EP1200586A2 - Menschliche nervensystem angewandte proteinen - Google Patents

Menschliche nervensystem angewandte proteinen

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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Withdrawn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
English (en)
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/de
Withdrawn legal-status Critical Current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
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    • A61P17/00—Drugs for dermatological disorders
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    • 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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