the same reading frame.
"Pepude nucleic acid*' (PNA) refers to an antisense molecule or anti-gene agent which comprises an ohgonucleotide of at least about 5 nucleotides in length linked to a peptide backbone of amino acid residues ending in lysine The terminal lysine confers solubility to the composition. PNAs preferentially bind complementary single stranded DNA or RNA and stop transcript elongation, and may be pegylated to extend their fespan in the cell
"Post-translational modification** of an NSPRT may involve hpidation, glycosylauon. phosphorylaϋon, acetylation. racemization, proteolytic cleavage, and other modifications known in the art These processes may occur synthetically or biochemically Biochemical modificaUons will vary by cell type depending on the enzymatic milieu of NSPRT
"Probe" reters to nucleic acid sequences encoding NSPRT. their complements, or fragments thereot. which are used to detect identical, allelic or related nucleic acid sequences Probes are isolated ohgonucleotides or polynucleotides attached to a detectable label or reporter molecule Typical labels include radioactive isotopes, hgands. chemiluminescent agents, and enzymes "Primers " are short nucleic acids, usually DNA ohgonucleotides. which mav be annealed to a target polynucleotide by WO 01/07470 PCT/USOO/l 9837
complementary base-pairing. The primer may then be extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification (and identification) of a nucleic acid sequence, e g., by the polymerase chain reaction (PCR).
Probes and primers as used in the present invention typically comprise at least 15 contiguous nucleotides of a known sequence. In order to enhance specificity, longer probes and primers may also be employed, such as probes and primers that comprise at least 20, 25, 30, 40, 50, 60. 70, 80, 90, 100, or at least 150 consecutive nucleotides of the disclosed nucleic acid sequences. Probes and primers may be considerably longer than these examples, and it is understood that any length supported by the specification, including the tables, figures, and Sequence Listing, may be used. Methods for preparing and using probes and primers are described in the references, for example Sambrook, J. et al., 1989, Molecular Clomng: A Laboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor Press, Plainview NY: Ausubel, F.M. et al.,1987, Cuπent Protocols in Molecular Biology. Greene Publ. Assoc. & Wiley-Intersciences, New York NY: Inrus, M. et al., 1990. PCR Protocols. A Guide to Methods and Applications. Academic Press, San Diego CA. PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge MA).
Ohgonucleotides for use as primers are selected using software known in the art tor such purpose. For example, OLIGO 4.06 software is useful for the selection of PCR primer pairs of up to 100 nucleotides each, and for the analysis of ohgonucleotides and larger polynucleotides of up to 5,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases. Similar primer selection programs have incorporated additional features for expanded capabilities. For example, the PπmOU primer selection program (available to the public from the Genome Center at Umversity of Texas South West Medical Center, Dallas TX) is capable ot choosing specific primers from megabase sequences and is thus useful for designing primers on a genome-wide scope. The Pπmer3 primer selection program (available to the public from the Whitehead Institute/MIT Center for Genome Research. Cambridge MA) allows the user to input a "mispπming library." in which sequences to avoid as primer binding sites are user-specified Pπmer3 is useful, in particular, for the selection ot ohgonucleotides for microaπays (The source code for the latter two primer selection programs may also be obtained from their respective sources and modified to meet the user's specific needs ) The PπmeGen program (available to the public from the UK Human Genome Mapping Pro]ect Resource Centre. Cambridge UK) designs primers based on multiple sequence alignments, thereby allowing selection ot primers that hybridize to either the most conserved or least conserved regions of aligned nucleic acid sequences Hence, this program is useful tor identification of both umque and conserved ohgonucleotides and WO 01/07470 PCTtUSOO/19837
polynucleotide fragments. The ohgonucleotides and polynucleotide fragments identified by any of the above selection methods are useful in hybridization technologies, for example, as PCR or sequencing primers, microarray elements, or specific probes to identify fully or partially complementary polynucleotides in a sample of nucleic acids Methods of ohgonucleotide selection are not limited to those described above.
A "recombinant nucleic acid"' is a sequence that is not naturally occumng or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence. This artificial combination is often accomphshed by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques such as those described m Sambrook, supra The term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid Frequently, a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence. Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a cell Alternatively, such recombinant nucleic acids may be part of a viral vector, e.g., based on a vaccima virus, that could be use to vaccinate a mammal wherein the recombinant nucleic acid is expressed, inducing a protective immunological response m the mammal.
A "regulatory element" refers to a nucleic acid sequence usually derived from untranslated regions of a gene and includes enhancers, promoters, introns, and 5' and 3' untranslated regions (UTRs). Regulatory elements interact with host or viral proteins which control transcription, translation, or RNA stability
"Reporter molecules" are chemical or biochemical moieties used for labeling a nucleic acid, amino acid, or antibody. Reporter molecules include radionuchdes: enzymes, fluorescent, chemiluminescent, or chromogemc agents, substrates: cofactors, inhibitors, magnetic particles, and other moieties known in the art An "RNA equivalent," in reference to a DNA sequence, is composed of the same linear sequence of nucleotides as the reference DNA sequence with the exception that all occuπences of the nitrogenous base thymine are replaced with uracil. and the sugar backbone is composed ot πbose instead of deoxyπbose
The term "sample" is used in its broadest sense A sample suspected ot contaimng nucleic acids encoding NSPRT. or fragments thereof, or NSPRT itself, may comprise a bodily fiuid. an extract trom a cell, chromosome, organelle. or membrane isolated from a cell, a cell, genomic DNA. RNA. or cDNA, in solution or bound to a substrate, a tissue, a tissue print, etc
The terms ""specific binding " and "specifically binding" refer to that interaction between a protein or peptide and an agomst. an antibody, an antagomst. a small molecule, or any natural or synthetic binding composition The interaction is dependent upon the presence of a particular structure of the protein, e.g., the antigenic determinant or epitope. recognized by the binding molecule. For example, if an antibody is specific for epitope "A,"* the presence of a polypeptide comprising the epitope A, or the presence of free unlabeled A. in a reaction contaimng free labeled A and the antibody will reduce the amount of labeled A that binds to the antibody
The term "substantially purified"* refers to nucleic acid or ammo acid sequences that are removed from their natural environment and are isolated or separated, and are at least 60% free, preferably at least 75% free, and most preferably at least 90% free from other components with which they are naturally associated A "substitution"" refers to the replacement of one or more amino acid residues or nucleotides by different amino acid residues or nucleotides. respectively
"Substrate" refers to any suitable rigid or semi-rigid support including membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries. The substrate can have a variety of surface forms, such as wells, trenches, pins, channels and pores, to which polynucleotides or polypeptides are bound
A "transcript image*' refers to the collective pattern of gene expression by a particular cell type or tissue under given conditions at a given time
"Transformation" describes a process by which exogenous DNA is introduced into a recipient cell. Transformation may occur under natural or artificial conditions according to various methods well known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell The method for transformation is selected based on the type of host cell being transformed and may include, but is not limited to. bacteπophage or viral infection, electroporation, heat shock, hpotection, and particle bombardment The term ""transformed"* cells includes stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as well as transiently transformed cells which express the inserted DNA or RNA for limited periods of time.
A "transgenic organism." as used herein, is any organism, including but not limited to animals and plants, in which one or more ot the cells of the orgamsm contains heterologous nucleic acid introduced by way of human intervention, such as by transgenic techmques well known in the art The nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way ot deliberate genetic manipulation, such as by mιcroιn|ectιon or by lntection with a recombinant virus The term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction ot a recombinant DNA molecule The transgenic organisms contemplated in accordance with the present invention include bacteria. cyanobacteπa. tungi. plants, and animals The isolated DNA ot the present invention can be WO 01/07470 PCT/USOO/l 9837
introduced into the host by methods known m the art, for example infection, transfection, transformation or transconjugation. Techmques for transfeπing the DNA of the present invention into such organisms are widely known and provided in references such as Sambrook et al. (1989), supra. A ""variant" of a particular nucleic acid sequence is defined as a nucleic acid sequence having at least 40% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the "BLAST 2 Sequences"* tool Version 2.0.9 (May-07-1999) set at default parameters. Such a pair of nucleic acids may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%. at least 90%, at least 95% or at least 98% or greater sequence identity over a certain defined length. A variant may be described as, for example, an "allelic" (as defined above), "splice,"' "species,** or ""polymorphic"* variant. A splice variant may have sigmficant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternative splicing of exons during mRNA processing. The corresponding polypeptide may possess additional functional domains or lack domains that are present in the reference molecule. Species variants are polynucleotide sequences that vary from one species to another. The resulting polypeptides generally will have sigmficant ammo acid identity relative to each other. A polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species. Polymorphic variants also may encompass "single nucleotide polymorphisms" (SNPs) in which the polynucleotide sequence varies by one nucleotide base The presence of SNPs may be indicative of. for example, a certain population, a disease state, or a propensity for a disease state.
A ""variant" of a particular polypeptide sequence is defined as a polypeptide sequence having at least 40% sequence identity to the particular polypeptide sequence over a certain length of one of the polypepude sequences using blastp with the "BLAST 2 Sequences" tool Version 2 0.9 (May-07-1999) set at default parameters Such a pair of polypeptides may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% or greater sequence identity over a certain defined length of one of the polypeptides THE INVENTION
The invention is based on the discovery of new human nervous system-associated proteins (NSPRT), the polynucleotides encoding NSPRT, and the use of these compositions for the diagnosis. treatment, or prevention of neurological, inflammatory, and cell prohferative disorders, including cancer
Table 1 lists the Incyte clones used to assemble full length nucleotide sequences encoding NSPRT Columns 1 and 2 show the sequence identification numbers (SEQ ID NOs) of the polypeptide and nucleotide sequences, respectively Column 3 shows the clone IDs ot the Incyte clones in which nucleic acids encoding each NSPRT were identified, and column 4 shows the cDNA libraries from which these clones were isolated. Column 5 shows Incyte clones and their coπesponding cDNA libraries. Clones for which cDNA libraries are not indicated were derived from pooled cDNA libraries. The Incyte clones in column 5 were used to assemble the consensus nucleotide sequence of each NSPRT and are useful as fragments in hybridization technologies. The columns of Table 2 show various properties of each of the polypeptides of the invention: column 1 references the SEQ ID NO; column 2 shows the number of amino acid residues in each polypeptide: column 3 shows potential phosphorylation sites; column 4 shows potential glycosylation sites; column 5 shows the amino acid residues comprising signature sequences and motifs: column 6 shows homologous sequences as identified by BLAST analysis; and column 7 shows analytical methods and in some cases, searchable databases to which the analytical methods were applied. The methods of column 7 were used to characterize each polypeptide through sequence homology and protein motifs.
The columns of Table 3 show the tissue-specificity and diseases, disorders, or conditions associated with nucleotide sequences encoding NSPRT. The first column of Table 3 lists the nucleotide SEQ ID NOs. Column 2 lists fragments of the nucleotide sequences of column 1. These fragments are useful, for example, in hybridization or amplification technologies to identify SEQ ID NO.5-8 and to distinguish between SEQ ID NO:5-8 and related polynucleotide sequences. The polypeptides encoded by these fragments are useful, for example, as immunogenic peptides. Column 3 lists tissue categories which express NSPRT as a fraction of total tissues expressing NSPRT. Column 4 lists diseases, disorders, or conditions associated with those tissues expressing NSPRT as a fraction of total tissues expressing NSPRT. Column 5 lists the vectors used to subclone each cDNA library. Of particular interest is the expression of SEQ ID NO:5 exclusively in nervous tissue (100%) and the expression of SEQ ID NO:7 predominately in nervous tissue (78.6%).
The columns of Table 4 show descriptions of the tissues used to construct the cDNA libraries from which cDNA clones encoding NSPRT were isolated. Column 1 references the nucleotide SEQ ID NOs, column 2 shows the cDNA libraries from which these clones were isolated, and column 3 shows the tissue origins and other descriptive information relevant to the cDNA libraries in column 2.
The invention also encompasses NSPRT variants. A prefeπed NSPRT variant is one which has at least about 80%, or alternatively at least about 90%, or even at least about 95% amino acid sequence identity to the NSPRT amino acid sequence, and which contains at least one functional or structural characteristic of NSPRT.
The invention also encompasses polynucleotides which encode NSPRT. In a particular embodiment, the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NO:5-8. which encodes NSPRT. The polynucleotide sequences of SEQ ID NO:5-8. as presented in the Sequence Listing, embrace the equivalent RNA sequences, wherein occurrences of the nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of πbose instead of deoxyπbose.
The invention also encompasses a variant ot a polynucleotide sequence encoding NSPRT. In particular, such a variant polynucleotide sequence will have at least about 70%, or alternatively at least about 85%, or even at least about 95% polynucleotide sequence identity to the polynucleotide sequence encoding NSPRT. A particular aspect of the invention encompasses a variant of a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NO.5-8 which has at least about 70%, or alternatively at least about 85%, or even at least about 95% polynucleotide sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO.5-8 Any one of the polynucleotide variants described above can encode an ammo acid sequence which contains at least one functional or structural characteristic of NSPRT.
It will be appreciated by those skilled in the art that as a result of the degeneracy of the genetic code, a multitude of polynucleotide sequences encoding NSPRT, some bearing minimal similarity to the polynucleotide sequences of any known and naturally occurring gene, may be produced. Thus, the invention contemplates each and every possible variation of polynucleotide sequence that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard triplet genetic code as applied to the polynucleotide sequence of naturally occurring NSPRT, and all such variations are to be considered as being specifically disclosed.
Although nucleotide sequences which encode NSPRT and its variants are generally capable of hybridizing to the nucleotide sequence of the naturally occurring NSPRT under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding NSPRT or its derivatives possessing a substantially different codon usage, e.g., inclusion ot non-naturally occumng codons. Codons may be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic host in accordance with the frequency with which particular codons are utilized by the host. Other reasons for substantially altering the nucleotide sequence encoding NSPRT and its derivatives without altering the encoded amino acid sequences include the production of RNA transcripts having more desirable properties, such as a greater half-life, than transcripts produced from the naturally occumng sequence
The invention also encompasses production of DNA sequences which encode NSPRT and NSPRT derivatives, or fragments thereof, entirely by synthetic chemistry After production, the synthetic sequence may be inserted into any of the many available expression vectors and cell systems using reagents well known in the art Moreover, synthetic chemistry may be used to introduce mutations into a sequence encoding NSPRT or any tragment thereof
Also encompassed by the invention are polynucleotide sequences that are capable ot WO 01/07470 PCT/USOO/l 9837
hybridizing to the claimed polynucleotide sequences, and, in particular, to those shown in SEQ ID NO:5-8 and fragments thereof under various conditions of stringency. (See, e.g., Wahl, G.M. and S.L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, AR. (1987) Methods Enzymol. 152:507- 511.) Hybridization conditions, including annealing and wash conditions, are described in "Definitions."
Methods for DNA sequencing are well known in the art and may be used to practice any of the embodiments of the invention. The methods may employ such enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase (PE Biosystems, Foster City CA), thermostable T7 polymerase (Amersham Pharmacia Biotech, Piscataway NJ), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE amplification system (Life Technologies, Gaithersburg MD). Preferably, sequence preparation is automated with machines such as the MICROLAB 2200 liquid transfer system (Hamilton, Reno NV), PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal cycler (PE Biosystems). Sequencing is then caπied out using either the ABI 373 or 377 DNA sequencing system (PE Biosystems), the MEGABACE 1000 DNA sequencing system (Molecular Dynamics, Sunnyvale CA), or other systems known in the art. The resulting sequences are analyzed using a variety of algorithms which are well known in the art. (See, e.g., Ausubel, F.M. (1997) Short Protocols in Molecular Biology, John Wiley & Sons, New York NY, unit 7.7; Meyers, R.A. (1995) Molecular Biology and Biotechnology. Wiley VCH, New York NY, pp. 856-853.) The nucleic acid sequences encoding NSPRT may be extended utilizing a partial nucleotide sequence and employing various PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements. For example, one method which may be employed, restriction-site PCR, uses universal and nested primers to amplify unknown sequence from genomic DNA within a cloning vector. (See, e.g., Sarkar, G. (1993) PCR Methods Applic. 2:318-322.) Another method, inverse PCR, uses primers that extend in divergent directions to amplify unknown sequence from a circularized template. The template is derived from restriction fragments comprising a known genomic locus and suπounding sequences. (See, e.g., Triglia, T. et al. (1988) Nucleic Acids Res. 16:8186.) A third method, capture PCR, involves PCR amplification of DNA fragments adjacent to known sequences in human and yeast artificial chromosome DNA. (See, e.g., Lagerstrom. M. et al. ( 1991 ) PCR Methods Applic. 1 :111-119.) In this method, multiple restriction enzyme digestions and ligations may be used to insert an engineered double-stranded sequence into a region of unknown sequence before performing PCR. Other methods which may be used to retrieve unknown sequences are known in the art. (See, e.g., Parker, J.D. et al. (1991) Nucleic Acids Res. 19:3055-3060). Additionally, one may use PCR. nested primers, and PROMOTERFINDER libraries (Clontech, Palo Alto CA) to walk genomic DNA. This procedure avoids the need to screen libraries and is useful in finding intron/exon junctions. For all PCR-based methods, primers may be designed using commercially available software, such as OLIGO 4.06 Primer Analysis software (National Biosciences, Plymouth MN) or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the template at temperatures of about 68 °C to 72°C.
When screening for full-length cDNAs, it is preferable to use libraries that have been size-selected to include larger cDNAs. In addition, random-primed libraries, which often include sequences containing the 5' regions of genes, are preferable for situations in which an oligo d(T) library does not yield a full-length cDNA. Genomic libraries may be useful for extension of sequence into 5' non-transcribed regulatory regions.
Capillary electrophoresis systems which are commercially available may be used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products. In particular, capillary sequencing may employ flowable polymers for electrophoretic separation, four different nucleotide- specific, laser-stimulated fluorescent dyes, and a charge coupled device camera for detection of the emitted wavelengths. Output/light intensity may be converted to electrical signal using appropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, PE Biosystems), and the entire process from loading of samples to computer analysis and electronic data display may be computer controlled. Capillary electrophoresis is especially preferable for sequencing small DNA fragments which may be present in limited amounts in a particular sample.
In another embodiment of the invention, polynucleotide sequences or fragments thereof which encode NSPRT may be cloned in recombinant DNA molecules that direct expression of NSPRT, or fragments or functional equivalents thereof, in appropriate host cells. Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence may be produced and used to express NSPRT.
The nucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter NSPRT-encoding sequences for a variety of purposes including, but not limited to, modification of the cloning, processing, and or expression of the gene product. DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic ohgonucleotides may be used to engineer the nucleotide sequences. For example, oligonucleotide- mediated site-directed mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, and so forth.
The nucleotides of the present invention may be subjected to DNA shuffling techniques such as MOLECULARB REEDING (Maxygen Inc., Santa Clara CA; described in U.S. Patent Number
23 WO 01/07470 PCT/USOO/l 9837
5,837,458; Chang, C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Chπstians, F.C. et al (1999) Nat. Biotechnol. 17.259-264. and Cramen, A. et al. (1996) Nat. Biotechnol. 14.315-319) to alter or improve the biological properties of NSPRT, such as its biological or enzymatic activity or its ability to bind to other molecules or compounds. DNA shuffling is a process by which a library of gene vaπants is produced using PCR-mediated recombination of gene fragments. The library is then subjected to selection or screemng procedures that identify those gene vaπants with the desired properties These prefeπed vaπants may then be pooled and further subjected to recursive rounds of DNA shuffling and selection/screening. Thus, genetic diversity is created through "artificial" breeding and rapid molecular evolution. For example, fragments of a single gene contaimng random point mutations may be recombined, screened, and then reshuffled until the desired properties are optimized. Alternatively, fragments of a given gene may be recombined with fragments of homologous genes in the same gene family, either from the same or different species, thereby maximizing the genetic diversity of multiple naturally occumng genes in a directed and controllable manner. In another embodiment, sequences encoding NSPRT may be synthesized, in whole or in part, using chemical methods well known in the art. (See, e.g., Caruthers, M.H. et al. (1980) Nucleic Acids Symp. Ser. 7.215-223, and Horn, T. et al. (1980) Nucleic Acids Symp. Ser. 7.225-232.) Alternatively, NSPRT itself or a fragment thereof may be synthesized using chemical methods. For example, peptide synthesis can be performed using various solution-phase or solid-phase techmques. (See, e.g., Creighton, T. (1984) Proteins. Structures and Molecular Properties, WH Freeman. New York NY, pp 55-60, and Roberge, J Y et al. (1995) Science 269.202-204.) Automated synthesis may be achieved using the ABI 431 A peptide synthesizer (PE Biosystems) Additionally, the ammo acid sequence of NSPRT, or any part thereof, may be altered during direct synthesis and/or combined with sequences from other proteins, or any part thereof, to produce a variant polypepude or a polypeptide having a sequence of a naturally occumng polypeptide.
The peptide may be substantially purified by preparative high performance liquid chromatography (See. e g., Chiez, R M. and F.Z. Regmer (1990) Methods Enzymol 182.392-421.) The composition of the synthetic peptides may be confirmed by amino acid analysis or by sequencing. (See, e g., Creighton, supra, pp 28-53 ) In order to express a biologically active NSPRT, the nucleotide sequences encoding NSPRT or derivatives thereof may be inserted into an appropriate expression vector, I e . a vector which contains the necessary elements for transcπptional and translational control ot the inserted coding sequence in a suitable host These elements include regulatory sequences, such as enhancers, constitutive and inducible promoters, and 5" and 3 " untranslated regions in the vector and in polynucleotide sequences encoding NSPRT Such elements may vary in their strength and specificity Specific initiation signals WO 01/07470 PCT/USOO/l 9837
may also be used to achieve more efficient translation of sequences encoding NSPRT. Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence. In cases where sequences encoding NSPRT and its initiation codon and upstream regulatory sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a fragment thereof, is inserted, exogenous translational control signals including an in-frame ATG initiation codon should be provided by the vector. Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers appropriate for the particular host cell system used. (See, e.g., Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162.)
Methods which are well known to those skilled in the art may be used to construct expression vectors containing sequences encoding NSPRT and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. (See, e.g., Sambrook, J. et al. (1989) Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Press, Plainview NY, ch. 4, 8, and 16-17; Ausubel, F.M. et al. (1995) Current Protocols in Molecular Biology. John Wiley & Sons, New York NY, ch. 9, 13, and 16.)
A variety of expression vector/host systems may be utilized to contain and express sequences encoding NSPRT. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors (e.g., baculovirus); plant cell systems transformed with viral expression vectors (e.g., cauliflower mosaic virus, CaMV. or tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems. (See, e.g., Sambrook, supra: Ausubel, supra: Van Heeke, G. and S.M. Schuster (1989) J. Biol. Chem. 264:5503-5509; Bitter, G.A. et al. (1987) Methods Enzymol. 153:516-544: Scorer, CA. et al. (1994) Bio/Technology 12:181-184; Engelhard, E.K. et al. (1994) Proc. Natl. Acad. Sci. USA 91 :3224-3227: Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-1945; Takamatsu, N. (1987) EMBO J. 6:307-311; Coruzzi, G. et al. (1984) EMBO J. 3: 1671-1680; Broghe. R. et al. (1984) Science 224:838-843: Winter, J. et al. (1991) Results Probl. Cell Differ. 17:85-105: The McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill. New York NY, pp. 191-196: Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. USA 81 :3655-3659; and Harrington, J.J. et al. (1997) Nat. Genet. 15:345-355.) Expression vectors derived from retroviruses. adenoviruses, or herpes or vaccinia viruses, or from various bacterial plasmids. may be used for delivery of nucleotide sequences to the targeted organ, tissue, or cell population. (See, e.g., Di Nicola, M. et al. (1998) Cancer Gen. Ther. 5(6):350-356: Yu. M. et al., (1993) Proc. Natl. Acad. Sci. USA 90(13):6340-6344: Buller. R.M. et al. (1985) Nature 317(6040):813-815; McGregor. D.P. et al. WO 01/07470 PCT/USOO/l 9837
(1994) Mol. Immunol. 31(3).219-226; and Verma, I.M. and N. Soπua (1997) Nature 389:239-242.) The invention is not limited by the host cell employed.
In bacterial systems, a number of clomng and expression vectors may be selected depending upon the use intended for polynucleotide sequences encoding NSPRT. For example, routine clomng, subclomng, and propagation of polynucleotide sequences encoding NSPRT can be achieved using a multifunctional E. coh vector such as PBLUESCRIPT (Stratagene, La Jolla CA) or PSPORT1 plasmid (Life Technologies). Ligation of sequences encoding NSPRT into the vector's multiple clomng site disrupts the lacL gene, allowing a coloπmetπc screemng procedure for identification of transformed bacteria contaimng recombinant molecules. In addition, these vectors may be useful for in vitro transcription, dideoxy sequencing, single strand rescue with helper phage, and creation of nested deletions in the cloned sequence. (See, e.g., Van Heeke, G. and S.M. Schuster (1989) J. Biol Chem. 264:5503-5509.) When large quantities of NSPRT are needed, e.g. for the production of antibodies, vectors which direct high level expression of NSPRT may be used. For example, vectors contaimng the strong, inducible T5 or T7 bacteriophage promoter may be used. Yeast expression systems may be used for production of NSPRT. A number of vectors contaimng constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH promoters, may be used in the yeast Saccharomyces cerevisiae or Pichia pastoπs. In addition, such vectors direct either the secretion or lntracellular retention of expressed proteins and enable integration of foreign sequences into the host genome for stable propagation. (See, e.g., Ausubel, 1995. supra; Bitter, supra; and Scorer, supra )
Plant systems may also be used for expression of NSPRT. Transcription of sequences encoding NSPRT may be driven viral promoters, e g , the 35S and 19S promoters of CaMV used alone or in combination with the omega leader sequence from TMV (Takamatsu, N (1987) EMBO J 6 307-311) Alternatively, plant promoters such as the small subumt of RUBISCO or heat shock promoters may be used (See, e g., Coruzzi, supra. Broghe. supra; and Winter, supra.) These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection (See, e.g.. The McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill. New York NY, pp 191-196 )
In mammalian cells, a number of viral-based expression systems may be utilized In cases where an adenovirus is used as an expression vector, sequences encoding NSPRT may be hgated into an adenovirus transcription/translation complex consisting ot the late promoter and tripartite leader sequence Insertion in a non-essential El or E3 region ot the viral genome may be used to obtain infective virus which expresses NSPRT in host cells (See. e g . Logan. J and T Shenk (1984) Proc Natl Acad Sci USA 81 3655-3659 ) In addition, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammahan host cells. SV40 or EBV- based vectors may also be used for high-level protein expression.
Human artificial chromosomes (HACs) may also be employed to deliver larger fragments of DNA than can be contained in and expressed from a plasmid HACs of about 6 kb to 10 Mb are constructed and delivered via conventional dehvery methods (hposomes, polycatiomc ammo polymers, or vesicles) for therapeutic purposes. (See. e.g., Hamngton, J.J. et al. (1997) Nat. Genet 15:345-355.) For long term production of recombinant proteins in mammahan systems, stable expression of NSPRT in cell lines is preferred. For example, sequences encoding NSPRT can be transformed into cell lines using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for about 1 to 2 days in enriched media before being switched to selective media. The purpose of the selectable marker is to confer resistance to a selective agent, and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clones of stably transformed cells may be propagated using tissue culture techmques appropπate to the cell type.
Any number of selection systems may be used to recover transformed cell lines. These include, but are not limited to, the heφes simplex virus thymidine kmase and ademne phosphoπbosyltransferase genes, for use in tk~ and apr cells, respectively. (See, e.g., Wigler, M. et al. (1977) Cell 11.223-232. Lowy, I. et al. (1980) Cell 22:817-823.) Also, antimetabo te, antibiotic, or herbicide resistance can be used as the basis for selection. For example, dhfr confers resistance to methotrexate. neo confers resistance to the aminoglycosides neomycin and G-418; and als and pat confer resistance to chlorsulfuron and phosphinotπcin acetyltransferase, respectively (See, e.g., Wigler, M et al. (1980) Proc. Natl. Acad. Sci. USA 77 3567-3570; Colbere-Garapm, F. et al (1981) J. Mol. Biol. 150.1-14 ) Additional selectable genes have been described, e g., trpB and hisD, which alter cellular requirements for metabohtes. (See, e.g., Hartman, S.C. and R.C. Mulligan (1988) Proc Natl. Acad Sci. USA 85.8047-8051 ) Visible markers, e g., anthocyanins, green fluorescent proteins (GFP. Clontech), β glucuromdase and its substrate β-glucuromde, or luciferase and its substrate lucifeπn may be used. These markers can be used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system (See. e g , Rhodes, C A (1995) Methods Mol Biol. 55 121-131 )
Although the presence/absence of marker gene expression suggests that the gene of interest is also present, the presence and expression of the gene may need to be confirmed For example, if the sequence encoding NSPRT is inserted within a marker gene sequence, transformed cells contaimng sequences encoding NSPRT can be identified by the absence of marker gene function Alternatively, a marker gene can be placed m tandem with a sequence encoding NSPRT under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.
In general, host cells that contain the nucleic acid sequence encoding NSPRT and that express NSPRT may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations, PCR amplification, and protein bioassay or lmmunoassay techmques which include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein sequences. Immunological methods for detecting and measuring the expression of NSPRT using either specific polyclonal or monoclonal antibodies are known m the art. Examples of such techmques include enzyme-linked lmmunosorbent assays (ELISAs), radioimmunoassays (RIAs), and fluorescence activated cell sorting (FACS). A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on NSPRT is preferred, but a competitive binding assay may be employed. These and other assays are well known in the art. (See, e.g., Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual, APS Press. St. Paul MN, Sect. IV: Cohgan. J.E. et al. (1997) Cuπent Protocols in Immunology, Greene Pub. Associates and Wiley-Interscience, New York NY; and Pound, J.D. (1998) Immunochemical Protocols, Humana Press, Totowa NJ.)
A wide variety of labels and conjugation techmques are known by those skilled in the art and may be used m various nucleic acid and ammo acid assays. Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding NSPRT include ohgolabehng, mck translation, end-labeling, or PCR amphtication using a labeled nucleotide. Alternatively, the sequences encoding NSPRT. or any fragments thereof, may be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides. These procedures may be conducted using a variety of commercially available kits, such as those provided by Amersham Pharmacia Biotech, Promega (Madison WI), and US Biochemical Suitable reporter molecules or labels which may be used for ease of detection include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogemc agents, as well as substrates, cot actors, inhibitors, magnetic particles, and the like Host cells transformed with nucleotide sequences encoding NSPRT may be cultured under conditions suitable for the expression and recovery of the protein from cell culture. The protein produced by a transformed cell may be secreted or retained intracellularly depending on the sequence and/or the vector used As will be understood by those of skill in the art. expression vectors contaimng polynucleotides which encode NSPRT may be designed to contain signal sequences which direct secretion of NSPRT through a prokaryotic or eukaryotic cell membrane.
In addition, a host cell strain may be chosen for its ability to modulate expression of the inserted sequences or to process the expressed protein in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing which cleaves a "prepro" or ""pro"' form of the protein may also be used to specify protein targeting, folding, and/or activity. Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are available from the American Type Culture Collection (ATCC, Manassas VA) and may be chosen to ensure the correct modification and processing of the foreign protein.
In another embodiment of the invention, natural, modified, or recombinant nucleic acid sequences encoding NSPRT may be ligated to a heterologous sequence resulting in translation of a fusion protein in any of the aforementioned host systems. For example, a chimeric NSPRT protein containing a heterologous moiety that can be recognized by a commercially available antibody may facilitate the screening of peptide libraries for inhibitors of NSPRT activity. Heterologous protein and peptide moieties may also facilitate purification of fusion proteins using commercially available affinity matrices. Such moieties include, but are not limited to, glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmoduUn binding peptide (CBP), 6-His, FLAG, c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable purification of their cognate fusion proteins on immobiUzed glutathione, maltose, phenylarsine oxide, calmoduUn, and metal-chelate resins, respectively. FLAG, c-myc, and hemagglutinin (HA) enable immunoaffinity purification of fusion proteins using commercially available monoclonal and polyclonal antibodies that specifically recognize these epitope tags. A fusion protein may also be engineered to contain a proteolytic cleavage site located between the NSPRT encoding sequence and the heterologous protein sequence, so that NSPRT may be cleaved away from the heterologous moiety following purification. Methods for fusion protein expression and purification are discussed in Ausubel (1995, supra, ch. 10). A variety of commercially available kits may also be used to facilitate expression and purification of fusion proteins.
In a further embodiment of the invention, synthesis of radiolabeled NSPRT may be achieved in vitro using the TNT rabbit reticulocyte lysate or wheat germ extract system (Promega). These systems couple transcription and translation of protein-coding sequences operably associated with the T7, T3, or SP6 promoters. Translation takes place in the presence of a radiolabeled amino acid precursor, for example. 35S-methionine.
NSPRT of the present invention or fragments thereof may be used to screen for compounds that specifically bind to NSPRT. At least one and up to a plurality of test compounds may be screened for specific binding to NSPRT Examples of test compounds include antibodies, ohgonucleotides, proteins (e.g., receptors), or small molecules
In one embodiment, the compound thus identified is closely related to the natural hgand of NSPRT, e.g., a hgand or fragment thereof, a natural substrate, a structural or functional mimetic, or a natural binding partner. (See, Cohgan, J.E. et al. (1991) Cuπent Protocols in Immunology 1(2) Chapter 5 ) Similarly, the compound can be closely related to the natural receptor to which NSPRT binds, or to at least a fragment of the receptor, e.g., the hgand binding site. In either case, the compound can be rationally designed using known techmques. In one embodiment, screemng for these compounds involves producing appropπate cells which express NSPRT, either as a secreted protein or on the cell membrane Prefeπed cells include cells from mammals, yeast, Drosophila, or E_ co . Cells expressing NSPRT or cell membrane fractions which contain NSPRT are then contacted with a test compound and binding, stimulation, or inhibition of activity of either NSPRT or the compound is analyzed.
An assay may simply test binding of a test compound to the polypeptide, wherein binding is detected by a fluorophore, radioisotope, enzyme conjugate, or other detectable label. For example, the assay may compπse the steps of combimng at least one test compound with NSPRT, either in solution or affixed to a solid support, and detecting the binding of NSPRT to the compound. Alternatively, the assay may detect or measure binding of a test compound in the presence of a labeled competitor. Additionally, the assay may be caπied out using cell-free preparations, chemical libraπes, or natural product mixtures, and the test compound(s) may be free in solution or affixed to a solid support
NSPRT of the present invention or fragments thereof may be used to screen for compounds that modulate the activity of NSPRT Such compounds may include agonists, antagonists, or partial or inverse agomsts. In one embodiment, an assay is performed under conditions permissive for NSPRT activity, wherein NSPRT is combined with at least one test compound, and the activity ot NSPRT in the presence of a test compound is compared with the activity of NSPRT in the absence of the test compound. A change in the activity of NSPRT in the presence of the test compound is indicative of a compound that modulates the activity of NSPRT Alternatively, a test compound is combined with an in vitro or cell-free system comprising NSPRT under conditions suitable for NSPRT activity, and the assay is performed In either of these assays, a test compound which modulates the activity of NSPRT may do so indirectly and need not come in direct contact with the test compound At least one and up to a plurality of test compounds may be screened
In another embodiment, polynucleotides encoding NSPRT or their mammalian homologs may be "knocked out" in an ammal model system using homologous recombination in embryonic stem (ES) cells Such techmques are well known in the art and are useful for the generation ot ammal
3: models of human disease. (See, e.g., U.S. Patent No. 5,175,383 and U.S. Patent No. 5,767,337.) For example, mouse ES cells, such as the mouse 129/SvJ cell line, are deπved from the early mouse embryo and grown in culture. The ES cells are transformed with a vector contaimng the gene of interest disrupted by a marker gene, e.g., the neomycin phosphotransferase gene (neo; Capecchi, M.R. (1989) Science 244:1288-1292). The vector integrates into the coπesponding region of the host genome by homologous recombination. Alternatively, homologous recombination takes place using the Cre-loxP system to knockout a gene of interest in a tissue- or developmental stage-specific manner (Marth, J.D. (1996) Clin. Invest. 97:1999-2002: Wagner, K.U. et al. (1997) Nucleic Acids Res. 25:4323-4330). Transformed ES cells are identified and microinjected into mouse cell blastocysts such as those from the C57BL/6 mouse strain. The blastocysts are surgically transferred to pseudopregnant dams, and the resulting chimeπc progeny are genotyped and bred to produce heterozygous or homozygous strains. Transgenic animals thus generated may be tested with potential therapeutic or toxic agents.
Polynucleotides encoding NSPRT may also be manipulated in vitro in ES cells deπved from human blastocysts. Human ES cells have the potential to differentiate into at least eight separate cell lineages including endoderm, mesoderm, and ectodermal cell types. These cell hneages differentiate into, for example, neural cells, hematopoietic lineages, and cardiomyocytes (Thomson, J.A. et al. (1998) Science 282.1145-1147).
Polynucleotides encoding NSPRT can also be used to create "knockin" humamzed animals (pigs) or transgenic animals (mice or rats) to model human disease. With knockin technology, a region of a polynucleotide encoding NSPRT is injected into ammal ES cells, and the injected sequence integrates into the ammal cell genome. Transformed cells are injected into blastulae, and the blastulae are implanted as described above. Transgemc progeny or inbred lines are studied and treated with potential pharmaceutical agents to obtain information on treatment of a human disease. Alternatively, a mammal inbred to overexpress NSPRT, e g., by secreting NSPRT in its milk, may also serve as a convement source of that protein (Janne, J. et al. (1998) Biotechnol. Annu. Rev. 4:55- 74) THERAPEUTICS
Chemical and structural similarity, e.g., in the context of sequences and motifs, exists between regions of NSPRT and human nervous system-associated proteins. In addition, the expression of NSPRT is closely associated with cancerous, proliferating, inflamed, nervous, reproductive, hematopoietic/immune, urologic, cardiovascular, and gastrointestinal tissue Therefore, NSPRT appears to play a role in neurological, inflammatory, and cell proliferative disorders, including cancer In the treatment of disorders associated with increased NSPRT expression or activity, it is desirable to decrease the expression or acuvity of NSPRT In the treatment of disorders associated with decreased NSPRT expression or activity, it is desirable to increase the expression or activity of NSPRT.
Therefore, in one embodiment, NSPRT or a fragment or derivative thereof may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of NSPRT. Examples of such disorders include, but are not limited to, a neurological disorder such as epilepsy, ischemic cerebrovascular disease, stroke, cerebral neoplasms, Alzheimer's disease, Pick's disease, Huntington s disease, dementia, Parkinson* s disease and other extrapyramidal disorders, amyottophic lateral sclerosis and other motor neuron disorders, progressive neural muscular atrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosis and other demyeUnating diseases, bacterial and viral meningitis, brain abscess, subdural empyema, epidural abscess, suppurative intracranial thrombophlebitis, myelitis and radicuUtis, viral central nervous system disease, prion diseases including kuru, Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker syndrome, fatal famiUal insomnia, nutritional and metaboUc diseases of the nervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinal hemangioblastomatosis, encephalotrigeminal syndrome, mental retardation and other developmental disorders of the central nervous system, cerebral palsy, neuroskeletal disorders, autonomic nervous system disorders, cranial nerve disorders, spinal cord diseases, muscular dystrophy and other neuromuscular disorders, peripheral nervous system disorders, dermatomyositis and polymyositis, inherited, metaboUc, endocrine, and toxic myopathies, myasthenia gravis, periodic paralysis, mental disorders including mood, anxiety, and schizophrenic disorders, seasonal affective disorder (SAD), akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia, dystonias, paranoid psychoses, postheφetic neuralgia, Tourette's disorder, progressive supranuclear palsy, corticobasal degeneration, and familial frontotemporal dementia; an inflammatory disorder such as acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyendocrinopathy- candidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins. erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves* disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis. Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren"s syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic puφura, ulcerative colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and extracoφoreal circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, and trauma: and a cell proliferative disorder such as actinic keratosis, arteπosclerosis, atherosclerosis, bursitis, ciπhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuπa, polycythemia vera, psoπasis, pπmary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia. gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, perns, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus
In another embodiment, a vector capable of expressing NSPRT or a fragment or derivative thereof may be admimstered to a subject to treat or prevent a disorder associated with decreased expression or activity of NSPRT including, but not Umited to, those described above
In a further embodiment, a pharmaceutical composition comprising a substantially purified NSPRT in conjunction with a suitable pharmaceutical carrier may be admimstered to a subject to treat or prevent a disorder associated with decreased expression or activity of NSPRT including, but not Umited to, those provided above In still another embodiment, an agomst which modulates the activity of NSPRT may be admimstered to a subject to treat or prevent a disorder associated with decreased expression or activity of NSPRT including, but not limited to, those listed above
In a further embodiment, an antagonist of NSPRT may be admimstered to a subject to treat or prevent a disorder associated with increased expression or activity of NSPRT Examples of such disorders include, but are not limited to, those neurological, inflammatory, and cell proliferative disorders, including cancer, described above In one aspect, an antibody which specifically binds NSPRT may be used directly as an antagonist or indirectly as a targeting or dehvery mechanism for bringing a pharmaceutical agent to cells or tissues which express NSPRT
In an additional embodiment, a vector expressing the complement of the polynucleotide encoding NSPRT may be admimstered to a subject to treat or prevent a disorder associated with increased expression or activity of NSPRT including, but not limited to, those described above
In other embodiments, any of the proteins, antagomsts, antibodies, ago sts, complementary sequences, or vectors of the invention may be admimstered in combination with other appropriate therapeutic agents Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skill in the art, according to conventional pharmaceutical principles The combination of therapeutic agents may act synergistically to effect the treatment or prevention of the various disorders described above Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent thus reducing the potential for adverse side effects
An antagomst of NSPRT may be produced using methods which are generally known in the art In particular, purified NSPRT mav be used to produce antibodies or to screen libraries ot pharmaceutical agents to identify those which specifically bind NSPRT. Antibodies to NSPRT may also be generated using methods that are well known in the art. Such antibodies may include, but are not Umited to, polyclonal, monoclonal, chimeπc, and single chain antibodies, Fab fragments, and fragments produced by a Fab expression Ubrary. Neutralizing antibodies (i.e., those which inhibit dimer formation) are generally prefeπed for therapeutic use.
For the production of antibodies, various hosts including goats, rabbits, rats, mice, humans, and others may be immunized by injection with NSPRT or with any fragment or ohgopeptide thereof which has immunogemc properties. Depending on the host species, various adjuvants may be used to increase immunological response. Such adjuvants include, but are not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluromc polyols, polyamons, peptides, oil emulsions, KLH, and dimtrophenol. Among adjuvants used in humans, BCG (bacilli Calmette-Gueπn) and Corvnebacteπum parvum are especially preferable.
It is prefeπed that the ohgopeptides, peptides, or fragments used to induce antibodies to NSPRT have an amino acid sequence consisting of at least about 5 amino acids, and generally will consist of at least about 10 amino acids. It is also preferable that these ohgopeptides, peptides, or fragments are identical to a portion of the am o acid sequence of the natural protein. Short stretches of NSPRT amino acids may be fused with those of another protein, such as KLH, and antibodies to the chimeπc molecule may be produced.
Monoclonal antibodies to NSPRT may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not Umited to, the hybπdoma technique, the human B-cell hybπdoma technique, and the EBV-hybπdoma technique. (See, e.g., Kohler, G. et al. (1975) Nature 256.495-497; Kozbor, D. et al. (1985) J. Immunol. Methods 81 31-42, Cote, R.J et al. (1983) Proc. Natl. Acad. Sci. USA 80.2026-2030; and Cole, S.P. et al. (1984) Mol. Cell Biol. 62.109-120.) In addition, techmques developed for the production of "chimeπc antibodies."' such as the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropπate antigen specificity and biological activity, can be used. (See, e.g., Moπison, S.L. et al. (1984) Proc Natl. Acad. Sci. USA 81.6851-6855: Neuberger, M.S. et al. (1984) Nature 312.604-608: and Takeda, S. et al. (1985) Nature 314452-454 ) Alternatively, techmques described for the production of single chain antibodies may be adapted, using methods known in the art, to produce NSPRT-specific single chain antibodies Antibodies with related specificity, but of distinct ldiotypic composition, may be generated by chain shuffling from random combinatorial immunoglobulin libraries (See. e.g., Burton. D R (1991) Proc. Natl. Acad. Sci. USA 88 10134-10137 )
Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobuUn libraries or panels of highly specific binding reagents as disclosed in the Uterature. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. USA 86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299.)
Antibody fragments which contain specific binding sites for NSPRT may also be generated. For example, such fragments include, but are not Umited to, F(ab")2 fragments produced by pepsin digestion of the antibody molecule and Fab fragments generated by reducing the disulfide bridges of the F(ab*)2 fragments. Alternatively, Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity. (See, e.g., Huse, W.D. et al. (1989) Science 246:1275-1281.) Various immunoassays may be used for screening to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are well known in the art. Such immunoassays typically involve the measurement of complex formation between NSPRT and its specific antibody. A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering NSPRT epitopes is generally used, but a competitive binding assay may also be employed (Pound, supra).
Various methods such as Scatchard analysis in conjunction with radioimmunoassay techniques may be used to assess the affinity of antibodies for NSPRT. Affinity is expressed as an association constant, IC,, which is defined as the molar concentration of NSPRT-antibody complex divided by the molar concentrations of free antigen and free antibody under equilibrium conditions. The K, determined for a preparation of polyclonal antibodies, which are heterogeneous in their affinities for multiple NSPRT epitopes, represents the average affinity, or avidity, of the antibodies for NSPRT. The K^ determined for a preparation of monoclonal antibodies, which are monospecific for a particular NSPRT epitope, represents a true measure of affinity. High-affinity antibody preparations with K, ranging from about 109 to 1012 L/mole are prefeπed for use in immunoassays in which the NSPRT-antibody complex must withstand rigorous manipulations. Low-affinity antibody preparations with K, ranging from about 106 to 107 L/mole are prefeπed for use in immunopurification and similar procedures which ultimately require dissociation of NSPRT, preferably in active form, from the antibody (Catty, D. (1988) Antibodies. Volume I: A Practical Approach, IRL Press, Washington DC: Liddell, J.E. and A. Cryer (1991) A Practical Guide to Monoclonal Antibodies. John Wiley & Sons, New York NY).
The Uter and avidity of polyclonal antibody preparations may be further evaluated to determine the quality and suitability of such preparations for certain downstream applications. For example, a polyclonal antibody preparation containing at least 1-2 mg specific antibody/ml, preferably 5-10 mg specific antibody/ml, is generally employed in procedures requiring precipitation of NSPRT-antibody complexes. Procedures for evaluating antibody specificity, titer, and avidity, and guideUnes for antibody quaUty and usage in various appUcations, are generally available. (See, e.g., Catty, supra, and CoUgan et al., supra.)
In another embodiment of the invention, the polynucleotides encoding NSPRT, or any fragment or complement thereof, may be used for therapeutic puφoses. In one aspect, modifications of gene expression can be achieved by designing complementary sequences or antisense molecules (DNA, RNA. PNA, or modified oligonucleotides) to the coding or regulatory regions of the gene encoding NSPRT. Such technology is well known in the art, and antisense ohgonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding NSPRT. (See, e.g., Agrawal, S., ed. (1996) Antisense Therapeutics, Humana Press Inc., Totawa NJ.) In therapeutic use, any gene dehvery system suitable for introduction of the antisense sequences into appropriate target cells can be used. Antisense sequences can be delivered intracellularly in the form of an expression plasmid which, upon transcription, produces a sequence complementary to at least a portion of the cellular sequence encoding the target protein. (See, e.g., Slater, J.E. et al. (1998) J. Allergy Clin. Immunol. 102(3):469-475; and Scanlon, K.J. et al. (1995) 9(13): 1288- 1296.) Antisense sequences can also be introduced intracellularly through the use of viral vectors, such as retrovirus and adeno-associated virus vectors. (See, e.g., Miller, A.D. (1990) Blood 76:271; Ausubel, supra; Uckert, W. and W. Walther (1994) Pharmacol. Ther. 63(3):323-347.) Other gene dehvery mechanisms include liposome-derived systems, artificial viral envelopes, and other systems known in the art. (See, e.g., Rossi, J.J. (1995) Br. Med. Bull. 51(l):217-225; Boado, R.J. et al. (1998) J. Pharm. Sci. 87(11):1308-1315; and Morris, M.C. et al. (1997) Nucleic Acids Res. 25(14):2730-2736.)
In another embodiment of the invention, polynucleotides encoding NSPRT may be used for somatic or germhne gene therapy. Gene therapy may be performed to (i) coπeet a genetic deficiency (e.g., in the cases of severe combined immunodeficiency (SCID)-Xl disease characterized by X-linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672), severe combined immunodeficiency syndrome associated with an inherited adenosine deaminase (ADA) deficiency (Blaese, R.M. et al. (1995) Science 270:475-480; Bordignon, C. et al. (1995) Science 270:470-475), cystic fibrosis (Zabner, J. et al. (1993) Cell 75:207-216; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:643-666; Crystal, R.G. et al. (1995) Hum. Gene Therapy 6:667-703), thalassamias, famiUal hypercholesterolemia. and hemophilia resulting from Factor VIII or Factor IX deficiencies (Crystal, R.G. (1995) Science 270:404-410; Verma, I.M. and Somia, N. (1997) Nature 389:239-242)), (ii) express a conditionally lethal gene product (e.g., in the case of cancers which result from unregulated cell proUferation), or (iii) express a protein which affords protection against intracellular parasites (e.g., against human retroviruses. such as human immunodeficiency virus (HIV) (Baltimore. D. (1988) Nature 335:395-396; Poeschla, E. et al. (1996) Proc. Natl. Acad. Sci. USA. 93:11395-11399), hepatitis B or C virus (HBV, HCV); fungal parasites, such as Candida albicans and Paracoccidioides brasiliensis; and protozoan parasites such as Plasmodium falciparum and Trypanosoma cruzi). In the case where a genetic deficiency in NSPRT expression or regulation causes disease, the expression of NSPRT from an appropriate population of transduced cells may alleviate the clinical manifestations caused by the genetic deficiency.
In a further embodiment of the invention, diseases or disorders caused by deficiencies in NSPRT are treated by constructing mammaUan expression vectors encoding NSPRT and introducing these vectors by mechanical means into NSPRT-deficient cells. Mechanical transfer technologies for use with cells in vivo or ex vitro include (i) direct DNA microinjection into individual cells, (ii) balUstic gold particle delivery, (iii) liposome-mediated transfection, (iv) receptor-mediated gene transfer, and (v) the use of DNA transposons (Morgan, R.A. and W.F. Anderson (1993) Annu. Rev. Biochem. 62:191- 217; Ivies, Z. (1997) Cell 91 :501-510; Boulay, J-L. and H. Recipon (1998) Cuπ. Opin. Biotechnol. 9:445-450). Expression vectors that may be effective for the expression of NSPRT include, but are not limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX vectors (Invitrogen, Carlsbad CA), PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La Jolla CA), and PTET-OFF, PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto CA). NSPRT may be expressed using (i) a constitutively active promoter, (e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or β-actin genes), (ii) an inducible promoter (e.g., the tetracycline-regulated promoter (Gossen, M. and H. Bujard (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551 : Gossen, M. et al. (1995) Science 268:1766-1769; Rossi, F.M.V. and H.M. Blau (1998) Cuπ. Opin. Biotechnol. 9:451-456), commercially available in the T-REX plasmid (Invitrogen)); the ecdysone-inducible promoter (available in the plasmids PVGRXR and PIND; Invitrogen); the FK506/rapamycin inducible promoter; or the RU486/mifepristone inducible promoter (Rossi, F.M.V. and H.M. Blau, supra)), or (iii) a tissue-specific promoter or the native promoter of the endogenous gene encoding NSPRT from a normal individual.
Commercially available liposome transformation kits (e.g., the PERFECT LIPID TRANSFECTION KIT, available from Invitrogen) allow one with ordinary skill in the art to deliver polynucleotides to target cells in culture and require minimal effort to optimize experimental parameters. In the alternative, transformation is performed using the calcium phosphate method (Graham. F.L. and A.J. Eb (1973) Virology 52:456-467), or by electroporation (Neumann. E. et al. (1982) EMBO J. 1 :841-845). The introduction of DNA to primary cells requires modification of these standardized mammalian transfection protocols. In another embodiment of the invention, diseases or disorders caused by genetic defects with respect to NSPRT expression are treated by constructing a refrovirus vector consisting of (i) the polynucleotide encoding NSPRT under the control of an independent promoter or the refrovirus long terminal repeat (LTR) promoter, (u) appropriate RNA packaging signals, and (in) a Rev-responsive element (RRE) along with additional refrovirus αs-acting RNA sequences and coding sequences required for efficient vector propagation. Refrovirus vectors (e.g., PFB and PFBNEO) are commercially available (Stratagene) and are based on pubhshed data (Riviere, I. et al. (1995) Proc. Natl. Acad. Sci USA 92:6733-6737), incoφorated by reference herein. The vector is propagated in an appropriate vector producing cell line (VPCL) that expresses an envelope gene with a tropism for receptors on the target cells or a promiscuous envelope protein such as VSVg (Armentano, D. et al (1987) J. Virol. 61 :1647-1650; Bender, M.A et al. (1987) J Virol. 61.1639-1646. Adam, M.A. and A.D. Miller (1988) J. Virol. 62.3802-3806. Dull, T. et al. (1998) J Virol 72.8463-8471 , Zufferey, R et al. (1998) J. Virol 72.9873-9880). U.S. Patent Number 5,910,434 to Rigg ("Method for obtaining refrovirus packaging cell Unes producing high transducing efficiency refroviral supernatant") discloses a method for obtaining refrovirus packaging cell lines and is hereby incoφorated by reference
Propagation of refrovirus vectors, transduction of a population of cells (e.g., CD4+ T-cells), and the return of transduced cells to a patient are procedures well known to persons skilled in the art of gene therapy and have been well documented (Ranga, U. et al. (1997) J. Virol. 71 :7020-7029: Bauer, G. et al. (1997) Blood 89:2259-2267: Bonyhadi, M L (1997) J. Virol. 71 :4707-4716, Ranga. U et al (1998) Proc. Natl. Acad. Sci. USA 95:1201-1206: Su, L (1997) Blood 89:2283-2290)
In the alternative, an adenovirus-based gene therapy delivery system is used to deliver polynucleotides encoding NSPRT to cells which have one or more genetic abnormalities with respect to the expression of NSPRT The construction and packaging of adenovirus-based vectors are well known to those with ordinary skill in the art. Replication defective adenovirus vectors have proven to be versatile for importing genes encoding lmmunoregulatory proteins into intact islets in the pancreas (Csete, M.E. et al. (1995) Transplantation 27 263-268) Potentially useful adenoviral vectors are described in U.S. Patent Number 5,707,618 to Armentano ("'Adenovirus vectors for gene therapy"'), hereby incoφorated by reference For adenoviral vectors, see also Antinozzi. P A et al. (1999) Annu Rev. Nutr. 19 511-544. and Verma, I.M and N Somia (1997) Nature 18 389.239-242. both incoφorated by reference herein
In another alternative, a heφes-based. gene therapy delivery system is used to deliver polynucleotides encoding NSPRT to target cells which have one or more genetic abnormalities with respect to the expression of NSPRT The use of heφes simplex virus (HSV)-based vectors may be especially valuable tor introducing NSPRT to cells of the central nervous system, for which HSV has a WO 01/07470 PCT/USOO/l 9837
tropism. The construction and packaging of heφes-based vectors are well known to those with ordinary skill in the art. A replication-competent heφes simplex virus (HSV) type 1 -based vector has been used to deliver a reporter gene to the eyes of primates (Liu, X. et al (1999) Exp. Eye Res.169:385-395). The construction of a HSV-1 virus vector has also been disclosed in detail in U.S Patent Number 5.804,413 to DeLuca ("Heφes simplex virus strains tor gene transfer"'), which is hereby incoφorated by reference. U S. Patent Number 5,804,413 teaches the use of recombinant HSV d92 which consists of a genome contaimng at least one exogenous gene to be transfeπed to a cell under the control of the appropriate promoter for puφoses including human gene therapy. Also taught by this patent are the construction and use of recombinant HSV strains deleted for ICP4, ICP27 and ICP22. For HSV vectors, see also Goms, W.F. et al. (1999) J Virol 73.519-532 and Xu. H. et al. (1994) Dev Biol. 163.152-161, hereby incoφorated by reference. The manipulation of cloned heφesvirus sequences, the generation of recombinant virus following the transfection of multiple plasmids contaimng different segments of the large heφesvirus genomes, the growth and propagation of heφesvirus. and the infection of cells with heφesvirus are techmques well known to those of ordinary skill in the art.
In another alternative, an alphavirus (positive, single-stranded RNA virus) vector is used to deliver polynucleotides encoding NSPRT to target cells. The biology of the prototypic alphavirus, Semliki Forest Virus (SFV), has been studied extensively and gene transfer vectors have been based on the SFV genome (Garoff, H. and K.-J. Li (1998) Cuπ. Opin Biotech. 9 464-469). During alphavirus RNA rephcation. a subgenormc RNA is generated that normally encodes the viral capsid proteins This subgenomic RNA replicates to higher levels than the full-length genomic RNA, resulting in the oveφroduction of capsid proteins relative to the viral proteins with enzymatic activity (e.g , protease and polymerase) Similarly, inserting the coding sequence for NSPRT into the alphavirus genome in place of the capsid-coding region results in the production of a large number of NSPRT-coding RNAs and the synthesis of high levels of NSPRT in vector transduced cells. While alphavirus infection is typically associated with cell lysis within a few days, the ability to establish a persistent infection in hamster normal kidney cells (BHK-21) with a variant of Sindbis virus (SIN) indicates that the lyuc replication of alphaviruses can be altered to suit the needs of the gene therapy application (Dryga, S A et al. (1997) Virology 228 74-83) The wide host range of alphaviruses will allow the introduction of NSPRT into a variety of cell types The specific transduction ot a subset of cells in a population ma> require the sorting of cells prior to transduction The methods ot manipulating infectious cDNA clones of alphaviruses. performing alphavirus cDNA and RNA transtections. and performing alphavirus infections, are well known to those with ordinary skill in the art
Ohgonucleotides derived trom the transcription initiation site, e g . between about positions - 10 and +10 from the start site, may also be employed to inhibit gene expression. Similarly, inhibition can be achieved using triple helix base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the abihty of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been described in the Uterature. (See, e.g., Gee, J.E. et al. (1994) m Huber, B.E. and B.I. Can, Molecular and Immunologic Approaches. Futura PubUshing, Mt. Kisco NY, pp. 163-177 ) A complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to πbosomes.
Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific cleavage of RNA. The mechamsm ot ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage For example, engineered hammerhead motif ribozyme molecules may specifically and efficiently catalyze endonucleolytic cleavage of sequences encoding NSPRT
Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, including the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides, coπesponding to the region of the target gene contaimng the cleavage site, may be evaluated for secondary structural features which may render the ohgonucleotide inoperable. The suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary ohgonucleotides using πbonuclease protection assays.
Complementary πbonucleic acid molecules and ribozymes of the invention may be prepared by any method known in the art for the synthesis of nucleic acid molecules. These include techmques for chemically synthesizing ohgonucleotides such as solid phase phosphoramidite chemical synthesis Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding NSPRT Such DNA sequences may be incoφorated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6. Alternatively, these cDNA constructs that synthesize complementary RNA, constitutively or mducibly, can be introduced into cell lines, cells, or tissues.
RNA molecules may be modified to increase lntracellular stability and half-lite Possible modifications include, but are not limited to, the addition of flanking sequences at the 5 ' and or 3' ends of the molecule, or the use of phosphorothioate or 2' 0-methyl rather than phosphodiesterase linkages within the backbone of the molecule This concept is inherent in the production ot PNAs and can be extended in all of these molecules by the inclusion ot nontraditional bases such as inosine. queosine. and wybutosine, as well as acetyl-. methyl-, thio-. and similarly modified forms ot ademne. cytidine, guanine, thymine, and uridine which are not as easily recognized by endogenous endonucleases. An additional embodiment of the invention encompasses a method for screening for a compound which is effective in altering expression of a polynucleotide encoding NSPRT. Compounds which may be effective in altering expression of a specific polynucleotide may include, but are not limited to, oligonucleotides, antisense oligonucleotides, triple helix-forming ohgonucleotides, transcription factors and other polypeptide transcriptional regulators, and non- macromolecular chemical entities which are capable of interacting with specific polynucleotide sequences. Effective compounds may alter polynucleotide expression by acting as either inhibitors or promoters of polynucleotide expression. Thus, in the treatment of disorders associated with increased NSPRT expression or activity, a compound which specifically inhibits expression of the polynucleotide encoding NSPRT may be therapeutically useful, and in the treament of disorders associated with decreased NSPRT expression or activity, a compound which specifically promotes expression of the polynucleotide encoding NSPRT may be therapeutically useful.
At least one, and up to a plurality, of test compounds may be screened for effectiveness in altering expression of a specific polynucleotide. A test compound may be obtained by any method commonly known in the art, including chemical modification of a compound known to be effective in altering polynucleotide expression; selection from an existing, commercially- avail able or proprietary library of naturally-occurring or non-natural chemical compounds; rational design of a compound based on chemical and or structural properties of the target polynucleotide; and selection from a library of chemical compounds created combinatorially or randomly. A sample comprising a polynucleotide encoding NSPRT is exposed to at least one test compound thus obtained. The sample may comprise, for example, an intact or permeabilized cell, or an in vitro cell-free or reconstituted biochemical system. Alterations in the expression of a polynucleotide encoding NSPRT are assayed by any method commonly known in the art. Typically, the expression of a specific nucleotide is detected by hybridization with a probe having a nucleotide sequence complementary to the sequence of the polynucleotide encoding NSPRT. The amount of hybridization may be quantified, thus forming the basis for a comparison of the expression of the polynucleotide both with and without exposure to one or more test compounds. Detection of a change in the expression of a polynucleotide exposed to a test compound indicates that the test compound is effective in altering the expression of the polynucleotide. A screen for a compound effective in altering expression of a specific polynucleotide can be carried out. for example, using a Schizosaccharomyces pombe gene expression system (Atkins, D. et al. (1999) U.S. Patent No. 5.932,435: Arndt, G.M. et al. (2000) Nucleic Acids Res. 28:E15) or a human cell line such as HeLa cell (Clarke, M.L. et al. (2000) Biochem. Biophys. Res. Commun. 268:8-13). A particular embodiment of the present invention involves screening a combinatorial library of oligonucleotides (such as deoxyribonucleotides, ribonucleotides. peptide nucleic acids, and modified ohgonucleotides) for antisense activity against a specific polynucleotide sequence (Bnuce, T.W. et al. (1997) U.S. Patent No. 5,686,242, Bruice, T.W. et al. (2000) U.S. Patent No. 6,022,691).
Many methods for introducing vectors into cells or tissues are available and equally smtable tor use m vivo, in vitro, and ex vivo For ex vivo therapy, vectors may be introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient Dehvery by transfection, by hposome injections, or by polycatiomc amino polymers may be achieved using methods which are well known in the art. (See. e g., Goldman, C K. et al. (1997) Nat Biotechnol. 15-462-466.) Any of the therapeutic methods described above may be applied to any subject in need of such therapy, including, for example, mammals such as humans, dogs, cats, cows, horses, rabbits, and monkeys.
An additional embodiment of the invention relates to the admimstration of a pharmaceutical composition which generally comprises an active ingredient formulated with a pharmaceutically acceptable excipient. Excipients may include, for example, sugars, starches, celluloses, gums, and proteins. Various formulations are commonly known and are thoroughly discussed in the latest edition of Remington's Pharmaceutical Sciences (Maack PubUshing, Easton PA) Such pharmaceutical compositions may consist of NSPRT, antibodies to NSPRT, and mimetics, agomsts. antagomsts, or inhibitors of NSPRT. The pharmaceutical compositions utilized in this invention may be admimstered by any number of routes including, but not limited to, oral, intravenous, intramuscular, lntra-arteπal. lntramedullary. lntrathecal, lntraventπcular, pulmonary, transdermal. subcutaneous, lntrapeπtoneal. intranasal, enteral. topical, sub ngual. or rectal means
Pharmaceutical compositions for pulmonary admimstration may be prepared in liquid or dry powder form. These compositions are generally aerosolized immediately prior to inhalation by the patient. In the case of small molecules (e.g. traditional low molecular weight orgamc drugs), aerosol delivery of fast-acting formulations is well-known in the art. In the case of macromolecules (e.g larger peptides and proteins), recent developments in the field of pulmonary delivery via the alveolar region ot the lung have enabled the practical delivery of drugs such as insulin to blood circulation (see. e.g., Patton, J S et al., U S Patent No 5.997.848) Pulmonary delivery has the advantage of admimstration without needle injection, and obviates the need for potentially toxic penetration enhancers
Pharmaceutical compositions suitable tor use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended puφose The determination of an effective dose is well within the capability of those skilled in the art WO 01/07470 PCT/USOO/l 9837
Specialized forms of pharmaceutical compositions may be prepared for direct intracellular dehvery of macromolecules comprising NSPRT or fragments thereof. For example, Uposome preparations contaimng a cell-impermeable macromolecule may promote cell fusion and intracellular deUvery of the macromolecule. Alternatively, NSPRT or a fragment thereof may be )θined to a short cationic N-terminal portion from the HIV Tat-1 protein. Fusion proteins thus generated have been found to transduce into the cells of all tissues, including the brain, in a mouse model system (Schwarze. S.R. et al. (1999) Science 285.1569-1572)
For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of neoplastic cells, or in ammal models such as mice, rats, rabbits, dogs, monkeys. or pigs An ammal model may also be used to determine the appropriate concentration range and route of admimstration. Such information can then be used to determine useful doses and routes tor admimstration in humans
A therapeutically effective dose refers to that amount ot active ingredient, for example NSPRT or fragments thereof, antibodies of NSPRT, and agomsts, antagomsts or inhibitors of NSPRT, which ameliorates the symptoms or condition. Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or with experimental ammals, such as by calculating the ED50 (the dose therapeutically effective in 50% of the population) or LDS0 (the dose lethal to 50% of the population) statistics. The dose ratio of toxic to therapeutic effects is the therapeutic index, which can be expressed as the ratio. Pharmaceutical compositions which exhibit large therapeutic indices are prefeπed. The data obtained from cell culture assays and ammal studies are used to formulate a range of dosage for human use The dosage contained in such compositions is preferably within a range of circulating concentrations that includes the ED,0 with little or no toxicity The dosage varies within this range depending upon the dosage form employed, the sensitivity of the patient, and the route of admimstration. The exact dosage will be determined by the practitioner, in light of factors related to the subject requiring treatment. Dosage and admimstration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect Factors which may be taken into account include the severity of the disease state, the general health of the subject, the age. weight, and gender of the sub)ect, time and frequency ot admimstration. drug combιnatιon(s). reaction sensitivities, and response to therapy Long-acting pharmaceutical compositions may be admimstered every 3 to 4 days, every week, or biweekly depending on the half-life and clearance rate of the particular formulation
Normal dosage amounts may vary trom about 0 1 μg to 100.000 μg, up to a total dose of about 1 gram, depending upon the route ot admimstration Guidance as to particular dosages and methods ot delivery is provided in the literature and generally available to practitioners in the art Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, dehvery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc. DIAGNOSTICS In another embodiment, antibodies which specifically bind NSPRT may be used for the diagnosis of disorders characterized by expression of NSPRT, or in assays to momtor patients being treated with NSPRT or agomsts. antagomsts, or inhibitors of NSPRT. Antibodies useful for diagnostic puφoses may be prepared in the same manner as described above for therapeutics. Diagnostic assays for NSPRT include methods which utilize the antibody and a label to detect NSPRT in human body fluids or in extracts of cells or tissues. The antibodies may be used with or without modification, and may be labeled by covalent or non-covalent attachment of a reporter molecule. A wide variety of reporter molecules, several of which are described above, are known in the art and may be used.
A variety of protocols tor measuring NSPRT, including ELIS As. RIAs, and FACS, are known in the art and provide a basis for diagnosing altered or abnormal levels of NSPRT expression. Normal or standard values for NSPRT expression are estabhshed by combimng body fluids or cell extracts taken from normal mammalian subjects, for example, human subjects, with antibody to NSPRT under conditions suitable for complex formation. The amount of standard complex formation may be quantitated by various methods, such as photometric means. Quantities of NSPRT expressed in subject, control, and disease samples from biopsied tissues are compared with the standard values Deviation between standard and subject values establishes the parameters for diagnosing disease.
In another embodiment of the invention, the polynucleotides encoding NSPRT may be used for diagnostic puφoses The polynucleotides which may be used include ohgonucleotide sequences, complementary RNA and DNA molecules, and PNAs The polynucleotides may be used to detect and quantify gene expression in biopsied tissues in which expression of NSPRT may be coπelated with disease The diagnostic assay may be used to determine absence, presence, and excess expression of NSPRT, and to momtor regulation of NSPRT levels during therapeutic intervention
In one aspect, hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding NSPRT or closely related molecules may be used to identify nucleic acid sequences which encode NSPRT The specificity of the probe, whether it is made from a highly specific region, e g., the 5 * regulatory region, or from a less specific region, e g., a conserved motif, and the stringency ot the hybridization or amplification will determine whether the probe identifies only naturally occumng sequences encoding NSPRT, allelic variants, or related sequences
Probes may also be used for the detection of related sequences, and may have at least 50% WO 01/07470 PCT/USOO/l 9837
sequence identity to any of the NSPRT encoding sequences. The hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ ID NO:5-8 or from genomic sequences including promoters, enhancers, and introns of the NSPRT gene
Means for producing specific hybridization probes for DNAs encoding NSPRT include the clomng of polynucleotide sequences encoding NSPRT or NSPRT derivatives into vectors for the production ot mRNA probes. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA polymerases and the appropriate labeled nucleotides. Hybridization probes may be labeled by a variety of reporter groups, for example, by radionuchdes such as 32P or 3,S, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin biotin coupling systems, and the like.
Polynucleotide sequences encoding NSPRT may be used for the diagnosis of disorders associated with expression of NSPRT. Examples of such disorders include, but are not limited to, a neurological disorder such as epilepsy, lschemic cerebrovascular disease, stroke, cerebral neoplasms, Alzheimer's disease, Pick's disease, Huntmgton's disease, dementia, Parkinson's disease and other exttapyramidal disorders, amyottophic lateral sclerosis and other motor neuron disorders, progressive neural muscular atrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosis and other demyehnating diseases, bacterial and viral meningitis, brain abscess, subdural empyema, epidural abscess, suppurative lntracranial thrombophlebitis, myelitis and radiculitis, viral central nervous system disease, pπon diseases including kuru, Creutzfeldt-Jakob disease, and Gerstmann-Sttaussler-Scheinker syndrome, fatal familial insomnia, nutritional and metabolic diseases of the nervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinal hemangioblastomatosis, encephalotπgeminal syndrome, mental retardation and other developmental disorders ot the central nervous system, cerebral palsy, neuroskeletal disorders, autonomic nervous system disorders, cranial nerve disorders, spinal cord diseases, muscular dystrophy and other neuromuscular disorders, peripheral nervous system disorders. dermatomyositis and polymyositis, inherited, metabolic, endocrine, and toxic myopathies, myasthema gravis, periodic paralysis, mental disorders including mood, anxiety, and schizophrenic disorders, seasonal affective disorder (SAD), akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia. dystomas, paranoid psychoses, postheφetic neuralgia, Tourette's disorder, progressive supranuclear palsy, corticobasal degeneration, and familial frontotemporal dementia, an inflammatory disorder such as acquired immunodeficiency syndrome (AIDS), Addison" s disease, adult respiratory distress syndrome, allergies, ankylosing spondyhtis. amyloidosis. anemia, asthma, atherosclerosis, autoimmune hemolyϋc anemia, autoimmune thyroiditis. autoimmune polyendocπnopathy- candidiasis-ectodermal dystrophy (APECED). bronchitis, cholecystitis, contact dermatitis. Crohn's disease, atopic dermautis. dermatomyositis. diabetes melhtus. emphysema, episodic lymphopema with lymphocytotoxins. erythroblastosis fetahs. erythema nodosum. atropfuc gastritis. glomerulonephπtis, Goodpasture's syndrome, gout. Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, lrπtable bowel syndrome, multiple sclerosis, myasthema gravis, myocardial or peπcardial inflammation, osteoarthπtis, osteoporosis, pancreatitis, polymyositis, psoπasis. Reiter's syndrome, rheumatoid arthπtis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopemc puφura, ulcerative colitis, uveius, Werner syndrome, complications of cancer, hemodialysis. and extracoφoreal circulation, viral, bacteπal, fungal, parasitic, protozoal, and helminthic infections, and trauma; and a cell proliferative disorder such as actinic keratosis, arteπosclerosis, atherosclerosis, bursitis, ciπhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuπa. polycythemia vera, psoπasis, pnmary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers ot the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, perns, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus . The polynucleotide sequences encoding NSPRT may be used in Southern or northern analysis, dot blot, or other membrane-based technologies: in PCR technologies; m dipstick, p , and multiformat ELISA-hke assays: and in microaπays utilizing fluids or tissues from patients to detect altered NSPRT expression. Such qualitative or quantitative methods are well known in the art.
In a particular aspect, the nucleotide sequences encoding NSPRT may be useful in assays that detect the presence of associated disorders, particularly those mentioned above. The nucleotide sequences encoding NSPRT may be labeled by standard methods and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes After a suitable incubation period, the sample is washed and the signal is quantified and compared with a standard value. If the amount of signal in the patient sample is significantly altered in comparison to a control sample then the presence of altered levels of nucleotide sequences encoding NSPRT in the sample indicates the presence of the associated disorder. Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in ammal studies, in clinical trials, or to momtor the treatment of an individual patient
In order to provide a basis for the diagnosis of a disorder associated with expression of NSPRT. a normal or standard profile tor expression is established This may be accomplished by combimng body fluids or cell extracts taken from normal sub|ects. either ammal or human, with a sequence, or a fragment thereof, encoding NSPRT. under conditions suitable for hybridization or amplification Standard hybridization may be quanufied by comparing the values obtained trom normal subjects with values from an experiment in which a known amount of a substantially purified polynucleotide is used Standard values obtained in this manner may be compared with values obtained WO 01/07470 PCT/USOO/l 9837
from samples from patients who are symptomatic for a disorder Deviation from standard values is used to estabhsh the presence of a disorder
Once the presence of a disorder is established and a treatment protocol is initiated hybridization assays may be repeated on a regular basis to determine if the level of expression in the patient begins to approximate that which is observed in the normal subject The results obtained from successive assays may be used to show the efficacy ot treatment over a period ranging from several days to months
With respect to cancer, the presence of an abnormal amount of transcript (either under- or overexpressed) in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual chmcal symptoms A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer
Additional diagnostic uses for ohgonucleotides designed from the sequences encoding NSPRT may involve the use of PCR These ohgomers may be chemically synthesized, generated enzymaticall> or produced in vitro Ohgomers will preferably contain a fragment ot a polynucleotide encoding NSPRT, or a fragment of a polynucleotide complementary to the polynucleotide encoding NSPRT, and will be employed under optimized conditions for identification of a specific gene or condition Ohgomers may also be employed under less stringent conditions for detection or quantification of closely related DNA or RNA sequences
In a particular aspect, oligonucleotide primers derived from the polynucleotide sequences encoding NSPRT may be used to detect single nucleotide polymoφhisms (SNPs) SNPs are substitutions, insertions and deletions that are a frequent cause of inherited or acquired genetic disease in humans Methods of SNP detection include, but are not limited to, single-stranded conformation polymoφhism (SSCP) and fluorescent SSCP (fSSCP) methods In SSCP, oligonucleotide primers derived from the polynucleotide sequences encoding NSPRT are used to amplify DNA using the polymerase chain reaction (PCR) The DNA may be derived, for example, from diseased or normal tissue, biopsy samples, bodily fluids, and the like SNPs in the DNA cause differences in the secondary and tertiary structures of PCR products in single-stranded form, and these differences are detectable using gel electrophoresis in non-denaturing gels In fSCCP the o gonucleotide primers are fluorescently labeled which allows detection of the amphmers in high-throughput equipment such as DNA sequencing machines Additionally, sequence database analysis methods, termed in sihco SNP (isSNP), are capable ot identifying polymorphisms by comparing the sequence of individual overlapping DNA fragments which assemble into a common consensus sequence These computer- based methods filter out sequence variations due to laboratory preparation of DNA and sequencing errors using statistical models and automated analyses of DNA sequence chromatograms. In the alternative, SNPs may be detected and characterized by mass spectrometry using, for example, the high throughput MASSARRAY system (Sequenom, Inc., San Diego CA) Methods which may also be used to quantify the expression of NSPRT include radiolabehng or biotinylating nucleotides, coamplification of a control nucleic acid, and inteφolating results from standard curves (See, e.g , Melby, P C et al. (1993) J. Immunol Methods 159 235-244, Duplaa, C et al (1993) Anal. Biochem. 212.229-236 ) The speed of quantitation of multiple samples may be accelerated by running the assay in a high-throughput format where the ohgomer or polynucleotide of interest is presented in various dilutions and a specfrophotometπc or coloπmetπc response gives rapid quantitation
In further embodiments, ohgonucleotides or longer fragments derived from any of the polynucleotide sequences described herein may be used as elements on a microaπay The microarray can be used in transcript imaging techmques which momtor the relative expression levels of large numbers of genes simultaneously as described in Seilhamer, J.J. et al., "Comparative Gene Transcπpt Analysis," U.S. Patent No 5,840,484, incoφorated herein by reference. The microaπay may also be used to identify genetic variants, mutations, and polymoφhisms This information may be used to determine gene function, to understand the genetic basis of a disorder, to diagnose a disorder, to momtor progression/regression of disease as a function of gene expression, and to develop and momtor the activities of therapeutic agents in the treatment of disease. In particular, this information may be used to develop a pharmacogenomic profile of a patient in order to select the most appropriate and effective treatment regimen for that patient For example, therapeutic agents which are highly effective and display the fewest side effects may be selected for a patient based on his/her pharmacogenomic profile In another embodiment, antibodies specific tor NSPRT, or NSPRT or fragments thereof may be used as elements on a microaπay The microaπay may be used to momtor or measure protein- protein interactions, drug-target interactions, and gene expression profiles, as described above.
A particular embodiment relates to the use of the polynucleotides of the present invention to generate a transcript image of a tissue or cell type A transcript image represents the global pattern of gene expression by a particular tissue or cell type Global gene expression patterns are analyzed by quantifying the number ot expressed genes and their relative abundance under given conditions and at a given time (See Seilhamer et al . '"Comparative Gene Transcript Analysis. * U S Patent Number 5,840,484. expressly incoφorated by reference herein ) Thus a transcript image may be generated by hybridizing the polynucleotides of the present invention or their complements to the totality of transcripts or reverse transcripts of a particular tissue or cell type In one embodiment, the hybridization takes place in high-throughput format, wherein the polynucleotides of the present invenuon or their complements comprise a subset of a plurality of elements on a microaπay The resultant transcript image would provide a profile of gene activity
Transcript images may be generated using transcripts isolated from tissues, cell lines, biopsies, or other biological samples The transcript image may thus reflect gene expression m vivo, as in the case of a tissue or biopsy sample, or in vitro, as in the case ot a cell line
Transcript images which profile the expression of the polynucleotides of the present invention may also be used in conjunction with in vitro model systems and preclinical evaluation of pharmaceuticals, as well as toxicological testing of industrial and naturally-occurring environmental compounds All compounds induce characteristic gene expression patterns, frequently termed molecular fingeφπnts or toxicant signatures, which are indicative of mechanisms of action and toxicity (Nuwaysir, E F et al (1999) Mol Carcinog 24 153-159 Sterner, S and N L Anderson (2000) Toxicol Lett 112- 113 467-471 expressly incoφorated by reference herein) If a test compound has a signature similar to that of a compound with known toxicity, it is likely to share those toxic properties These fingeφπnts or signatures are most useful and refined when they contain expression information from a large number of genes and gene famihes Ideally, a genome-wide measurement of expression provides the highest quality signature Even genes whose expression is not altered by any tested compounds are important as well, as the levels of expression of these genes are used to normalize the rest of the expression data The normalization procedure is useful for comparison of expression data after treatment with different compounds While the assignment of gene function to elements of a toxicant signature aids in inteφretation of toxicity mechanisms, knowledge of gene function is not necessary for the statistical matching of signatures which leads to prediction of toxicity (See. for example, Press Release 00-02 from the National Institute of Environmental Health Sciences released February 29, 2000, available at http //www niehs mh gov/oc/news/toxchip htm ) Therefore it is important and desirable in toxicological screemng using toxicant signatures to include all expressed gene sequences
In one embodiment, the toxicity ot a test compound is assessed by treating a biological sample contaimng nucleic acids with the test compound Nucleic acids that are expressed in the treated biological sample are hybπdized with one or more probes specific to the polynucleotides ot the present invention so that transcπpt levels coπesponding to the polynucleotides of the present invention mav be quantified The transcπpt levels in the treated biological sample are compared with levels in an untreated biological sample Differences in the transcπpt levels between the two samples are indicative of a toxic response caused by the test compound in the treated sample
Microaπays may be prepared used and analyzed using methods known in the art (See, e g Brennan T M et al (1995) U S Patent No 5 474 796 Schena M et l (1996) Proc Natl Acad Sci WO 01/07470 PCT/USOO/l 9837
USA 93.10614-10619; Baldeschweiler et al. (1995) PCT application W095/251116: Shalon. D. et al (1995) PCT appUcation WO95/35505; Heller, R.A. et al. (1997) Proc. Natl. Acad. Sci. USA 94.2150- 2155; and Heller, M.J. et al. (1997) U.S. Patent No. 5,605.662.) Various types of microarrays are well known and thoroughly described in DNA Microaπavs A Practical Approach. M. Schena. ed. (1999) Oxford Umversity Press, London, hereby expressly incoφorated by reference
In another embodiment of the mvention, nucleic acid sequences encoding NSPRT may be used to generate hybridization probes useful in mapping the naturally occumng genomic sequence Either coding or noncoding sequences may be used, and in some instances, noncoding sequences may be preferable over coding sequences. For example, conservation of a coding sequence among members of a multi-gene family may potentially cause undesired cross hybπdization duπng chromosomal mapping. The sequences may be mapped to a particular chromosome, to a specific region of a chromosome, or to artificial chromosome constructions, e.g., human artificial chromosomes (HACs). yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial PI constructions, or single chromosome cDNA libraries. (See, e.g., Harrington, J.J. et al. (1997) Nat. Genet. 15.345-355; Price, CM. (1993) Blood Rev. 7.127-134, and Trask, B.J. (1991) Trends Genet. 7:149-154.) Once mapped, the nucleic acid sequences of the invention may be used to develop genetic linkage maps, for example, which correlate the inheπtance of a disease state with the inheπtance of a particular chromosome region or restriction fragment length polymoφhism (RFLP) (See. e.g., Lander, E.S. and D. Botstein (1986) Proc. Natl. Acad. Sci. USA 83.7353-7357.) Fluorescent in situ hybridization (FISH) may be coπelated with other physical and genetic map data. (See, e.g., Heinz- Ulπch, et al (1995) in Meyers, supra, pp 965-968 ) Examples ot genetic map data can be found in various scientific journals or at the Online Mendehan Inheritance in Man (OMIM) World Wide Web site Coπelation between the location of the gene encoding NSPRT on a physical map and a specific disorder, or a predisposition to a specific disorder, may help define the region of DNA associated with that disorder and thus may further positional clomng efforts
In si hybridization of chromosomal preparations and physical mapping techmques. such as linkage analysis using established chromosomal markers, may be used for extending genetic maps Often the placement of a gene on the chromosome of another mammalian species, such as mouse, ma\ reveal associated markers even if the exact chromosomal locus is not known. This inf ormauon is valuable to investigators searching for disease genes using positional clomng or other gene discovery techmques Once the gene or genes responsible for a disease or syndrome have been crudeh localized by genetic linkage to a particular genomic region, e g . ataxia-telangiectasia to 1 lq22-23. any sequences mapping to that area may represent associated or regulatory genes tor further investigation (See. e g . Gatti, R A et al (1988) Nature 336 577-580 ) The nucleotide sequence of the instant invenuon mav also be used to detect differences in the chromosomal location due to translocation. inversion, etc . among normal, caπier, or affected individuals
In another embodiment of the invention, NSPRT, its catalytic or immunogemc fragments, or ohgopeptides thereof can be used for screemng libraries of compounds in any of a variety of drug screemng techniques The tragment employed in such screemng may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly The formation of binding complexes between NSPRT and the agent being tested may be measured
Another technique for drug screemng provides for high throughput screemng of compounds having suitable binding affinity to the protein of interest (See, e g , Geysen, et al (1984) PCT application WO84/03564 ) In this method, large numbers ot different small test compounds are synthesized on a solid substtate The test compounds are reacted with NSPRT, or fragments thereof, and washed Bound NSPRT is then detected by methods well known in the art Purified NSPRT can also be coated directly onto plates for use in the aforementioned drug screemng techmques Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support In another embodiment, one may use competitive drug screemng assays in which neutralizing antibodies capable of binding NSPRT specifically compete with a test compound for binding NSPRT In this manner, antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants with NSPRT
In additional embodiments, the nucleotide sequences which encode NSPRT may be used in any molecular biology techmques that have yet to be developed, provided the new techmques rely on properties of nucleotide sequences that are cuπently known, including, but not limited to, such properties as the triplet genetic code and specific base pair interactions
Without further elaboration, it is believed that one skilled in the art can. using the preceding descπption, utilize the present invention to its fullest extent The following prefeπed specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever
Without further elaboration, it is believed that one skilled in the art can. using the preceding descπption, utilize the present invention to its fullest extent The following preteπed specific embodiments are, therefore, to be construed as merely illustrative and not limitative of the remainder of the disclosure in any way whatsoever
The disclosures of all patents, applications, and publications mentioned above and below in particular U S Ser No 60/144 994. are hereby expressly incoφorated by reference
EXAMPLES I. Construction of cDNA Libraπes RNA was purchased from Clontech or isolated from tissues described in Table 4 Some tissues were homogenized and lysed in guanidinium lsothiocyanate, while others were homogenized and lysed in phenol or in a suitable mixture of denaturants, such as TRIZOL (Life Technologies), a monophasic solution of phenol and guamdine lsothiocyanate The resulting lysates were centπfuged over CsCl cushions or extracted with chloroform RNA was precipitated from the lysates with either isopropanol or sodium acetate and ethanol, or by other routine methods
Phenol extraction and precipitation of RNA were repeated as necessary to increase RNA purity In some cases. RNA was treated with DNase For most libraries, poly(A+) RNA was isolated using o go d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex particles (QIAGEN, Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN) Alternatively, RNA was isolated directly from tissue lysates using other RNA isolation kits, e g . the POLY(A)PURE mRNA purification kit (Ambion, Austin TX)
In some cases, Sfratagene was provided with RNA and constructed the coπesponding cDNA libraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed with the UNIZAP vector system (Sttatagene) or SUPERSCRIPT plasmid system (Life Technologies), using the recommended procedures or similar methods known in the art (See, e g , Ausubel, 1997, supra, umts 5 1-6.6 ) Reverse transcription was initiated using ohgo d(T) or random primers Synthetic oligonucleotide adapters were hgated to double stranded cDNA, and the cDNA was digested with the appropriate restriction enzyme or enzymes For most libraries, the cDNA was size-selected (300-1000 bp) using SEPHACRYL SI 000, SEPHAROSE CL2B, or SEPH AROSE CL4B column chromatography (Amersham Pharmacia Biotech) or preparative agarose gel electrophoresis cDNAs were hgated into compatible restriction enzyme sites of the polylinker of a suitable plasmid, e g , PBLUESCRIPT plasmid (Sttatagene), PSPORT1 plasmid (Life Technologies), pcDNA2 1 plasmid (Invitrogen, Carlsbad CA), or pINCY plasmid (Incyte Genomics Palo Alto CA) Recombinant plasmids were transformed into competent E coli cells including XL 1 -Blue, XLl-BlueMRF. or SOLR from Sttatagene or DH5α, DH10B, or ElectroMAX DH10B from Life Technologies II. Isolation of cDNA Clones
Plasmids obtained as described in Example I were recovered from host cells by in ivo excision using the UNIZAP vector system (Sttatagene) or by cell lysis Plasmids were purified using at least one of the following a Magic or WIZARD Minipreps DNA purification system (Promega). an AGTC Mimprep purification kit (Edge Biosystems, Gaithersburg MD), and QIAWELL 8 Plasmid. QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the R E A L PREP 96 plasmid purification kit from QIAGEN Following precipitation, plasmids were resuspended in 0 1 ml of distilled water and stored, with or without lyophihzation, at 4°C
Alternatively plasmid DNA was amplified trom host cell lysates using direct link PCR in a WO 01/07470 PCT/USOO/l 9837
high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216.1-14). Host cell lysis and thermal cychng steps were carried out in a single reaction mixture. Samples were processed and stored in 384- well plates, and the concentration of amplified plasmid DNA was quantified fluorometπcally using PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN II fluorescence scanner (Labsystems Oy, Helsinki, Finland). III. Sequencing and Analysis
Incyte cDNA recovered in plasmids as descπbed in Example II were sequenced as follows. Sequencing reactions were processed using standard methods or high-throughput instrumentation such as the ABI CATALYST 800 (PE Biosystems) thermal cycler or the PTC-200 thermal cycler (MJ Research) in conjunction with the HYDRA microdispenser (Robbins Scientific) or the MICROLAB 2200 (Hamilton) liquid transfer system. cDNA sequencing reactions were prepared using reagents provided by Amersham Pharmacia Biotech or supplied in ABI sequencing kits such as the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (PE Biosystems) Electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides were earned out using the MEGABACE 1000 DNA sequencing system (Molecular Dynamics); the ABI PRISM 373 or 377 sequencing system (PE Biosystems) in conjunction with standard ABI protocols and base calling software, or other sequence analysis systems known in the art. Reading frames within the cDNA sequences were identified using standard methods (reviewed in Ausubel, 1997, supra, umt 7.7). Some of the cDNA sequences were selected for extension using the techmques disclosed in Example V. The polynucleotide sequences derived from cDNA sequencing were assembled and analyzed using a combination of software programs which utilize algorithms well known to those skilled in the art. Table 5 summarizes the tools, programs, and algorithms used and provides applicable descriptions, references, and threshold parameters. The first column of Table 5 shows the tools, programs, and algorithms used, the second column provides brief descriptions thereof, the third column presents appropriate references, all of which are incoφorated by reference herein in their entirety, and the fourth column presents, where applicable, the scores, probability values, and other parameters used to evaluate the strength of a match between two sequences (the higher the score, the greater the homology between two sequences) Sequences were analyzed using MACDNASIS PRO software (Hitachi Software Engineering, South San Francisco CA) and LASERGENE software (DNASTAR). Polynucleotide and polypeptide sequence alignments were generated using the default parameters specified by the clustal algorithm as incoφorated into the MEGALIGN multisequence alignment program (DNASTAR), which also calculates the percent identity between aligned sequences
The polynucleotide sequences were validated by removing vector, linker, and polyA sequences and by masking ambiguous bases, using algorithms and programs based on BLAST, dynamic
:>:> programing, and dinucleotide nearest neighbor analysis The sequences were then queried against a selection of public databases such as the GenBank primate, rodent, mammahan, vertebrate, and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM, and PFAM to acquire annotation using programs based on BLAST, FASTA, and BLIMPS The sequences were assembled into full length polynucleotide sequences using programs based on Phred, Phrap, and Consed, and were screened for open reading frames using programs based on GeneMark. BLAST, and FASTA The full length polynucleotide sequences were translated to derive the coπesponding full length amino acid sequences, and these full length sequences were subsequently analyzed by querying against databases such as the GenBank databases (described above), SwissProt, BLOCKS, PRINTS, DOMO, PRODOM. Prosite, and Hidden Markov Model (HMM)-based protein family databases such as PFAM HMM is a probabilistic approach which analyzes consensus primary structures of gene families (See. e g , Eddy, S R (1996) Cuπ Opin Struct Biol 6 361-365 )
The programs described above for the assembly and analysis of full length polynucleotide and ammo acid sequences were also used to identify polynucleotide sequence fragments from SEQ ID NO 5-8 Fragments from about 20 to about 4000 nucleotides which are useful in hybridization and amplification technologies were described in The Invention section above IV. Analysis of Polynucleotide Expression
Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled nucleotide sequence to a membrane on which RNAs from a particular cell type or tissue have been bound (See, e g , Sambrook. supra, ch 7 Ausubel 1995, supra, ch 4 and 16 )
Analogous computer techmques applying BLAST were used to search for identical or related molecules in cDNA databases such as GenBank or LIFESEQ (Incyte Genormcs) This analvsis is much faster than multiple membrane-based hybridizations In addition, the sensitivity of the computer search can be modified to determine whether any particular match is categorized as exact or similar The basis of the search is the product score, which is defined as
BLAST Score x Percent Identity
5 x minimum {length(Seq 1) length(Seq 2)}
The product score takes into account both the degree of similarity between two sequences and the length of the sequence match The product score is a normalized value between 0 and 100 and is calculated as follows the BLAST score is multiplied by the percent nucleotide identity and the product is divided by (5 times the length of the shorter of the two sequences) The BLAST score is calculated by assigmng a score of +5 for every base that matches in a high-scoring segment pair (HSP), and -4 for
36 every mismatch Two sequences may share more than one HSP (separated by gaps) If there is more than one HSP, then the pair with the highest BLAST score is used to calculate the product score The product score represents a balance between fractional overlap and quality in a BLAST alignment For example, a product score of 100 is produced only for 100% identity over the entire length of the shorter of the two sequences being compared A product score of 70 is produced either by 100% identity and 70% overlap at one end, or by 88% identity and 100% overlap at the other A product score of 50 is produced either by 100% identity and 50% overlap at one end. or 79% identity and 100% overlap
The results of northern analyses are reported as a percentage distribution of libraries in which the transcript encoding NSPRT occuπed Analysis involved the categorization of cDNA libraries by organ/tissue and disease The organ/tissue categories included cardiovascular, dermatologic, developmental endocrine, gastrointestinal, hematopoietic/immune, musculoskeletal. nervous reproductive, and urologic The disease/condition categories included cancer, inflammation, trauma, cell proliferation, neurological, and pooled For each category, the number of libraries expressing the sequence of interest was counted and divided by the total number of libraries across all categories Percentage values of tissue-specific and disease- or condition-specific expression are reported in Table 3 V. Extension of NSPRT Encoding Polynucleotides
The full length nucleic acid sequences of SEQ ID NO 5-8 were produced by extension of an appropriate fragment of the full length molecule using oligonucleotide primers designed from this fragment One primer was synthesized to initiate 5 " extension of the known fragment, and the other primer, to initiate 3* extension of the known fragment The initial primers were designed using OLIGO 406 software (National Biosciences), or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about 68 °C to about 72°C Any stretch of nucleotides which would result in haiφin structures and primer-primer dimeπzations was avoided
Selected human cDNA libraries were used to extend the sequence If more than one extension was necessary or desired, additional or nested sets of primers were designed
High fidelity amplification was obtained by PCR using methods well known in the art PCR was performed in 96-well plates using the PTC-200 thermal cycler (MJ Research, Inc ) The reaction mix contained DNA template. 200 nmol of each primer, reaction buffer contaimng Mg"+, (NH4)2S04 and β-mercaptoethanol Taq DNA polymerase (Amersham Pharmacia Biotech), ELONGASE enzyme (Life Technologies) and Phi DNA polymerase (Sttatagene) with the following parameters for primer pair PCI A and PCI B Step 1 94°C 3 mm Step 2 94°C 15 sec Step 3 6()°C 1 m Step 4 68°C 2 mm Step 5 Steps 2 3 and 4 repeated 20 times Step 6 68 °C 5 mm Step 7 storage at 4 °C In the alternative, the parameters for primer pair T7 and SK+ were as follows Step 1 94°C, 3 mm, Step 2 94°C, 15 sec, Step 3 57 °C, 1 mm, Step 4 68 °C, 2 mm. Step 5 Steps 2, 3, and 4 repeated 20 times, Step 6 68 °C, 5 mm, Step 7 storage at 4°C
The concentration of DNA in each well was determined by dispensing 100 μl PICOGREEN quantitation reagent (0 25% (v/v) PICOGREEN, Molecular Probes, Eugene OR) dissolved in IX TE and 0 5 μl of undiluted PCR product into each well of an opaque fluoπmeter plate (Corning Costar, Acton MA), allowing the DNA to bind to the reagent The plate was scanned in a Fluoroskan II (Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample and to quantify the concentration of DNA A 5 μl to 10 μl ahquot of the reaction mixture was analyzed by electrophoresis on a 1 % agarose mini-gel to determine which reactions were successful in extending the sequence The extended nucleotides were desalted and concentrated, ttansfeπed to 384-well plates, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison WI), and somcated or sheared prior to rehgation into pUC 18 vector (Amersham Pharmacia Biotech) For shotgun sequencing, the digested nucleotides were separated on low concentration (0 6 to 0 8%) agarose gels, fragments were excised, and agar digested with Agar ACE (Promega) Extended clones were rehgated using T4 hgase (New England Biolabs, Beverly MA) into pUC 18 vector (Amersham Pharmacia Biotech), treated with Pfu DNA polymerase (Sttatagene) to fill-in restriction site overhangs, and ttansfected into competent E coh cells Transformed cells were selected on antibiotic-containing media, and individual colomes were picked and cultured overnight at 37 °C in 384-well plates in LB/2x carb liquid media
The cells were lysed. and DNA was amplified by PCR using Taq DNA polymerase (Amersham Pharmacia Biotech) and Pfu DNA polymerase (Sttatagene) with the following parameters Step 1 94°C, 3 mm. Step 2 94°C, 15 sec. Step 3 60°C, 1 mm Step 4 72°C 2 mm. Step 5 steps 2, 3, and 4 repeated 29 times. Step 6 72°C 5 mm. Step 7 storage at 4°C DNA was quanufied by PICOGREEN reagent (Molecular Probes) as described above Samples with low DNA recoveries were reamplified using the same conditions as described above Samples were diluted with 20% dimethysulfoxide (1 2, v/v), and sequenced using DYENAMIC energy transfer sequencing primers and the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (PE Biosystems) In like manner, the polynucleotide sequences of SEQ ID NO 5-8 are used to obtain 5' regulatory sequences using the procedure above along with ohgonucleotides designed tor such extension, and an appropriate genomic library VI. Labeling and Use of Individual Hybridization Probes
Hybridization probes derived from SEQ ID NO 5-8 are employed to screen cDNAs. genomic WO 01/07470 PCT/USOO/l 9837
DNAs. or mRNAs. Although the labehng of ohgonucleotides, consisting of about 20 base pairs, is specifically described, essentially the same procedure is used with larger nucleotide fragments. Ohgonucleotides are designed using state-of-the-art software such as OLIGO 4.06 software (National Biosciences) and labeled by combimng 50 pmol of each ohgomer, 250 μCi of [γ-3 P] adenosine tπphosphate (Amersham Pharmacia Biotech), and T4 polynucleotide kinase (DuPont NEN, Boston MA). The labeled oligonucleotides are substantially purified using a SEPHADEX G-25 superfine size exclusion dexttan bead column (Amersham Pharmacia Biotech). An aliquot contaimng 107 counts per minute of the labeled probe is used in a typical membrane-based hybridization analysis of human genomic DNA digested with one of the following endonucleases: Ase I, Bgl II, Eco RI. Pst I. Xba I, or Pvu II (DuPont NEN)
The DNA from each digest is fractionated on a 0.7% agarose gel and ttansfeπed to nylon membranes (Nyttan Plus, Schleicher & Schuell, Durham NH). Hybridization is caπied out tor 16 hours at 40°C To remove nonspecific signals, blots are sequentially washed at room temperature under conditions of up to, for example, 0.1 x saline sodium citrate and 0.5% sodium dodecyl sulfate. Hybridization patterns are visualized using autoradiography or an alternative imaging means and compared. VII. Microarrays
The Unkage or synthesis of aπay elements upon a microaπay can be achieved utiUzing photolithography, piezoelectric printing (ink-jet printing, See, e.g., Baldeschweiler, supra), mechanical microspotting technologies, and derivatives thereof The substrate in each of the aforementioned technologies should be umform and sohd with a non-porous surface (Schena (1999), supra) Suggested substtates include silicon, silica, glass slides, glass chips, and silicon wafers Alternatively, a procedure analogous to a dot or slot blot may also be used to aπange and link elements to the surface of a substtate using thermal, UV, chemical, or mechanical bonding procedures. A typical aπay may be produced using available methods and machines well known to those of ordinary skill in the art and may contain any appropriate number of elements (See, e g , Schena, M. et al. (1995) Science 270467-470. Shalon, D et al ( 1996) Genome Res 6 639-645. Marshall, A. and J. Hodgson (1998) Nat Biotechnol 16.27-31 )
Full length cDNAs. Expressed Sequence Tags (ESTs), or fragments or ohgomers thereof may comprise the elements of the microaπay. Fragments or ohgomers suitable for hybridization can be selected using software well known in the art such as LASERGENE software (DNASTAR) The aπay elements are hybridized with polynucleotides in a biological sample The polynucleotides in the biological sample are conjugated to a fluorescent label or other molecular tag for ease oi detection After hybridization, nonhybπdized nucleotides trom the biological sample are removed, and a fluorescence scanner is used to detect hybridization at each aπay element. Alternatively, laser desorbtion and mass specttometty may be used for detection of hybridization. The degree of complementarity and the relative abundance of each polynucleotide which hybridizes to an element on the microaπay may be assessed. In one embodiment, microaπay preparation and usage is described in detail below.
Tissue or Cell Sample Preparation
Total RNA is isolated from tissue samples using the guanidinium thiocyanate method and poly(A)+ RNA is puπfied using the ohgo-(dT) cellulose method. Each poly(A)+ RNA sample is reverse ttanscπbed using MMLV reverse-transcπptase, 0.05 pg/μl olιgo-(dT) primer (21mer), IX first strand buffer, 0 03 umts/μl RNase inhibitor, 500 μM dATP. 500 μM dGTP, 500 μM dTTP. 40 μM dCTP, 40 μM dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Pharmacia Biotech). The reverse transcπption reaction is performed in a 25 ml volume contaimng 200 ng poly(A)+ RNA with GEMB RIGHT kits (Incyte). Specific control poly(A)+ RNAs are synthesized by in vitro transcription from non-coding yeast genomic DNA. After incubation at 37 °C for 2 fir, each reaction sample (one with Cy3 and another with Cy5 labeling) is treated with 2.5 ml of 0.5M sodium hydroxide and incubated for 20 minutes at 85 °C to the stop the reaction and degrade the RNA. Samples are purified using two successive CHROMA SPIN 30 gel filtration spin columns (CLONTECH Laboratories, Inc. (CLONTECH), Palo Alto CA) and after combimng, both reaction samples are ethanol precipitated using 1 ml of glycogen (1 mg/ml), 60 ml sodium acetate, and 300 ml of 100% ethanol. The sample is then dried to completion using a SpeedVAC (Savant Instruments Inc., Holbrook NY) and resuspended in 14 μl 5X SSC/0.2% SDS Microaπav Preparation
Sequences of the present invenuon are used to generate aπay elements Each aπay element is amplified from bacteπal cells contaimng vectors with cloned cDNA inserts PCR amplification uses primers complementary to the vector sequences flanking the cDNA insert Aπay elements are amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a final quantity greater than 5 μg. Amplified array elements are then purified using SEPHACRYL-400 (Amersham Pharmacia Biotech)
Purified aπay elements are immobilized on polymer-coated glass slides Glass microscope slides (Cormng) are cleaned by ultrasound in 0 1 % SDS and acetone, with extensive distilled water washes between and after treatments Glass shdes are etched in 4% hydrofluoric acid (VWR Scientific Products Coφoration (VWR), West Chester PA), washed extensively in distilled water, and coated with 0 05% aminopropyl silane (Sigma) in 95% ethanol Coated slides are cured in a 110°C oven Aπay elements are applied to the coated glass substrate using a procedure described in US Patent No. 5.807,522, incoφorated herein by reference. 1 μl of the aπay element DNA. at an average concentration of 100 ng/μl, is loaded into the open capillary pπnting element by a high-speed robotic apparatus. The apparatus then deposits about 5 nl of aπay element sample per slide.
Microaπays are UV-crosshnked using a STRATALINKER UV-crosshnker (Sttatagene). Microaπays are washed at room temperature once in 0.27c SDS and three times in distilled water. Non-specific binding sites are blocked by incubation of microaπays in 0 2% casein in phosphate buffered saline (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60 °C followed by washes in 0 2% SDS and distilled water as before. Hybndization Hybndization reactions contain 9 μl of sample mixture consisting of 0 2 μg each of Cy3 and
Cy5 labeled cDNA synthesis products in 5X SSC, 0.27c SDS hybndization buffer The sample mixture is heated to 65 °C for 5 minutes and is ahquoted onto the microaπay surface and covered with an 1.8 cm2 covershp. The aπays are ttansfeπed to a wateφroot chamber having a cavity just slightly larger than a microscope slide. The chamber is kept at 100% humidity internally by the addition of 140 μl of 5X SSC in a corner of the chamber. The chamber contaimng the aπays is incubated for about 6.5 hours at 60°C The aπays are washed for 10 mm at 45 °C in a first wash buffer (IX SSC, 0.17c SDS), three times tor 10 minutes each at 45 °C in a second wash buffer (0 IX SSC), and dried. Detection
Reporter-labeled hybridization complexes are detected with a microscope equipped with an Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of generating specttal hnes at 488 nm for excitation ot Cy3 and at 632 nm for excitation ot Cy5 The excitation laser light is focused on the aπay using a 20X microscope objective (Nikon. Inc., Melville NY) The slide contaimng the aπay is placed on a computer-controlled X-Y stage on the microscope and raster- scanned past the objective The 1.8 cm x 1.8 cm array used in the present example is scanned with a resolution of 20 micrometers
In two separate scans, a mixed gas multiline laser excites the two tluorophores sequentially Emitted light is split, based on wavelength, into two photomulϋplier tube detectors (PMT R1477, Hamamatsu Photomcs Systems. Bndgewater NJ) coπesponding to the two tluorophores Appropπate filters positioned between the aπay and the photomultipher tubes are used to filter the signals The emission maxima ot the tluorophores used are 565 nm for Cy3 and 650 nm tor Cy5 Each aπay is typically scanned twice, one scan per fluorophore using the appropriate filters at the laser source, although the apparatus is capable of recording the spectra trom both tluorophores simultaneously The sensitivity ot the scans is typically calibrated using the signal intensity generated by a cDNA control species added to the sample mixture at a known concentration A specific location on the aπay contains a complementary DNA sequence, allowing the intensity ot the signal at that WO 01/07470 PCT/USOO/l 9837
location to be coπelated with a weight ratio of hybπdizing species of 1 100,000 When two samples from different sources (e g , representing test and control cells), each labeled with a different fluorophore. are hybπdized to a single aπay for the puφose of identifying genes that are differentially expressed, the calibration is done by labeling samples of the cahbrating cDNA with the two fluorophores and adding identical amounts of each to the hybndization mixture
The output of the photomultiplier tube is digitized using a 12-bit RTI-835H analog-to-digital (A/D) conversion board (Analog Devices. Inc.. Norwood MA) installed in an IBM-compatible PC computer The digitized data are displayed as an image where the signal intensity is mapped using a linear 20-color transformation to a pseudocolor scale ranging from blue (low signal) to red (high signal) The data is also analyzed quantitatively Where two different fluorophores are excited and measured simultaneously, the data are first coπected for optical crosstalk (due to overlapping emission spectra) between the fluorophores using each fluorophore* s emission spectrum
A grid is supeπmposed over the fluorescence signal image such that the signal from each spot is centered in each element of the gπd The fluorescence signal within each element is then integrated to obtain a numeπcal value coπesponding to the average intensity of the signal The software used for signal analysis is the GEMTOOLS gene expression analysis program (Incyte)
VIII. Complementary Polynucleotides
Sequences complementary to the NSPRT-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occumng NSPRT Although use of ohgonucleotides comprising from about 15 to 30 base pairs is described, essentially the same procedure is used with smaller or with larger sequence fragments Appropriate ohgonucleotides are designed using OLIGO 4 06 software (National Biosciences) and the coding sequence of NSPRT To inhibit transcription, a complementary ohgonucleotide is designed from the most umque 5 " sequence and used to prevent promoter binding to the coding sequence To inhibit translation, a complementary oligonucleotide is designed to prevent πbosomal binding to the NSPRT-encoding transcript
IX. Expression of NSPRT
Expression and purification of NSPRT is achieved using bacterial or virus-based expression systems For expression of NSPRT in bacteria, cDNA is subcloned into an appropriate vector contaimng an antibiotic resistance gene and an inducible promoter that directs high levels of cDNA transcription Examples of such promoters include, but are not limited to. the trp-lac (tac) hybrid promoter and the T5 or T7 bacteriophage promoter in conjunction with the lac operator regulatory element Recombinant vectors are transformed into suitable bacterial hosts, e g . BL21(DE3) Antibiotic resistant bacteria express NSPRT upon induction with isopropyl beta-D- thiogalactopyranoside (IPTG) Expression of NSPRT in eukaryotic cells is achieved by infecting insect or mammalian cell lines with recombinant Autograp uca cahiornica nuclear polyhedrosis virus (AcMNPV), commonly known as baculovirus The nonessential polyhedπn gene of baculovirus is replaced with cDNA encoding NSPRT by either homologous recombination or bacterial-mediated transposition involving transfer plasmid intermediates Viral mfectivity is maintained and the strong polyhedrin promoter drives high levels of cDNA transcription Recombinant baculovirus is used to infect Spodoptera frugiperda (Sf9) insect cells in most cases, or human hepatocytes, in some cases Infection of the latter requires additional genetic modifications to baculovirus (See Engelhard, E K et al (1994) Proc Natl Acad Sci USA 91 3224-3227, Sandig, V et al (1996) Hum Gene Ther 7 1937-1945 )
In most expression systems, NSPRT is synthesized as a fusion protein with, e g , glutathione S- transferase (GST) or a peptide epitope tag. such as FLAG or 6-His, permitting rapid, single-step, affimty-based purification of recombinant fusion protein from crude cell lysates GST, a 26-kιlodalton enzyme from Schistosoma lapomcum. enables the purification of fusion proteins on immobilized glutathione under conditions that maintain protein activity and antigenicity (Amersham Pharmacia Biotech) Following purification, the GST moiety can be proteolytically cleaved from NSPRT at specifically engineered sites FLAG, an 8-amιno acid peptide, enables immunoaffimty purification using commercially available monoclonal and polyclonal anti-FLAG antibodies (Eastman Kodak) 6- His, a stretch of six consecutive histidine residues, enables purification on metal-chelate resins (QIAGEN) Methods for protein expression and purification are discussed in Ausubel (1995, supra, ch 10 and 16) Purified NSPRT obtained by these methods can be used directly in the assays shown in Examples X and XIV
X. Demonstration of NSPRT Activity
NSPRT, or biologically active fragments thereof, are labeled with 121 Bolton-Hunter reagent (See, e g , Bolton et al (1973) Biochem J 133 529 ) Candidate molecules previously arrayed in the wells of a multi-well plate are incubated with the labeled NSPRT, washed, and any wells with labeled NSPRT complex are assayed Data obtained using different concentrations of NSPRT are used to calculate values for the number, affinity, and association of NSPRT with the candidate molecules
XI. Functional Assays
NSPRT function is assessed by expressing the sequences encoding NSPRT at physiologically elevated levels in mammalian cell culture systems cDNA is subcloned into a mammahan expression vector contaimng a strong promoter that drives high levels of cDNA expression Vectors of choice include pCMV SPORT plasmid (Life Technologies) and pCR3 1 plasmid (Invitrogen), both of which contain the cytomegalovirus promoter 5-10 μg ot recombinant vector are transiently transfected into a human cell line lor example, an endothehal or hematopoietic cell line, using either liposome formulations or electroporation 1-2 μg of an additional plasmid contaimng sequences encoding a marker protein are co-ttansfected Expression of a marker protein provides a means to distinguish ttansfected cells from nontransfected cells and is a reliable predictor of cDNA expression from the recombinant vector Marker proteins of choice include, e g . Green Fluorescent Protein (GFP, Clontech), CD64, or a CD64-GFP fusion protein Row cytometry (FCM). an automated, laser optics- based technique, is used to identity ttansfected cells expressing GFP or CD64-GFP and to evaluate the apoptotic state of the cells and other cellular properties FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with cell death These events include changes in nuclear DNA content as measured by staining of DNA with propidium iodide, changes in cell size and granularity as measured by forward light scatter and 90 degree side light scatter, down- regulation of DNA synthesis as measured by decrease in bromodeoxyuπdine uptake, alterations in expression of cell surface and intracellular proteins as measured by reactivity with specific antibodies, and alterations in plasma membrane composition as measured by the binding of fluorescem-conjugated Annex V protein to the cell surface Methods in flow cytometry are discussed in Ormerod. M G (1994) Flow Cytometry, Oxford, New York NY The influence of NSPRT on gene expression can be assessed using highly purified populations of cells ttansfected with sequences encoding NSPRT and either CD64 or CD64-GFP CD64 and CD64-GFP are expressed on the surface of ttansfected cells and bind to conserved regions of human immunoglobulin G (IgG) Transfected cells are efficiently separated from nontransfected cells using magnetic beads coated with either human IgG or antibody against CD64 (DYNAL, Lake Success NY) mRNA can be purified from the cells using methods well known by those of skill in the art Expression of mRNA encoding NSPRT and other genes of interest can be analyzed by northern analysis or microaπay techmques XII. Production of NSPRT Specific Antibodies
NSPRT substantially purified using polyacrylarmde gel electrophoresis (PAGE, see e g , Hamngton, M G (1990) Methods Enzymol 182 488-495), or other purification techmques, is used to immumze rabbits and to produce antibodies using standard protocols
Alternatively, the NSPRT ammo acid sequence is analyzed using LASERGENE software (DNASTAR) to determine regions of high lmmunogenicity, and a coπesponding ohgopeptide is synthesized and used to raise antibodies by means known to those of skill in the art Methods for selection of appropriate epitopes such as those near the C-terminus or in hydrophihc regions are well described in the art (See, e g , Ausubel, 1995, supra ch 11 )
Typically, ohgopeptides of about 15 residues in length are synthesized using an ABI 431 A peptide synthesizer (PE Biosystems) using FMOC chemistry and coupled to KLH (Sigma- Aldrich St Louis MO) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increase immunogemcity. (See, e.g., Ausubel, 1995, supra.) Rabbits are immumzed with the ohgopeptide- KLH complex in complete Freunds adjuvant. Resulting antisera are tested for antipeptide and anti-NSPRT activity by, for example, binding the peptide or NSPRT to a substtate. blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting with radio-iodinated goat anti-rabbit IgG. XIII. Purification of Naturally Occurring NSPRT Using Specific Antibodies
Naturally occurring or recombinant NSPRT is substantially purified by immunoaffimty chromatography using antibodies specific for NSPRT. An immunoaffimty column is constructed by covalently couphng anti-NSPRT antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech) After the coupling, the resin is blocked and washed according to the manufacturer "s instructions.
Media contaimng NSPRT are passed over the lmmunoatfinity column, and the column is washed under conditions that allow the preferential absorbance of NSPRT (e.g., high lomc strength buffers in the presence of detergent) The column is eluted under conditions that disrupt antibody/NSPRT binding (e.g., a buffer of pH 2 to pH 3, or a high concentration of a chaotrope, such as urea or thiocyanate ion), and NSPRT is collected
XIV. Identification of Molecules Which Interact with NSPRT
Molecules interacting with NSPRT are analyzed using the yeast two-hybπd system as descπbed in Fields, S. and O. Song (1989, Nature 340:245-246), or using commercially available kits based on the two-hybπd system, such as the MATCHMAKER system (Clontech). NSPRT may also be used in the PATHCALLING process (CuraGen Coφ.. New Haven CT) which employs the yeast two-hybrid system in a high-throughput manner to determine all interactions between the proteins encoded by two large libraries of genes (Nandabalan. K. et al (2000) U S. Patent No. 6,057.101)
Various modifications and variations of the described methods and systems of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention Although the invention has been described in connection with certain embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications ot the described modes for caπying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims Table 1
O
Table 2
Table 3
Table 4
Table 5
Program Description Reference Parameter Threshold
ABI I ACTURA A program that removes vector sequences and PE Biosystems, Foster City CA masks ambiguous bases in nucleic acid sequences
ABI/PARACEI FDF A Tast Data Finder uselul in comparing and PE Biosystems, Foster City, CA, Mismatch <50% annotating ammo acid or nucleic acid sequences Paraccl Inc , Pasadena CA
ABI AuloAssembler A program that assembles nucleic acid sequences PE Biosystems, Foster City CA
BI AS T A Basic Local Alignment Search Tool uselul in Allschul, S F et αl ( 1990) J Mol Biol ESTs Probability value= 1 0E 8 sequence similarity search lor ammo acid and 215 403 410, Allschul, S F et al ( 1997) or less nucleic acid sequences BLAST includes live Nucleic Acids Res 25 3389 3402 Full Length sequences Probability lunclions blastp, blastn, blaslx, tblastn, and tblastx value= 1 0E 10 or less
I ASTA A Peai son and Lipman algorithm that searches tor Pearson, W R and D J Lipman ( 1988) Proc ESTs |jsla E value= l 06E-6 similarity between a query sequence and a group ol Natl Acad Sci USA 85 2444 2448, Pearson, Assembled ESTs tasta Identιty= sequences ol the same type FASTA comprises as W R ( 1990) Methods Enzymol 183 63 98, 95% or greater and least l ive lunclions lastα, llasta lastx, tlastx, and and Smith, T F and M S Waterman ( 1981 ) Match length=200 bases or greater, sseaich Adv Appl Math 2 482 489 lastx E vαlue= l 0E 8 or less
Full Length sequences fastx scorc= I 00 or greater
BI IMPS A BLocks lMProvcd Scαrchei that matches α Henιkol l , S and J G Hemkol l ( 1991 ) Nucleic Scores 1 00 or greater, sequence against those in BLOCKS, PRINTS, Acids Res 19 6565-6572, Hemkofl, J G and Ratio of Score/Strength = 0 75 or DOMO, PRODOM, and PFAM databases to search S Hemkoll ( 1996) Methods Enzymol larger, and, if applicable, lor gene families, sequence homology, and structural 266 88 105, and Attwood, T K et al ( 1997) J Probability value= 1 0E-3 or less I mgei print regions Chem Inl Comput Sci 37 417 424
HMMLR An algorithm lor searching a query sequence against Krogh, A et al ( 1994) J Mol Biol Score= 10-50 bits lor PFAM hits, hidden Markov model (HMM) based databases of 235 1501 - 1531 , Sonnhammer, E L L et αl depending on individual protein protein lamily consensus sequences, such as PFAM ( 1988) Nucleic Acids Res 26 320-322 families
Table 5 (cont.)
Program Description Reference Parameter 1 reshold
PiolilcScαn An dlgoiilhin that seaiches lor sli uclural and Gribskov, M et al ( 1988) CABIOS 4 61 66, N imdli/cd quality scoιe>GCG sequence molils m protein sequences that match Gribskov, M et al ( 1989) Methods Enzymol specilied "HIGH" value lor thai sequence patterns del med in Prosite 183 146 159, Bαiroch, A et al ( 1997) pdrticuldr Prosite mod I Nucleic Acids Res 25 217 221 Generally, score= l 4 2 I 5 Phicd A base calling algorithm that examines automated Ewmg, B ct αl ( 1998) Genome Res sequencer traces with high sensitivity and 8 175 185, Ewing, B and P Green probability ( 1998) Genome Res 8 186 194
Phrαp A Phils Revised Assembly Program including Smith, T F and M S Waterman ( 1981 ) Ad v Scorc= 120 or gredler, SWAT and CrossMalch, programs based on Appl Math 2 482 489, Smith, T F and M S Mdlch lenglh= 56 or gredler el licienl implementation ol the Smith Waterman Waterman ( 1981 ) J Mol Biol 147 195 197, algorithm, uselul in searching sequence homology and Green, P , University ol Washington, and assembling DNA sequences Seattle, WA
Consed A graphical tool lor viewing and editing Phrαp Gordon, D et al ( 1998) Genome assemblies Res 8 195 202 S
SPScdii A weight nidlrix andlysis piogrdin thdt scans protein Niclson, H et al ( 1997) Protein Engineering Scoιc=3 5 or gredler sequences lor the presence ol secretory signal 10 I 6, Ctdvcπc J M and S Audιc ( l 997) peptides CABIOS 12 431 439
Molds A program that searches ammo acid sequences lor Bairoch, A et al ( 1997) Nucleic Acids R s pallet ns that iiidlchcd those delincd in Piosite 25 217-221 , Wisconsin Package Program Mdnudl, version 9, pdge M51 59, Genetics Computer Group, Mddtson, WI

Claims

What is claimed is:
1. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of: a) an amino acid sequence selected from the group consisting of SEQ ID NO: 1 -4, b) a naturally occurring amino acid sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-4, c) a biologically active fragment of an amino acid sequence selected from the group consisting of SEQ ID NO : 1 -4, and d) an immunogenic fragment of an amino acid sequence selected from the group consisting of SEQ ID NO: l-4.
2. An isolated polypeptide of claim 1 selected from the group consisting of SEQ ID NO: 1 -4.
3. An isolated polynucleotide encoding a polypepude of claim 1.
4. An isolated polynucleotide encoding a polypeptide of claim 2.
5. An isolated polynucleotide of claim 4 selected from the group consisting of SEQ ID NO:5-8.
6. A recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide of claim 3.
7. A cell transformed with a recombinant polynucleotide of claim 6.
8. A transgenic organism comprising a recombinant polynucleotide of claim 6.
9. A method for producing a polypeptide of claim 1, the method comprising: a) culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide, and said recombinant polynucleotide comprises a promoter sequence operably linked to a polynucleotide encoding the polypeptide of claim 1, and b) recovering the polypeptide so expressed.
10. An isolated antibody which specifically binds to a polypeptide of claim 1.
11. An isolated polynucleotide compπsing a polynucleotide sequence selected from the group consisting of a) a polynucleotide sequence selected from the group consisting of SEQ ID NO.5-8, b) a naturally occurring polynucleotide sequence having at least 70% sequence identity to a polynucleotide sequence selected from the group consisting of SEQ ID NO.5-8, c) a polynucleotide sequence complementary to a), d) a polynucleotide sequence complementary to b), and e) an RNA equivalent of a)-d)
12. An isolated polynucleotide compπsing at least 60 contiguous nucleotides of a polynucleotide of claim 11
13. A method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 11 , the method comprising: a) hybπdizing the sample with a probe comprising at least 20 contiguous nucleotides compπsing a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybndization complex, and, optionally, if present, the amount thereof.
14 A method of claim 1 . wherein the probe compπses at least 60 contiguous nucleotides
15 A method for detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 11, the method comprising a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof, and. optionally, if present, the amount thereof
16 A pharmaceutical composition comprising an effective amount of a polypeptide of claim 1 and a pharmaceutically acceptable excipient
17. A pharmaceutical composition of claim 16, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1-4.
18. A method for treating a disease or condition associated with decreased expression of functional NSPRT, comprising administering to a patient in need of such treatment the pharmaceutical composition of claim 16.
19. A method for screening a compound for effectiveness as an agonist of a polypeptide of claim 1, the method comprising: a) exposing a sample comprising a polypeptide of claim 1 to a compound, and b) detecting agonist activity in the sample.
20. A pharmaceutical composition comprising an agonist compound identified by a method of claim 19 and a pharmaceutically acceptable excipient.
21. A method for treating a disease or condition associated with decreased expression of functional NSPRT, comprising administering to a patient in need of such treatment a pharmaceutical composition of claim 20.
22. A method for screening a compound for effectiveness as an antagonist of a polypeptide of claim 1, the method comprising: a) exposing a sample comprising a polypeptide of claim 1 to a compound, and b) detecting antagonist activity in the sample.
23. A pharmaceutical composition comprising an antagonist compound identified by a method of claim 22 and a pharmaceutically acceptable excipient.
24. A method for treating a disease or condition associated with overexpression of functional NSPRT, comprising administering to a patient in need of such treatment a pharmaceutical composition of claim 23.
25. A method of screening for a compound that specifically binds to the polypeptide of claim 1, said method comprising the steps of: a) combining the polypeptide of claim 1 with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide of claim 1 to the test compound, thereby identifying a compound that specifically binds to the polypeptide of claim 1
26 A method of screemng for a compound that modulates the activity of the polypeptide of claim 1, said method compπsing a) combimng the polypeptide of claim 1 with at least one test compound under conditions permissive for the activity of the polypeptide of claim 1 , b) assessing the activity of the polypepude of claim 1 in the presence of the test compound, and c) companng the activity of the polypeptide of claim 1 in the presence of the test compound with the activity of the polypeptide of claim 1 in the absence of the test compound, wherein a change in the activity of the polypeptide of claim 1 in the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide of claim 1
27 A method for screemng a compound for effectiveness in alteπng expression of a target polynucleotide, wherein said target polynucleotide compπses a sequence of claim 5, the method compnsing a) exposing a sample compπsing the target polynucleotide to a compound, and b) detecting altered expression of the target polynucleotide
28 A method for assessing toxicity of a test compound, said method comprising a) treating a biological sample contaimng nucleic acids with the test compound, b) hybπdizing the nucleic acids of the treated biological sample with a probe compnsing at least 20 contiguous nucleotides of a polynucleotide of claim 11 under conditions whereby a specific hybndization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide compπsing a polynucleotide sequence of a polynucleotide of claim 11 or fragment thereof, c) quantifying the amount of hybridization complex, and d) compaπng the amount of hybridization complex in the treated biological sample with the amount of hybndization complex in an untreated biological sample, wherein a difference in the amount of hybndization complex in the treated biological sample is indicative of toxicity ot the test compound
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WO2001007470A3 (en) 2001-10-11
WO2001007470A2 (en) 2001-02-01

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